Background
S-phase kinase-associated protein 2 (Skp2), a mammalian F-box protein, displays S-phase-promoting function, through ubiquitin-mediated proteolysis of the CDK inhibitor p27. Skp2 has been shown to regulate cellular proliferation by targeting several cell cycle-regulated proteins for ubiquitination and degradation. Skp2 has also been demonstrated to display an oncogenic function since its overexpression has been observed in many human cancers
1
. High expression of Skp2 was reported to correlate with reduced overall survival in patients with myxofibrosarcoma
2
3
. Di Vizio et al.
4
found that Skp2 expression correlates with poor prognosis in gastrointestinal stromal tumors (GIST). Oliveira found that Skp2 expression is associated with cell proliferation and a worse prognosis in 182 soft tissue sarcomas
5
. In a previous study we showed that high expression of Skp2 was a negative prognostic factor for DSS
6
. Interestingly, this correlation was statistically significant in females only, not in males. This may be related to differences in expression of sexual hormone receptors (ER and PGR) in male and female STS patients
7
8
. In previous studies, we have shown the prognostic value of female steroid hormone receptors in STSs, both alone and in coexpression with TGF-β, fascin and Akt isoforms
7
8
9
. Such prognostic impact is not surprising, since both ER and PGR regulate growth and cell differentiation upon ligand-dependent and ligand-independent activation and are in essence growth factors. However, the prognostic significance of Skp2 related to ER and PGR in STS has not been sufficiently investigated.
The purpose of this study is to clarify the prognostic significance of expression of Skp2 related to age, gender and female steroid hormone receptors (ER and PGR) in non-gastrointestinal stromal tumor (non-GIST) STS. To achieve this, we analyzed the expression of these markers in 193 patients with non-GIST STS in relation to demographic and other clinicopathological variables. Our major hypothesis is that a different prognostic significance of Skp2 in men and women exists and is related to diverse gender expressions of ER and PGR.
Methods
Primary tumor tissues from patients diagnosed with STS at the University Hospital of North Norway (UNN) from 1973 to 2006 and the Hospitals of Arkhangelsk region, Russia, were used in this retrospective study. In total, 496 potentially suitable patient records were identified from the hospitals’ databases. Of these, 247 patients were excluded due to missing clinical data (n = 86) or inadequate material for histological examination (n = 161). In addition, 33 were excluded because of metastasis at the time of the diagnosis, 13 were excluded because they had no surgery, and 10 patients had both metastasis and no surgery, leaving a total of 193 patients eligible for this study. This report includes data for 131 Norwegian patients and 62 Russian patients followed until September 2009. The median follow-up was 38 (range 0–392) months. Complete demographic and clinical data were collected retrospectively. Formalin-fixed and paraffin-embedded tumor specimens were obtained from the archives of the Departments of Pathology at UNN and Arkhangelsk. The tumors were graded according to the French Fédération Nationales des Centres de Lutte Contre le Cancer (FNCLCC) system [WHO Tumors of Soft Tissue and bone, 2002]. Wide resection margins were defined as wide local resection with free microscopic margins or amputation of the affected limb or organ. Non-wide resection margins were defined as either marginal or intralesional resection margins.
Microarray construction
Two pathologists (AV and SWS) reviewed the histology of all soft tissue sarcoma cases. Tissue microarrays (TMAs) were constructed for high-throughput molecular pathology research
10
. The most representative areas of viable tumor cells were carefully selected and marked on the hematoxylin and eosin (HE) slides for the corresponding donor blocks and sampled for the tissue microarray collector blocks. The TMAs were assembled using a tissue-arraying instrument (Beecher Instruments).
Studies suggest that punching multiple 0.6 mm cores from different regions captures the heterogeneity of the tumors more accurately than a single 2 to 4 mm core
11
. We therefore chose to use two 0.6-mm cores of viable neoplastic tissue. After reviewing all original sections of the tumor and taking heterogeneity into consideration, the two cores were selected to be as representative as possible (different areas). To include all core samples, 12 tissue array blocks were constructed. Multiple 4-μm sections were cut with a Micron microtome (HM355S) and stained with specific antibodies for immunohistochemistry (IHC).
Immunohistochemistry (IHC)
The applied antibodies were subjected to in-house validation by the manufacturer of IHC analysis on paraffin-embedded material. All staining was performed in the Ventana Benchmark XT automated slide stainer (Ventana Medical System, Illkirch, France). Before staining, the sections were incubated over night at 60 degrees Celsius. Tissue sections were incubated with primary mouse monoclonal antibodies recognizing Skp2 (Zymed, catalog number 18–0307, 1:10), ER (Ventana, catalog number 790–4324, ready to use) and PGR (Ventana, catalog number 790–4296). The incubation periods were 40 minutes for Skp2, 32 minutes for ER and 24 min for PGR. This was followed by application of liquid diaminobenzidine as substrate-chromogen, yielding a brown reaction product at the site of the target antigen (Ventana iView DAB Detection Kit, catalog number 760–091). iVIEW DAB Detection Kit is an indirect biotin streptavidin system for detecting mouse and rabbit primary antibodies. The DAB chromogen produces a dark brown precipitate that is readily visualized by light microscopy. All reagents are provided pre-diluted by the manufacturer for use in Ventana Benchmark XT. Finally, slides were counterstained with hematoxylin to visualize the nuclei. For each antibody, including negative controls, all TMA staining were performed in a single experiment. In the TMA we also used cores from carcinomas and normal tissue as positive and negative controls.
Scoring of IHC
The ARIOL imaging system (Genetix, San Jose, CA) was used to scan the slides for antibody staining of the TMAs. The specimens were scanned at a low resolution (1.25×) and a high resolution (20×) using an Olympus BX 61 microscope with an automated platform (Prior). The slides were loaded in the automated slide loader (Applied Imaging SL 50). Representative and viable tissue sections were scored manually on a computer screen semi-quantitatively for nuclear and/or cytoplasmic staining. The expression of Skp2, ER and PGR was scored as: 0, negative; 1, weak; 2, intermediate and 3, strong (Figure 1). The score for each patient was based on the mean scoring of cores from one or several biopsies. To achieve maximal reproducibility in all cases, every staining was dichotomized (negative and positive expression). Positive expression was defined as mean score > 0. All samples were anonymized and independently scored by two pathologists (AV and SWS). In case of disagreement, the slides were re-examined and the observers reached a consensus. When assessing a variable for a given score, the scores of the other variables and the outcome were hidden from the observers.
<p>Figure 1</p>Pictures of cores
Pictures of cores. Immunohistochemistry microscopic pictures of tissue micro array of soft tissue sarcoma representing different expression of Skp2 and ER. (A) Skp2 low score; (B) Skp2 high score; (C) ER low score; (D) ER high score; (E) PGR low score; (F) PGR high score; Original magnification ×100 and ×400.
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Statistical methods
All statistical analysis was performed using the statistical package SPSS (Chicago, IL), version 18. The IHC scores from each observer were compared for inter-observer reliability by use of a two-way random effects model with absolute agreement definition. The intra-class correlation coefficient (reliability coefficient) was obtained from these results.
Chi-square and Fisher exact tests were used to examine the association between molecular marker expression and various clinicopathological parameters. Univariate analyses were done using the Kaplan-Meier method, and statistical significance between survival curves was assessed by the log rank test. Disease-specific survival (DSS) was determined from the date of histologically confirmed STS diagnosis. Correlation of marker expression was done using the Pearson correlation (2-tailed) at the 0.05 and 0.01 levels.
Multivariate analysis was carried out using the Cox proportional hazards model to assess the specific impact of each pre-treatment variable on survival in the presence of other variables. Variables of significant value from the univariate analysis were entered into the Cox regression analysis. Probability for stepwise entry and removal was set at 0.05 and 0.10, respectively. The significance level used was p < 0.05.
Consent
The National Cancer Data Inspection Board and The Regional Committee for Research Ethics (REK nord) approved the study. The material was collected from our approved biobank for paraffin embedded material and slides. The Regional Committee approved that written consent from the patients for their information to be stored in the hospital database and used for research was not needed because most of the material was more than 10 years old, and most of the patients being dead. The ethics committee specifically waived the need for consent. Data were analyzed anonymously.
Results
Clinicopathological variables
Demographic, clinical, and histopathological variables are shown in Table 1. Patient age ranged from 0–89 years (mean 55 years), and 42% of patients (81/193) were male. Treatment for all patients included surgery: 104 patients received surgery only; 52 patients received surgery and radiotherapy; 28 patients received surgery and chemotherapy; 9 patients received surgery, radiotherapy and chemotherapy. The 5-year survival for patients with wide and non-wide resection margins was 66% and 46% respectively, Table 1.
<p>Table 1</p>
Prognostic clinicopathological variables as predictors for disease-specific survival of soft tissue sarcomas (univariate analysis, log rank test), N = 193
Characteristic
Patients (n)
Patients (%)
Median survival (months)
5-Year survival (%)
P
Abbreviations: MF/MFT, malignant fibroblastic/myofibroblastic tumors; MPNST, malignant peripheral nerve sheath tumor; STS, soft tissue sarcomas; NR, not reached; NOS, non specified.
Age
<20 years
17
9
190
47
0.064
20–59 years
85
44
235
63
≥60 years
91
47
111
51
Gender
Male
81
42
235
60
0.087
Female
112
58
180
53
Nationality
Norwegian
131
68
228
62
0.005
Russian
62
32
81
44
Histology
Pleomorphic sarcoma
57
30
52
45
0.031
Leiomyosarcoma
47
24
89
64
Liposarcoma
32
17
NR
71
MF/MFT
16
8
123
56
Angiosarcoma
8
4
10
38
Rhabdomyosarcoma
9
5
NR
67
MPNST
9
5
NR
56
Synovial sarcoma
12
6
31
30
Other STS
3
2
NR
-
Tumor localization
Extremities
78
40
201
56
0.922
Trunk
37
19
214
53
Retroperitoneum
27
14
135
51
Head/Neck
13
7
191
58
Visceral
38
20
202
62
Tumor size
<5 cm
57
30
257
69
0.026
5–9 cm
73
38
183
54
≥10 cm
61
32
127
48
Missing
2
1
Malignancy grade FNCLCC
1
54
28
NR
81
<0.001
2
76
39
80
55
3
63
33
28
36
Surgical margins
Wide
97
50
254
66
<0.001
Non-wide
96
50
128
46
Chemotherapy
No
156
81
207
57
0.669
Yes
37
19
180
51
Radiotherapy
No
132
68
216
58
0.190
Yes
61
32
152
52
Inter-observer variability
There was good scoring agreement between the two investigating pathologists. The IHC scores from each observer were compared using a two-way random effects model with absolute agreement definition. The intra-class correlation coefficients (reliability coefficients, r) obtained from these results were 0.94 for Skp2 (p < 0.001), 0.92 for ER (p < 0.001) and 0.96 for PGR (p < 0.001).
Univariate analyses
Nationality, histology, tumor size, malignancy grade and surgical margins were all significant indicators for disease-specific survival (DSS) in univariate analyses (Table 1). Table 2 shows the percentage of high expression of ER, PGR and Skp2 in the different histological subtypes. Chi-square test showed no differences in overall expression of ER, PGR and Skp2 with respect to the different histological subtypes.
<p>Table 2</p>
Percentage of high expression of ER, PGR and Skp2 in the different histological subtypes N = 193
Histology
N
ER (%)*
PGR (%)**
Skp2 (%)***
* Chi 8.516, p = 0.385.
** Chi 10.238, p = 0.249.
*** Chi 8.596, p = 0.377.
Abbreviations: MF/MFT, malignant fibroblastic/myofibroblastic tumors; MPNST, malignant peripheral nerve sheath tumor.
Chi-square test showed no differences in percentage of high expression of ER, PGR and Skp2 in the different histological subtypes.
Pleomorphic sarcoma
57
40
26
37
Leiomyosarcoma
47
50
43
40
Liposarcoma
32
35
23
21
MF/MFT
16
27
29
36
Angiosarcoma
8
25
13
29
Rhabdomyosarcoma
9
50
56
67
MPNST
9
11
11
44
Synovial sarcoma
12
40
27
50
Other STS
3
67
33
67
Total
193
39
30
38
In univariate analyses, increased expression of Skp2 (p = 0.050) correlated significantly with reduced DSS, (Table 3 and Figure 2). No such relationship was apparent for ER and PGR when males and females were combined in one group.
<p>Figure 2</p>Survival plots ER and PGR
Survival plots ER and PGR. Disease-specific survival curves for high and low expression of ER and PGR in male (N = 81) and female (N = 112) patients with soft tissue sarcomas.
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<p>Table 3</p>
Expression of markers, gender and their prediction for disease-specific survival in patients with soft tissue sarcomas (univariate analysis; log-rank test), All = 193, Males = 81, Females = 112
Marker expression
Patients (n)
Patients (%)
Median survival (months)
5-year survival (%)
P
Abbreviations: NR, not reached.
Skp2, all
Low
109
56
NR
63
0.050
High
67
45
59
50
Missing
17
9
Skp2, men
Low
50
62
NR
63
0.577
High
23
28
67
61
Missing
8
10
Skp2, women
Low
59
53
NR
63
0.066
High
44
39
49
44
Missing
9
8
ER, all
Low
112
58
123
57
0.725
High
72
67
91
57
Missing
9
5
ER, men
Low
49
60
NR
69
0.089
High
29
36
58
49
Missing
3
4
ER, women
Low
63
56
57
47
0.041
High
43
38
NR
62
Missing
6
5
PGR, all
Low
132
68
NR
62
0.101
High
57
30
52
46
Missing
4
2
PGR, men
Low
64
79
NR
69
0.010
High
15
19
41
33
Missing
2
2
PGR, women
Low
68
61
80
55
0.832
High
42
38
74
51
Missing
2
2
In subgroup analyses (Tables 3 and 4), increased PGR expression in men (p = 0.010) and in patients older than 60 years (p = 0.043) was associated with a reduced DSS. Increased ER expression in women was associated with longer DSS (p = 0.041). High expression of ER were associated with favorable survival in patients with rhabdomyosarcoma (N = 9, p = 0.040). High expression of ER was associated with poor survival in patients with synovial sarcoma (N = 12, p = 0.010). There were no significant differences in survival according to high or low expression of Skp2 in any of the histological subtypes (data not shown).
<p>Table 4</p>
Expression of markers, age and their prediction for disease-specific survival in patients with soft tissue sarcomas (univariate analysis; log-rank test)
Marker expression
Patients (n)
Patients (%)
Median survival (months)
5-Year survival (%)
P
Abbreviations: NR, not reached.
Skp2, <60 years, N = 99
Low
52
53
NR
71
0.074
High
38
38
67
56
Missing
9
9
Skp2, ≥60 years, N = 94
Low
57
61
80
57
0.188
High
29
31
36
42
Missing
8
9
ER, <60 years, N = 99
Low
55
56
127
59
0.197
High
40
40
NR
67
Missing
4
4
ER, ≥60 years, N = 94
Low
57
61
80
55
0.293
High
32
34
52
44
Missing
5
5
PGR, <60 years, N = 99
Low
63
64
NR
67
0.488
High
34
34
NR
55
Missing
2
2
PGR, ≥60 years, N = 94
Low
69
73
91
57
0.043
High
23
24
39
32
Missing
2
2
In patients with low expression of ER (N = 112), men had better 5-year survival (69%) compared to women (47%, p = 0.002), while there were no differences (p = 0.376) between men and women in patients with high expression of ER (N = 72). In patients with low expression of PGR (N = 132), men had better 5-year survival (69%) compared to women (55%, p = 0.013), while there were no differences (p = 0.271) between men and women in patients with high expression of PGR (N = 57). There were no differences in survival between men and women in univariate analyses of patients with low (N = 109, p = 0.529) or high (N = 67, p = 0.233) expression of Skp2 (data not shown).
In co-expression analyses (Table 5) Skp2 negative / ER negative profile was associated with longer DSS (p = 0.049). In women a Skp2 positive and ER negative profile was associated with reduced DSS (p = 0.021), Table 5 and Figure 3. In men a double negative ER/PGR profile was associated with longer DSS (p = 0.013) while in women a double positive ER/PGR was associated with longer DSS (p = 0.001). In patients younger than 60 years the combination ER negative and PGR positive was associated with shorter DSS. In the whole cohort of patients a triple positive expression of ER, PGR and Skp2 was associated with longer DSS (p = 0.005), Figure 3. Triple negative expression of ER, PGR and Skp2 was also associated with longer DSS, but not statistically significant (p = 0.068), Figure 3. ER negative / PGR positive co-expression was associated with shorter DSS regardless of Skp2 expression, Table 6.
<p>Figure 3</p>Survival plots co-expression
Survival plots co-expression. Disease-specific survival curves for co-expression of Skp2, ER or PGR in males (N = 81), females (N = 112) and co-expression of ER and PGR in Skp2 negative (N = 109) and Skp2 positive (N = 67) patients.
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<p>Table 5</p>
Co-expression of Skp2/ER, Skp2/PGR and their prediction for disease-specific survival in patients with soft tissue sarcomas (univariate analysis; log-rank test), All = 193, Men = 81, Women = 112
Co-expression
Patients (n)
Patients (%)
Median survival (months)
5-Year survival (%)
P
Abbreviations: NR, not reached.
Skp2 / ER, all
Low/low
66
34
NR
67
0.049
Low/high
39
20
91
59
High/low
35
18
57
44
High/high
30
16
NR
58
Missing
23
12
Skp2 / ER, men
Low/low
33
41
NR
72
0.427
Low/high
16
20
37
50
High/low
11
14
NR
72
High/high
11
14
63
58
Missing
10
12
Skp2 / ER, women
Low/low
33
29
127
61
0.021
Low/high
23
21
91
65
High/low
24
21
31
32
High/high
19
17
NR
58
Missing
13
12
Skp2 / PGR, all
Low/low
80
41
NR
71
0.056
Low/high
25
13
54
46
High/low
40
21
59
49
High/high
27
14
67
51
Missing
21
11
Skp2 / PGR, men
Low/low
41
51
NR
73
0.141
Low/high
7
9
26
29
High/low
18
22
NR
61
High/high
5
6
67
60
Missing
10
12
Skp2 / PGR, women
Low/low
39
35
NR
68
0.234
Low/high
18
16
75
54
High/low
22
20
29
39
High/high
22
20
57
49
Missing
11
10
<p>Table 6</p>
Co-expression of ER/PGR and their prediction for disease-specific survival in patients with soft tissue sarcomas (univariate analysis; log-rank test)
Co-expression
Patients (n)
Patients (%)
Median survival (months)
5-year survival (%)
P
Abbreviations: NR, not reached.
ER / PGR, all, N = 193
Low/low
84
44
NR
69
<0.001
Low/high
26
13
38
24
High/low
41
21
62
52
High/high
31
16
NR
64
Missing
11
6
ER / PGR, men, N = 81
Low/low
39
48
NR
79
0.013
Low/high
9
11
41
33
High/low
23
28
63
53
High/high
6
7
37
33
Missing
4
5
ER / PGR, women, N = 121
Low/low
45
40
89
59
0.001
Low/high
17
15
31
19
High/low
18
16
29
50
High/high
25
22
NR
72
Missing
7
6
ER / PGR, <60 years, N = 99
Low/low
41
41
NR
72
0.001
Low/high
13
13
31
23
High/low
19
19
NR
58
High/high
21
21
NR
76
Missing
5
5
ER / PGR, ≥60 years, N = 94
Low/low
43
46
NR
64
0.052
Low/high
13
14
39
26
High/low
22
23
58
47
High/high
10
11
37
40
Missing
6
6
ER / PGR, Skp2 low, N = 109
Low/low
55
50
NR
76
0.068
Low/high
9
8
68
25
High/low
23
21
91
61
High/high
16
15
75
56
Missing
6
6
ER / PGR, Skp2 high, N = 67
Low/low
21
31
89
55
0.005
Low/high
14
21
31
29
High/low
17
25
29
42
High/high
13
19
NR
77
Missing
2
3
Taking into consideration the possible distortion of results by gender-related sarcomas (i.e. leiomyosarcoma in uterus) we have attempted to exclude these sarcomas and recalculate all analyses. There were no significant differences in the results compared to those obtained without exclusion of gender-related sarcomas (data not shown).
Multivariate analyses
Significant demographic, clinicopathological and expression variables from the univariate analyses were entered into the multivariate Cox regression analysis (Table 7). In the multivariate analyses, age (p = 0.012), malignancy grade (p < 0.001), wide resection margins (p = 0.010), ER negative / PGR positive co-expression (p = 0.002) and ER positive / PGR negative co-expression (p = 0.015) were independent negative prognostic factors for DSS. In women, expression of Skp2 (p = 0.006) was associated with reduced DSS. In women, tumor size (p = 0.020) and nationality (p = 0.014) were independent prognostic factors for DSS, Table 7. In multivariate analyses co-expression of Skp2/ER or Skp2/PGR were not stronger prognosticators for DSS than single expression of Skp2, ER and PGR (data not shown).
<p>Table 7</p>
Results of Cox regression analysis summarizing prognostic factors in patients with soft tissue sarcomas
All patients, N = 193
Men, N = 81
Women, N = 112
Factor
Hazard ratio
95% CI
P
Hazard ratio
95% CI
P
Hazard ratio
95% CI
P
* Overall significance as a prognostic factor. The difference between the individual p-value and total p-value in the multivariate analysis is relevant in cases where there are more than two categories for a given variable. Overall p-value is calculated based on a general assessment of all categories for the given variable, but the individual p-value only calculates the significance of a given category versus the reference category.
Age
0–59 years
1.00
1.00
1.00
≥60 years
1.84
1.15-2.95
0.012
1.69
0.65-4.41
0.282
1.51
0.83-2.77
0.179
Nationality
Norwegian
1.00
1.00
1.00
Russian
1.49
0.88-2.52
0.143
1.39
0.41-4.66
0.598
2.51
1.20-5.21
0.014
Tumor size
<5 cm
1.00
0.138*
1.00
0.668*
1.00
0.020*
5–9 cm
1.47
0.79-2.73
0.226
1.68
0.54-5.25
0.372
1.71
0.77-3.77
0.187
≥10 cm
1.91
1.01-3.60
0.047
1.32
0.40-4.39
0.652
3.14
1.38-7.15
0.006
Malignancy grade FNCLCC
1
1.00
<0.001*
1.00
<0.001
1.00
0.004*
2
2.72
1.36-5.46
0.005
3.07
0.86-10.96
0.084
4.33
1.76-10.67
0.001
3
4.61
2.26-9.40
<0.001
15.47
4.36-54.97
<0.001
4.23
1.64-10.89
0.003
Resection margins
Wide
1.00
1.00
1.00
Non-wide
1.87
1.16-3.02
0.010
7.69
2.67-22.16
<0.001
0.81
0.42-1.54
0.512
Skp2
Low
1.00
1.00
1.00
High
1.48
0.87-2.52
0.151
0.46
0.19-1.12
0.088
2.52
1.31-4.85
0.006
ER / PGR
Low/low
1.00
0.006*
1.00
0.004*
1.00
0.216*
Low/high
2.64
1.43-4.85
0.002
4.99
1.31-18.97
0.018
1.91
0.89-4.11
0.097
High/low
2.07
1.15-3.73
0.015
8.35
2.55-27.36
<0.001
1.76
0.79-3.93
0.170
High/high
1.16
0.57-2.38
0.682
4.50
0.96-21.13
0.056
0.92
0.36-2.35
0.868
Discussion
In this large-scale study, we evaluated the prognostic significance of expression of Skp2 related to age, gender, ER and PGR in 193 STS patients. Our hypothesis was confirmed. We found diverse prognostic DSS impacts from gender related expression of Skp2, ER, PGR and DSS in STS. In men, but not women, an ER positive/PGR negative co-expression profile was an independent negative prognostic factor for DSS. In women, but not men, high expression of Skp2 was associated with reduced DSS. High expression of ER reduced the negative impact of Skp2 in women. While women with the Skp2 positive / ER positive phenotype had favorable survival, women with the Skp2 positive / ER negative phenotype had poor survival. To the best of our knowledge, this is the first prognostic evaluation of Skp2 related to the female hormone receptors ER and PGR in STS.
Expression of ER and PGR is a routinely investigated indicator of endocrine therapy success in breast cancer
12
13
and a modest, but significantly better overall survival of anti-estrogen receptor therapy has been documented
14
. ER and PGR are also reported to be positive prognosticators of uterine leiomyosarcomas
15
. However, extra-uterine sarcomas have barely been explored in this context. The distribution and prognostic value of expression of these steroid hormone receptors in STS are therefore of great scientific interest. In our study, in the univariate analyses, ER showed a significantly favorable influence on survival in female patients, but not in males. PGR was an unfavorable prognosticator for men, but not for women. In multivariate analysis ER positive / PGR negative co-expression is an independent negative prognostic factor for DSS in males, but not in females.
We have modified the Allred score for STS and used 1% positivity as cut-off value
7
16
. The strong and moderate (score 3 and 2, respectively) hormone receptor expression occurred mostly in sarcomas of uterus, pelvis and breast, while the weak (score 1) expression of both ER and PGR was surprisingly evenly distributed among location, gender and age. Generally, 39% of the tumors expressed ER and 30% expressed PGR in our material. Roughly half of the patients expressed at least one of these receptors. The findings are in partial agreement with findings of Chaudhuri et al.
17
who found ER to be positive in 24% of STS.
Huang et al. suggested that the therapeutic strategies designed to reduce Skp2 may play an important clinical role in treatment of breast cancer cells, especially ER/HER2 negative breast cancers
18
. Voduc et al. found cyclin E and Skp2 to be prognostic for breast cancer-specific survival in univariate analyses. Double positive expression of cyclin E / Skp2 was associated with young age at diagnosis, grade 3 tumors, ER-negative status and HER2 negative status
19
. Zheng et al. found that higher levels of Skp2 were detected more frequently in ER-negative breast cancer tumors and tumors metastatic to the axillary lymph nodes
20
. Signoretti et al. also found that higher levels of Skp2 are present more frequently in ER-negative tumors than in ER-positive cases. The subset of Skp2 positive / ER negative breast carcinomas were also characterized by high tumor grade and HER2 negative
21
. In our material, the five year DSS in Skp2 positive / ER negative women with STS was 32% compared to 58% in Skp2 positive / ER positive women (P = 0.021).
In our previous work we have shown that ER and PGR expression possess variable prognostic significance depending on gender, both per se and in co-expression with TGF-β, fascin and Akt isoforms
7
8
9
. In the present study, the prognostic diversity of Skp2, ER and PGR in men and women was seen in the different co-expression profiles: female patients with Skp2 positive / ER negative profile had decreased survival rates. For men, the Skp2 negative / ER negative profile was the most favorable phenotype. PGR expression in men, but not women, was associated with a shorter DSS. ER expression in women, but not men, was associated with a longer DSS. The ER negative / PGR positive profile was a significantly unfavorable factor for the whole patient cohort both in univariate and multivariate analysis. Interestingly, such a profile occurred in only 2% of patients in one large-scale study based on 3000 breast cancer cases
22
, while in our STS study this profile was seen in 13% of tumors.
The data collection introduced problems in identifying adequate numbers of similar patients with similar tumors and with the same treatment traditions. These are well known problems when conducting STS studies. Our findings are in large hypothesis generating, and to be more conclusive future STS studies must be based on large, multi-institutional and multinational studies with possibilities to establish adequately sized STS patient cohorts of homogenous tumor groups. However, all tumors investigated herein had mesenchymal derivation and belong to the same generic group.
Conclusions
In conclusion, there were different prognostic impacts of expression of Skp2, ER, PGR and DSS in male and female patients with STS. In men, but not in women, ER positive / PGR negative co-expression was an independent negative prognostic factor for DSS. In women, but not in men, expression of Skp2 was associated with reduced DSS.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SWS, TK, AV, TD, RMB and LTB participated in the design of the study. TK, ES and AV collected clinical information. SWS and AV reviewed all the histological diagnosis, histological grading, selected and marked the slides for TMA construction. SWS, TK and AV performed the experiments. SWS, TK, AV, TD, RMB and LTB performed the statistical analysis. SWS, TK, AV, TD, ES, KAS and LTB contributed reagents/materials/analysis tools. SWS, TD, ES, KAS, RMB and LTB drafted the manuscript. All authors read and approved the final manuscript.