Jpn J Clin Oncol 2014;44(5)486 – 492 doi:10.1093/jjco/hyu020 Advance Access Publication 18 March 2014
Prognostic Impact of Fatty Acid Synthase Expression in Upper Urinary Tract Urothelial Carcinoma Shinsuke Hamada1,*, Akio Horiguchi1, Takako Asano1, Kenji Kuroda1, Junichi Asakuma1, Keiichi Ito1, Tomohiko Asano1, Kosuke Miyai2 and Keiichi Iwaya2 1
Department of Urology, National Defense Medical College, Tokorozawa, Saitama and 2Department of Basic Pathology, National Defense Medical College, Tokorozawa, Saitama, Japan
Received November 6, 2013; accepted February 10, 2014
Objective: Fatty acid synthase has been shown to be highly expressed in various types of cancers with increased tumour aggressiveness. In this study we examined the level of fatty acid synthase expression in surgically resected upper urinary tract urothelial carcinoma specimens and evaluated the relations between fatty acid synthase expression and the patients’ pathological features and clinical outcomes. Methods: Sections of paraffin-embedded tumour specimens from 113 patients who underwent surgical treatment for upper urinary tract urothelial carcinoma were immunostained with a polyclonal fatty acid synthase antibody, and a tumour was considered to have high fatty acid synthase expression if .50% of the cancer cells stained with moderate-to-strong intensity. Associations between fatty acid synthase expression and the patients’ pathological parameters and survival were analyzed statistically. Results: During the follow-up time (median: 46.8 months), 61 patients (54.0%) had recurrence and 17 (15.0%) died of upper urinary tract urothelial carcinoma. High fatty acid synthase expression was significantly associated with high tumour grade (P ¼ 0.0273). Patients with high fatty acid synthase expression had significantly worse recurrence-free survival and extravesicalrecurrence-free survival than those with low fatty acid synthase expression (P ¼ 0.0171, P ¼ 0.0228, respectively). In multivariate analysis, high fatty acid synthase expression was an independent predictor of shortened recurrence-free survival (P ¼ 0.0220, hazard ratio (HR) ¼ 1.970). Conclusions: Fatty acid synthase expression in upper urinary tract urothelial carcinoma is an independent predictor for tumour recurrence. Patients with high fatty acid synthase expression in upper urinary tract urothelial carcinoma should be followed carefully and adjuvant therapy for them should be considered.Keywords: fatty acid synthase, upper urinary tract urothelial carcinoma, prognosis Key words: fatty acid synthase – upper urinary tract urothelial carcinoma – prognosis
INTRODUCTION Fatty acid synthase (FAS) is a biosynthetic enzyme for lipogenesis and the production of long-chain fatty acids from acetyl-coenzyme A (CoA) and malonyl-CoA (1,2). Fatty acids are tightly regulated by diet, hormones and growth factors;
and FAS expression and activity levels are low in normal cells except those in liver, endometrium, mammary glands and adipose tissue (3). On the other hand, FAS is highly expressed in various cancers (breast, colorectum, ovary, oesophagus, stomach, lung, head and neck and thyroid cancers and soft
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*For reprints and all correspondence: Shinsuke Hamada, Department of Urology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. E-mail: [email protected]
Jpn J Clin Oncol 2014;44(5)
PATIENTS AND METHODS PATIENTS We reviewed the records of 125 patients who underwent surgical treatment for UTUC at the National Defense Medical College between March 1999 and December 2010. Patients with carcinoma in situ and patients with histology results showing cancer other than UC were excluded from this study. Consequently, a total of 113 patients were enrolled in this study: 65 (57.5%) who had undergone open nephroureterectomy and partial cystectomy, 41 (36.3%) who had undergone laparoscopic nephroureterectomy and partial cystectomy, three (2.7%) who had undergone nephroureterectomy and total cystectomy, two (1.8%) who had undergone nephroureterectomy, and two (1.8%) who had undergone partial ureterectomy and ureterocystoneostomy. Regional lymph node dissection was performed in patients whose preoperative evaluation indicated a T3 tumour or the possibility nodal involvement. The follow-up period, calculated using the date of the operation and the date of the last recorded follow-up, was 3.9 – 152.3 months (median: 46.8 months). The patients with pT3 tumours were recommended to receive two or three cycles of adjuvant gemcitabin and cisplatin (GC) or methotrexate, vinblastine, doxorubicin and cisplatin (M-VAC) regimens. Patients were assessed by cystoscopy and urine cytology every 3 months for 2 years after the operation, and computed tomography or magnetic resonance imaging was conducted every 6 months. Intravesical recurrence was considered to have occurred if UC was detected in residual bladder
by cystoscopy, and extravesical recurrence was considered to have occurred if any nodal and/or visceral metastasis was evident on radiograph examination. Recurrence-free survival (RFS) was calculated as the period from the operation to the appearance of intravesical or extravesical recurrence. Cancer-specific survival (CSS) was calculated as the period from the operation to the date of death from UTUC. All pathologic specimens were reviewed by two expert genitourinary pathologists (K.M. and K.I.). Tumour stages were classified according to the 2002 TNM classification. Tumour grades were classified into two levels (high and low) based on the World Health Organization (WHO) grading system. This study was approved by the National Defense Medical College ethical committee. IMMUNOHISTOCHEMISTRY Sections 4 mm thick were cut from formalin-fixed, paraffinembedded blocks of UTUC, deparaffinized in xylene and rehydrated through graded ethanols. Endogenous peroxidase activity was quenched by immersing the sections in DAKO Peroxidase Blocking Reagent (Dako Corporation) for 10 min. The sections were incubated in 10% normal goat serum in phosphate buffered saline (PBS) for 60 min at room temperature. After the sections were incubated 60 min in a 1:50 dilution of rabbit polyclonal anti-FAS antibody (Immuno-Biological Laboratories Co., Fujioka, Japan) in PBS, the slides were stained by using a Simple Stain Max PO kit (Nichirei Corporation, Tokyo, Japan) according to the manufacturer’s instructions. Reaction products were visualized by immersing the slides in diaminobenzidine tetrahydrochloride (DAB) for 2 min. The sections were finally counterstained with haematoxylin. Periureteral adipose tissues served as positive internal controls for FAS immunoreactivity. Sections stained with unimmunized-rabbit serum instead of the primary antibody were used as negative controls. Cytoplasmic immunoreactivity was taken into account for FAS protein expression. Intensities of FAS immunoreactivity were classified semiquantitatively into four categories: nonstaining (negative), weak (score 1þ), moderate (score 2þ) and strong (score 3þ) (7,13). Immunoreactivity in the tumour was considered to indicate high FAS expression if .50% of the tumour cells evaluated in the slides showed moderate (2þ) to strong (3þ) immunoreactivity. The staining intensities in the entire slide of each tumour were evaluated and scores were assigned according to the highest intensity. The cut-off value was determined on the basis of the following criteria: (i) the cut-off giving the best P values at testing and (ii) the cut-off used in previous reports (11). Immunohistochemistry data were evaluated independently by two pathologists (K.M. and K.I.) blinded from any clinical data. Any discrepancies between the assessments of the two observers were resolved by conferring at a multiviewer microscope. In all examined case, there was no discrepancy affecting the assessment of expression status using the cut-off values described.
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tissue sarcoma) (1 – 3) and tends to be highly expressed in high-grade tumours (4,5). Increased FAS expression was found to be significantly associated with tumour aggressiveness and poor prognosis in renal cell carcinoma (5). FAS expression in prostate cancer was found to be an independent predictor for pathologic stage (6) and to be significantly associated with high-grade prostatic epithelial neoplasia (PIN) and high Gleason score (7). Upper urinary tract urothelial carcinoma (UTUC) is a relatively uncommon malignant tumour, accounting for 5 – 10% of all urological malignancies (8). Pathological stage and tumour grade are useful predictors of outcome in populations of patients with UTUC but are still insufficient for predicting the individual outcome (9,10). Although there are several reports that show the relations between FAS and pathological features and outcome in patients with bladder urothelial carcinoma (UC) (11,12), its relations with pathological features and outcome in patients with UTUC have not been clarified. Considering the relations between FAS and other types of cancer, we thought that FAS expression could be a novel predictor of outcome in patients with UTUC. In the present study, we examined the expression of FAS in surgically resected UTUC specimens and evaluated the relations between FAS expression and the patients’ pathological features and clinical outcomes.
487
488
FAS expression in UTUC
STATISTICAL ANALYSIS All statistical analyses were performed using the JMP 9.0 software system for Windows (SAS Institute Inc. Cary, NC, USA). A x 2 test was used to assess the association between pre-operative and pathological specimen parameters relative to the level of FAS expression. P values ,0.05 were considered to indicate statistical significance. The CSS rates, RFS rates, intravesical-recurrence-free survival (IRFS) rates and extravesical-recurrence-free survival (ERFS) rates were calculated by the Kaplan – Meier method and were compared by log-rank testing. The prognostic significance of parameters was assessed using the Cox proportional hazards regression model.
Table 1. Patient characteristics Parameters
Number of patients (%)
Overall
113
Median age (range)
68 (38–86)
Sex Male
86 (76.1)
Female
27 (23.9)
Tumour side Left
65 (57.5)
Right
48 (42.5)
RESULTS PATIENT CHARACTERISTICS Patient characteristics are listed in Table 1. The median age of patients was 68 years (range 38 – 86 years). Eighty-six of them were men and the other 27 were women. Pathologic analysis revealed that 57 patients (50.4%) had tumours in the renal pelvis and 56 (49.6%) had tumours in ureters. The pathological T stage was pTa in 23 cases (20.4%), pT1 in 21 cases (18.6%), pT2 in 16 cases (14.2%) and pT3 in 53 cases (46.9%). Tumour grade was low in 26 cases (23.0%) and high in 87 cases (77.0%). Thirty-four cases (30.1%) were positive for lymphovascular invasion (LVI), and nodal involvement was observed in six cases (5.3%). Adjuvant chemotherapy was used in 29 patients (25.7%). Follow-up times ranged from 3.9 to 152.3 months (median: 46.8 months). Sixty-one patients (54.0%) had recurrence: 44 (38.9%) had intravesical recurrence, 25 (22.2%) had extravesical recurrence and 8 (7.1%) had both intravesical and extravesical recurrence. Seventeen patients (15.0%) died of their cancer during follow-up.
Renal pelvis
57 (50.4)
Ureter
56 (49.6)
Pathologic T stage pTa
23 (20.4)
pT1
21 (18.6)
pT2
16 (14.2)
pT3
53 (46.8)
Tumour grade Low
26 (23.0)
High
87 (77.0)
Lymphovascular invasion Positive
34 (30.1)
Negative
79 (69.9)
Nodal involvement Unknown
8 (7.1)
pN0
99 (87.6)
pN1
2 (1.8)
pN2
4 (3.5)
Surgical margin
RELATIONS BETWEEN FAS EXPRESSION AND CLINICOPATHOLOGICAL PARAMETERS Figure 1 shows representative photographs of FAS immunostaining of UTUC. As in a previous study (5), most of the FAS immunostaining in tumour tissue was in cytoplasm with granular patterns. The staining intensity and stained proportion widely varied among tumours (Fig. 1A – D) and the areas of tumours (Fig. 2). No immunostaining was observed in the negative controls (data not shown). Thirty-one of the 113 tumours (27.4%) showed high FAS expression, and 82 (72.6%) showed low FAS expression. Associations between FAS expression status and clinicopathological parameters are shown by the values listed in Table 2. High FAS expression was significantly associated with high tumour grade (P ¼ 0.0273), and none of the other parameters (sex, age, pathological T stage, LVI and lymph node metastasis) were associated with FAS expression status. Of the 31 patients having tumours with high FAS expression, five had only superficial lesions ( pTa) and two had only deep lesions with little superficial lesions.
Positive
12 (10.6)
Negative
101 (89.4)
Adjuvant chemotherapy Yes
29 (25.7)
No
84 (74.3)
Evaluating the FAS staining patterns in the tumours of the other 24 patients having tumours with high FAS expression, we found that 10 (41.6%) had high FAS expression only in deep lesions, and 14 (58.4%) had high FAS expression in both deep and superficial lesions. None had high FAS expression only in superficial lesions.
IMPACT OF FAS EXPRESSION ON CLINICAL OUTCOME We next evaluated the impact of FAS expression on the clinical outcome in patients with UTUC. The Kaplan – Meier
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Main tumour location
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Table 2. Fatty acid synthase (FAS) expression and clinicopathological parameters Parameters
N
FAS expression
P value
Low (50%)
High (.50%)
Sex Male
86
65
21
Female
27
17
10
,70
61
43
18
70
52
39
13
Low
26
23
3
High
87
59
28
0.20
Age
Figure 2. Representative tumour showing heterogeneous FAS staining intensities. Note low FAS expression in the superficial lesion (arrows) and high FAS expression in the deeper lesion (filled triangles).
survival curves for CSS, RFS, IRFS and ERFS are shown in Fig. 3, where they are stratified by FAS expression. High FAS expression was significantly associated with worse RFS and ERFS rates (P ¼ 0.0171, 0.0228, respectively) but not with CSS (P ¼ 0.2327) or IRFS (P ¼ 0.0555). In univariate analysis, pathological T stage (P , 0.0001), tumour grade (P ¼ 0.007), LVI (P , 0.0001), lymph node metastasis (P , 0.0001), adjuvant chemotherapy (P ¼ 0.0003), surgical margin (P ¼ 0.0121) and high FAS expression (P ¼ 0.0313) were significant predictors of ERFS. In multivariate analysis, pathological T stage (P ¼ 0.0202, hazard ratio (HR) ¼ 3.831),
0.59
Tumour grade 0.0273
Pathological T stage ,pT3
60
43
17
pT3
53
39
14
0.81
Lymphovascular invasion Positive
34
21
13
Negative
79
61
18
,N1
107
78
29
N1
6
4
2
0.09
Nodal involvement 0.74
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Figure 1. Representative immunohistochemistry for fatty acid synthase (FAS) in upper urinary tract urothelial carcinoma: (A) no cytoplasmic immunoreactivity for FAS (score 0), (B) weak cytoplasmic immunoreactivity for FAS (score 1þ), (C) moderate cytoplasmic immunoreactivity for FAS (score 2þ) and (D) strong cytoplasmic immunoreactivity for FAS (score 3þ). Immunoperoxidase stain, original magnification 200.
490
FAS expression in UTUC
Table 3. Univariate and multivariate analysis for extravesical-recurrence-free survival (ERFS)
Table 4. Univariate and multivariate analysis for RFS Univariate Multivariate
Univariate Multivariate P value
Age (years) (,70 vs. 70) Sex (male vs. female) pT stage (,pT3 vs. pT3 )
0.94
HR (95% confidence interval (CI))
P value
1.06 (0.46–2.47)
0.89
Age (years) (,70 vs. 70)
0.32
1.42 (0.44–3.40)
0.44
,0.0001
3.83 (1.24–11.95)
0.0202
P value
HR (95 % CI)
P value
0.81
1.14 (0.66– 1.97)
0.62
Sex (male vs. female)
0.37
1.14 (0.67– 2.11)
0.67
pT stage (,pT3 vs. pT3 )
0.0050
2.00 (0.98– 3.96)
0.0564
Grade (low vs. high)
0.27
1.36 (0.62– 2.87)
0.42
0.0089
1.45 (0.71– 2.85)
0.29
0.007
1.34 (0.33–8.99)
0.70
Lymphovascular invasion (negative vs. positive)
Lymphovascular invasion (negative vs. positive)
,0.0001
3.70 (1.41–9.99)
0.0079
Nodal involvement (negative vs. positive)
0.0068
3.19 (1.10– 8.21)
0.0337
Nodal involvement (negative vs. positive)
,0.0001
4.83 (1.55–14.09)
0.0082
Adjuvant chemotherapy (yes vs. no)
0.0275
1.42 (0.62– 3.19)
0.39
Surgical margin (negative vs. positive)
0.0145
2.03 (0.95– 3.92)
0.06
FAS expression (low vs. high)
0.0235
1.97 (1.10– 3.43)
0.0220
Tumour grade (low vs. high)
Adjuvant chemotherapy (yes vs. no)
0.0003
0.88 (0.32–2.48)
0.22
Surgical margin (negative vs. positive)
0.0121
1.98 (0.74–4.77)
0.16
FAS expression (low vs. high)
0.0313
2.11 (0.94–4.68)
0.06
LVI (P ¼ 0.0079, HR ¼ 3.701) and lymph node metastasis (P ¼ 0.0082, HR ¼ 4.838) were independent predictors for ERFS but FAS was not (P ¼ 0.0677) (Table 3). As for RFS, pathological T stage (P ¼ 0.0050), LVI (P ¼ 0.0089),
lymph node metastasis (P ¼ 0.0068), adjuvant chemotherapy (P ¼ 0.0275), surgical margin (P ¼ 0.0145) and high FAS expression (P ¼ 0.0235) were significant predictors in univariate analysis and lymph node metastasis (P ¼ 0.0337, HR ¼ 3.196) and high FAS expression (P ¼ 0.0220, HR ¼ 1.970) were independent predictors in multivariate analysis (Table 4).
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Figure 3. Kaplan – Meier curves for (A) cancer-specific survival (CSS), (B) recurrence-free survival (RFS), (C) intravesical-recurrence-free survival (IRFS) and (D) extravesical-recurrence-free survival (ERFS) after surgery for UTUC in patients with high and low FAS expression.
Jpn J Clin Oncol 2014;44(5)
DISCUSSION
FAS functions as the key enzyme responsible for the de novo synthesis of fatty acids. The generated lipids are integrated into membrane lipid rafts and modulate membrane receptor tyrosine kinases, resulting in the stimulation of various key signalling pathways involved in the survival, proliferation and invasion of cancer cells (1 – 3,5). Inhibition of FAS activity has therefore emerged as a novel therapeutic strategy against cancer (1,2,15,18). FAS inhibition impairs the correct localization and functioning of tyrosine kinases such as EGFR and HER2 by inducing changes in the synthesis of membrane phospholipids and assembling of lipid rafts and thereby inhibits their downstream signals (1). The FAS inhibitor C75 suppresses EGFR and HER2 expression in renal cancer cells, concomitantly attenuating the STAT3 signalling pathway, which is constitutively activated and contributes to preventing apoptosis in renal cancer (18). The PI3K/ Akt signalling pathway in bladder cancer cells has been shown to be important for survival and contributes to cell cycle progression via cyclin D1 upregulation, and the FAS inhibitor cerulenin has been shown to cause inactivity of Akt and downregulation of cyclin D1, resulting in increased apoptosis and decreased proliferation (12). Moreover, FAS inhibition has been shown to reduce experimental metastasis of melanomas and colorectal cancer via inhibition of tumour angiogenesis and CD-44-associated signalling (19,20). The results of these studies make FAS inhibition an attractive strategy for UTUC treatment, one that should be examined in further studies. There are several limitations to the present study. Although we found high FAS expression to be significantly associated with poor RFS and ERFS, we found no significant association between FAS expression status and CSS. We think that the reasons are that the surgical treatment was performed by various surgeons and the lymphadenectomy region was not uniform. The cycles of chemotherapy after tumour recurrence depended on the patient’s general condition and varied among patients. Another limitation is that this study was a retrospective study based on a limited number of patients. Despite these limitations, we are confident that our results are very important and that the FAS expression in UTUC could be information useful to clinicians.
CONCLUSIONS FAS expression was high in 27.4% (31 of 113) of surgically resected UTUC specimens, and high FAS expression was significantly associated with high-grade tumours and poor RFS. Patients with high FAS expression in UTUC should be followed carefully and adjuvant therapy for them should be considered.
Funding This work was supported by a grant-in-aid for scientific research from the ministry of National Defense.
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In the present study we examined FAS expression in UTUC by immunohistochemistry and retrospectively evaluated the impact of FAS expression on pathological features and clinical outcome in 113 patients who underwent surgical resection. High FAS expression was significantly associated with tumour grade and poor RFS and ERFS in univariate analysis. Multivariate analysis of various clinicopathological parameters revealed that high FAS expression was an independent predictor of tumour recurrence. To the best of our knowledge, this is the first study examining FAS expression and evaluating the prognostic value of FAS expression in patients with UTUC. In contrast to normal tissue, which has a low level of FAS (1,2), various malignant tumours, especially a biologically aggressive subset, express increased levels of FAS (1 – 3,5). FAS was first identified as an oncogenic antigen in 1994 by Kuhajda et al. (14) who found it in breast cancer patients whose prognosis was extremely poor. As for urological malignancies, high FAS expression in radical prostatectomy specimens has been suggested to be an independent predictor of advanced pathological stage as well as higher Gleason score in prostate cancer (6). High FAS expression has been found in 82% of lethal castration-resistant prostate cancer cases examined at autopsy and appears to increase the risk of prostate cancer death 4.45-fold (15,16). In renal cell carcinoma, high FAS expression was associated with tumour aggressiveness (advanced pathological stage, nodal involvement, distant metastasis, higher tumour grade and microvascular invasion) and was an independent predictor of shortened CSS (5). High FAS expression in bladder UC has been reported to be significantly associated with higher pathological T stage and higher tumour grade (11), and in that study the high-score group for combined FAS and Ki-67 expression was significantly associated with poor CSS in univariate analysis. Moreover, high FAS expression in non-muscle-invasive bladder cancer has been reported to be associated with higher tumour grade and recurrence-free survival at 5 years (12). In our study, increased FAS expression was significantly associated with tumour grade in UTUC as well as other urological malignancies. Our results also showed that high FAS expression was significantly associated with poor RFS and ERFS. Of note, increased FAS expression was found predominantly in deeper lesions of the tumour, suggesting that FAS expression plays important roles in invasive and metastatic progression of UTUC. Surgical resection is the mainstay treatment for organ-confined UTUC, and 5-year survival after it has been reported to be 76.1% (9,10). A significant proportion of patients who undergo surgical resection eventually develop local recurrence and/or metastases, however, and the prognosis of patients with metastatic UTUC is poor (8). Local recurrence and metastasis are the major causes of mortality in patients with UTUC (8,17), so their accurate precise prediction is quite important. Patients with high FAS in surgical specimens should be followed carefully and might need adjuvant therapy.
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Conflict of interest statement None declared.
11.
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proposal and multi-institutional validation of a post-operative nomogram. Br J Cancer 2012;106:1083–8. Sugino T, Baba K, Hoshi N, Aikawa K, Yamaguchi O, Suzuki T. Overexpression of fatty acid synthase in human urinary bladder cancer and combined expression of the synthase and Ki-67 as a predictor of prognosis of cancer patients. Med Mol Morphol 2011;44:146– 50. Jiang B, Li EH, Lu YY, et al. Inhibition of fatty-acid synthase suppresses P-AKT and induces apoptosis in bladder cancer. Urology 2012;80:484 e9– 15. Migita T, Ruiz S, Fornari A, et al. Fatty acid synthase: a metabolic enzyme and candidate oncogene in prostate cancer. J Natl Cancer Inst 2009;101:519 –32. Kuhajda FP, Jenner K, Wood FD, et al. Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proc Natl Acad Sci U S A 1994;91:6379 –83. Pizer ES, Pflug BR, Bova GS, Han WF, Udan MS, Nelson JB. Increased fatty acid synthase as a therapeutic target in androgen-independent prostate cancer progression. Prostate 2001;47:102–10. Bandyopadhyay S, Pai SK, Watabe M, et al. FAS expression inversely correlates with PTEN level in prostate cancer and a PI 3-kinase inhibitor synergizes with FAS siRNA to induce apoptosis. Oncogene 2005;24:5389 –95. Margulis V, Shariat SF, Matin SF, et al. Outcomes of radical nephroureterectomy: a series from the upper tract urothelial carcinoma collaboration. Cancer 2009;115:1224–33. Horiguchi A, Asano T, Ito K, Sumitomo M, Hayakawa M. Pharmacological inhibitor of fatty acid synthase suppresses growth and invasiveness of renal cancer cells. J Urol 2008;180:729–36. Seguin F, Carvalho MA, Bastos DC, et al. The fatty acid synthase inhibitor orlistat reduces experimental metastases and angiogenesis in B16-F10 melanomas. Br J Cancer 2012;107:977– 87. Zaytseva YY, Rychahou PG, Gulhati P, et al. Inhibition of fatty acid synthase attenuates CD44-associated signaling and reduces metastasis in colorectal cancer. Cancer Res 2012;72:1504 –17.
Prognostic Impact of Fatty Acid Synthase Expression in Upper Urinary Tract Urothelial Carcinoma Shinsuke Hamada1,*, Akio Horiguchi1, Takako Asano1, Kenji Kuroda1, Junichi Asakuma1, Keiichi Ito1, Tomohiko Asano1, Kosuke Miyai2 and Keiichi Iwaya2 1
Department of Urology, National Defense Medical College, Tokorozawa, Saitama and 2Department of Basic Pathology, National Defense Medical College, Tokorozawa, Saitama, Japan
Received November 6, 2013; accepted February 10, 2014
Objective: Fatty acid synthase has been shown to be highly expressed in various types of cancers with increased tumour aggressiveness. In this study we examined the level of fatty acid synthase expression in surgically resected upper urinary tract urothelial carcinoma specimens and evaluated the relations between fatty acid synthase expression and the patients’ pathological features and clinical outcomes. Methods: Sections of paraffin-embedded tumour specimens from 113 patients who underwent surgical treatment for upper urinary tract urothelial carcinoma were immunostained with a polyclonal fatty acid synthase antibody, and a tumour was considered to have high fatty acid synthase expression if .50% of the cancer cells stained with moderate-to-strong intensity. Associations between fatty acid synthase expression and the patients’ pathological parameters and survival were analyzed statistically. Results: During the follow-up time (median: 46.8 months), 61 patients (54.0%) had recurrence and 17 (15.0%) died of upper urinary tract urothelial carcinoma. High fatty acid synthase expression was significantly associated with high tumour grade (P ¼ 0.0273). Patients with high fatty acid synthase expression had significantly worse recurrence-free survival and extravesicalrecurrence-free survival than those with low fatty acid synthase expression (P ¼ 0.0171, P ¼ 0.0228, respectively). In multivariate analysis, high fatty acid synthase expression was an independent predictor of shortened recurrence-free survival (P ¼ 0.0220, hazard ratio (HR) ¼ 1.970). Conclusions: Fatty acid synthase expression in upper urinary tract urothelial carcinoma is an independent predictor for tumour recurrence. Patients with high fatty acid synthase expression in upper urinary tract urothelial carcinoma should be followed carefully and adjuvant therapy for them should be considered.Keywords: fatty acid synthase, upper urinary tract urothelial carcinoma, prognosis Key words: fatty acid synthase – upper urinary tract urothelial carcinoma – prognosis
INTRODUCTION Fatty acid synthase (FAS) is a biosynthetic enzyme for lipogenesis and the production of long-chain fatty acids from acetyl-coenzyme A (CoA) and malonyl-CoA (1,2). Fatty acids are tightly regulated by diet, hormones and growth factors;
and FAS expression and activity levels are low in normal cells except those in liver, endometrium, mammary glands and adipose tissue (3). On the other hand, FAS is highly expressed in various cancers (breast, colorectum, ovary, oesophagus, stomach, lung, head and neck and thyroid cancers and soft
# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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*For reprints and all correspondence: Shinsuke Hamada, Department of Urology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. E-mail: [email protected]
Jpn J Clin Oncol 2014;44(5)
PATIENTS AND METHODS PATIENTS We reviewed the records of 125 patients who underwent surgical treatment for UTUC at the National Defense Medical College between March 1999 and December 2010. Patients with carcinoma in situ and patients with histology results showing cancer other than UC were excluded from this study. Consequently, a total of 113 patients were enrolled in this study: 65 (57.5%) who had undergone open nephroureterectomy and partial cystectomy, 41 (36.3%) who had undergone laparoscopic nephroureterectomy and partial cystectomy, three (2.7%) who had undergone nephroureterectomy and total cystectomy, two (1.8%) who had undergone nephroureterectomy, and two (1.8%) who had undergone partial ureterectomy and ureterocystoneostomy. Regional lymph node dissection was performed in patients whose preoperative evaluation indicated a T3 tumour or the possibility nodal involvement. The follow-up period, calculated using the date of the operation and the date of the last recorded follow-up, was 3.9 – 152.3 months (median: 46.8 months). The patients with pT3 tumours were recommended to receive two or three cycles of adjuvant gemcitabin and cisplatin (GC) or methotrexate, vinblastine, doxorubicin and cisplatin (M-VAC) regimens. Patients were assessed by cystoscopy and urine cytology every 3 months for 2 years after the operation, and computed tomography or magnetic resonance imaging was conducted every 6 months. Intravesical recurrence was considered to have occurred if UC was detected in residual bladder
by cystoscopy, and extravesical recurrence was considered to have occurred if any nodal and/or visceral metastasis was evident on radiograph examination. Recurrence-free survival (RFS) was calculated as the period from the operation to the appearance of intravesical or extravesical recurrence. Cancer-specific survival (CSS) was calculated as the period from the operation to the date of death from UTUC. All pathologic specimens were reviewed by two expert genitourinary pathologists (K.M. and K.I.). Tumour stages were classified according to the 2002 TNM classification. Tumour grades were classified into two levels (high and low) based on the World Health Organization (WHO) grading system. This study was approved by the National Defense Medical College ethical committee. IMMUNOHISTOCHEMISTRY Sections 4 mm thick were cut from formalin-fixed, paraffinembedded blocks of UTUC, deparaffinized in xylene and rehydrated through graded ethanols. Endogenous peroxidase activity was quenched by immersing the sections in DAKO Peroxidase Blocking Reagent (Dako Corporation) for 10 min. The sections were incubated in 10% normal goat serum in phosphate buffered saline (PBS) for 60 min at room temperature. After the sections were incubated 60 min in a 1:50 dilution of rabbit polyclonal anti-FAS antibody (Immuno-Biological Laboratories Co., Fujioka, Japan) in PBS, the slides were stained by using a Simple Stain Max PO kit (Nichirei Corporation, Tokyo, Japan) according to the manufacturer’s instructions. Reaction products were visualized by immersing the slides in diaminobenzidine tetrahydrochloride (DAB) for 2 min. The sections were finally counterstained with haematoxylin. Periureteral adipose tissues served as positive internal controls for FAS immunoreactivity. Sections stained with unimmunized-rabbit serum instead of the primary antibody were used as negative controls. Cytoplasmic immunoreactivity was taken into account for FAS protein expression. Intensities of FAS immunoreactivity were classified semiquantitatively into four categories: nonstaining (negative), weak (score 1þ), moderate (score 2þ) and strong (score 3þ) (7,13). Immunoreactivity in the tumour was considered to indicate high FAS expression if .50% of the tumour cells evaluated in the slides showed moderate (2þ) to strong (3þ) immunoreactivity. The staining intensities in the entire slide of each tumour were evaluated and scores were assigned according to the highest intensity. The cut-off value was determined on the basis of the following criteria: (i) the cut-off giving the best P values at testing and (ii) the cut-off used in previous reports (11). Immunohistochemistry data were evaluated independently by two pathologists (K.M. and K.I.) blinded from any clinical data. Any discrepancies between the assessments of the two observers were resolved by conferring at a multiviewer microscope. In all examined case, there was no discrepancy affecting the assessment of expression status using the cut-off values described.
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tissue sarcoma) (1 – 3) and tends to be highly expressed in high-grade tumours (4,5). Increased FAS expression was found to be significantly associated with tumour aggressiveness and poor prognosis in renal cell carcinoma (5). FAS expression in prostate cancer was found to be an independent predictor for pathologic stage (6) and to be significantly associated with high-grade prostatic epithelial neoplasia (PIN) and high Gleason score (7). Upper urinary tract urothelial carcinoma (UTUC) is a relatively uncommon malignant tumour, accounting for 5 – 10% of all urological malignancies (8). Pathological stage and tumour grade are useful predictors of outcome in populations of patients with UTUC but are still insufficient for predicting the individual outcome (9,10). Although there are several reports that show the relations between FAS and pathological features and outcome in patients with bladder urothelial carcinoma (UC) (11,12), its relations with pathological features and outcome in patients with UTUC have not been clarified. Considering the relations between FAS and other types of cancer, we thought that FAS expression could be a novel predictor of outcome in patients with UTUC. In the present study, we examined the expression of FAS in surgically resected UTUC specimens and evaluated the relations between FAS expression and the patients’ pathological features and clinical outcomes.
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FAS expression in UTUC
STATISTICAL ANALYSIS All statistical analyses were performed using the JMP 9.0 software system for Windows (SAS Institute Inc. Cary, NC, USA). A x 2 test was used to assess the association between pre-operative and pathological specimen parameters relative to the level of FAS expression. P values ,0.05 were considered to indicate statistical significance. The CSS rates, RFS rates, intravesical-recurrence-free survival (IRFS) rates and extravesical-recurrence-free survival (ERFS) rates were calculated by the Kaplan – Meier method and were compared by log-rank testing. The prognostic significance of parameters was assessed using the Cox proportional hazards regression model.
Table 1. Patient characteristics Parameters
Number of patients (%)
Overall
113
Median age (range)
68 (38–86)
Sex Male
86 (76.1)
Female
27 (23.9)
Tumour side Left
65 (57.5)
Right
48 (42.5)
RESULTS PATIENT CHARACTERISTICS Patient characteristics are listed in Table 1. The median age of patients was 68 years (range 38 – 86 years). Eighty-six of them were men and the other 27 were women. Pathologic analysis revealed that 57 patients (50.4%) had tumours in the renal pelvis and 56 (49.6%) had tumours in ureters. The pathological T stage was pTa in 23 cases (20.4%), pT1 in 21 cases (18.6%), pT2 in 16 cases (14.2%) and pT3 in 53 cases (46.9%). Tumour grade was low in 26 cases (23.0%) and high in 87 cases (77.0%). Thirty-four cases (30.1%) were positive for lymphovascular invasion (LVI), and nodal involvement was observed in six cases (5.3%). Adjuvant chemotherapy was used in 29 patients (25.7%). Follow-up times ranged from 3.9 to 152.3 months (median: 46.8 months). Sixty-one patients (54.0%) had recurrence: 44 (38.9%) had intravesical recurrence, 25 (22.2%) had extravesical recurrence and 8 (7.1%) had both intravesical and extravesical recurrence. Seventeen patients (15.0%) died of their cancer during follow-up.
Renal pelvis
57 (50.4)
Ureter
56 (49.6)
Pathologic T stage pTa
23 (20.4)
pT1
21 (18.6)
pT2
16 (14.2)
pT3
53 (46.8)
Tumour grade Low
26 (23.0)
High
87 (77.0)
Lymphovascular invasion Positive
34 (30.1)
Negative
79 (69.9)
Nodal involvement Unknown
8 (7.1)
pN0
99 (87.6)
pN1
2 (1.8)
pN2
4 (3.5)
Surgical margin
RELATIONS BETWEEN FAS EXPRESSION AND CLINICOPATHOLOGICAL PARAMETERS Figure 1 shows representative photographs of FAS immunostaining of UTUC. As in a previous study (5), most of the FAS immunostaining in tumour tissue was in cytoplasm with granular patterns. The staining intensity and stained proportion widely varied among tumours (Fig. 1A – D) and the areas of tumours (Fig. 2). No immunostaining was observed in the negative controls (data not shown). Thirty-one of the 113 tumours (27.4%) showed high FAS expression, and 82 (72.6%) showed low FAS expression. Associations between FAS expression status and clinicopathological parameters are shown by the values listed in Table 2. High FAS expression was significantly associated with high tumour grade (P ¼ 0.0273), and none of the other parameters (sex, age, pathological T stage, LVI and lymph node metastasis) were associated with FAS expression status. Of the 31 patients having tumours with high FAS expression, five had only superficial lesions ( pTa) and two had only deep lesions with little superficial lesions.
Positive
12 (10.6)
Negative
101 (89.4)
Adjuvant chemotherapy Yes
29 (25.7)
No
84 (74.3)
Evaluating the FAS staining patterns in the tumours of the other 24 patients having tumours with high FAS expression, we found that 10 (41.6%) had high FAS expression only in deep lesions, and 14 (58.4%) had high FAS expression in both deep and superficial lesions. None had high FAS expression only in superficial lesions.
IMPACT OF FAS EXPRESSION ON CLINICAL OUTCOME We next evaluated the impact of FAS expression on the clinical outcome in patients with UTUC. The Kaplan – Meier
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Main tumour location
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Table 2. Fatty acid synthase (FAS) expression and clinicopathological parameters Parameters
N
FAS expression
P value
Low (50%)
High (.50%)
Sex Male
86
65
21
Female
27
17
10
,70
61
43
18
70
52
39
13
Low
26
23
3
High
87
59
28
0.20
Age
Figure 2. Representative tumour showing heterogeneous FAS staining intensities. Note low FAS expression in the superficial lesion (arrows) and high FAS expression in the deeper lesion (filled triangles).
survival curves for CSS, RFS, IRFS and ERFS are shown in Fig. 3, where they are stratified by FAS expression. High FAS expression was significantly associated with worse RFS and ERFS rates (P ¼ 0.0171, 0.0228, respectively) but not with CSS (P ¼ 0.2327) or IRFS (P ¼ 0.0555). In univariate analysis, pathological T stage (P , 0.0001), tumour grade (P ¼ 0.007), LVI (P , 0.0001), lymph node metastasis (P , 0.0001), adjuvant chemotherapy (P ¼ 0.0003), surgical margin (P ¼ 0.0121) and high FAS expression (P ¼ 0.0313) were significant predictors of ERFS. In multivariate analysis, pathological T stage (P ¼ 0.0202, hazard ratio (HR) ¼ 3.831),
0.59
Tumour grade 0.0273
Pathological T stage ,pT3
60
43
17
pT3
53
39
14
0.81
Lymphovascular invasion Positive
34
21
13
Negative
79
61
18
,N1
107
78
29
N1
6
4
2
0.09
Nodal involvement 0.74
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Figure 1. Representative immunohistochemistry for fatty acid synthase (FAS) in upper urinary tract urothelial carcinoma: (A) no cytoplasmic immunoreactivity for FAS (score 0), (B) weak cytoplasmic immunoreactivity for FAS (score 1þ), (C) moderate cytoplasmic immunoreactivity for FAS (score 2þ) and (D) strong cytoplasmic immunoreactivity for FAS (score 3þ). Immunoperoxidase stain, original magnification 200.
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FAS expression in UTUC
Table 3. Univariate and multivariate analysis for extravesical-recurrence-free survival (ERFS)
Table 4. Univariate and multivariate analysis for RFS Univariate Multivariate
Univariate Multivariate P value
Age (years) (,70 vs. 70) Sex (male vs. female) pT stage (,pT3 vs. pT3 )
0.94
HR (95% confidence interval (CI))
P value
1.06 (0.46–2.47)
0.89
Age (years) (,70 vs. 70)
0.32
1.42 (0.44–3.40)
0.44
,0.0001
3.83 (1.24–11.95)
0.0202
P value
HR (95 % CI)
P value
0.81
1.14 (0.66– 1.97)
0.62
Sex (male vs. female)
0.37
1.14 (0.67– 2.11)
0.67
pT stage (,pT3 vs. pT3 )
0.0050
2.00 (0.98– 3.96)
0.0564
Grade (low vs. high)
0.27
1.36 (0.62– 2.87)
0.42
0.0089
1.45 (0.71– 2.85)
0.29
0.007
1.34 (0.33–8.99)
0.70
Lymphovascular invasion (negative vs. positive)
Lymphovascular invasion (negative vs. positive)
,0.0001
3.70 (1.41–9.99)
0.0079
Nodal involvement (negative vs. positive)
0.0068
3.19 (1.10– 8.21)
0.0337
Nodal involvement (negative vs. positive)
,0.0001
4.83 (1.55–14.09)
0.0082
Adjuvant chemotherapy (yes vs. no)
0.0275
1.42 (0.62– 3.19)
0.39
Surgical margin (negative vs. positive)
0.0145
2.03 (0.95– 3.92)
0.06
FAS expression (low vs. high)
0.0235
1.97 (1.10– 3.43)
0.0220
Tumour grade (low vs. high)
Adjuvant chemotherapy (yes vs. no)
0.0003
0.88 (0.32–2.48)
0.22
Surgical margin (negative vs. positive)
0.0121
1.98 (0.74–4.77)
0.16
FAS expression (low vs. high)
0.0313
2.11 (0.94–4.68)
0.06
LVI (P ¼ 0.0079, HR ¼ 3.701) and lymph node metastasis (P ¼ 0.0082, HR ¼ 4.838) were independent predictors for ERFS but FAS was not (P ¼ 0.0677) (Table 3). As for RFS, pathological T stage (P ¼ 0.0050), LVI (P ¼ 0.0089),
lymph node metastasis (P ¼ 0.0068), adjuvant chemotherapy (P ¼ 0.0275), surgical margin (P ¼ 0.0145) and high FAS expression (P ¼ 0.0235) were significant predictors in univariate analysis and lymph node metastasis (P ¼ 0.0337, HR ¼ 3.196) and high FAS expression (P ¼ 0.0220, HR ¼ 1.970) were independent predictors in multivariate analysis (Table 4).
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Figure 3. Kaplan – Meier curves for (A) cancer-specific survival (CSS), (B) recurrence-free survival (RFS), (C) intravesical-recurrence-free survival (IRFS) and (D) extravesical-recurrence-free survival (ERFS) after surgery for UTUC in patients with high and low FAS expression.
Jpn J Clin Oncol 2014;44(5)
DISCUSSION
FAS functions as the key enzyme responsible for the de novo synthesis of fatty acids. The generated lipids are integrated into membrane lipid rafts and modulate membrane receptor tyrosine kinases, resulting in the stimulation of various key signalling pathways involved in the survival, proliferation and invasion of cancer cells (1 – 3,5). Inhibition of FAS activity has therefore emerged as a novel therapeutic strategy against cancer (1,2,15,18). FAS inhibition impairs the correct localization and functioning of tyrosine kinases such as EGFR and HER2 by inducing changes in the synthesis of membrane phospholipids and assembling of lipid rafts and thereby inhibits their downstream signals (1). The FAS inhibitor C75 suppresses EGFR and HER2 expression in renal cancer cells, concomitantly attenuating the STAT3 signalling pathway, which is constitutively activated and contributes to preventing apoptosis in renal cancer (18). The PI3K/ Akt signalling pathway in bladder cancer cells has been shown to be important for survival and contributes to cell cycle progression via cyclin D1 upregulation, and the FAS inhibitor cerulenin has been shown to cause inactivity of Akt and downregulation of cyclin D1, resulting in increased apoptosis and decreased proliferation (12). Moreover, FAS inhibition has been shown to reduce experimental metastasis of melanomas and colorectal cancer via inhibition of tumour angiogenesis and CD-44-associated signalling (19,20). The results of these studies make FAS inhibition an attractive strategy for UTUC treatment, one that should be examined in further studies. There are several limitations to the present study. Although we found high FAS expression to be significantly associated with poor RFS and ERFS, we found no significant association between FAS expression status and CSS. We think that the reasons are that the surgical treatment was performed by various surgeons and the lymphadenectomy region was not uniform. The cycles of chemotherapy after tumour recurrence depended on the patient’s general condition and varied among patients. Another limitation is that this study was a retrospective study based on a limited number of patients. Despite these limitations, we are confident that our results are very important and that the FAS expression in UTUC could be information useful to clinicians.
CONCLUSIONS FAS expression was high in 27.4% (31 of 113) of surgically resected UTUC specimens, and high FAS expression was significantly associated with high-grade tumours and poor RFS. Patients with high FAS expression in UTUC should be followed carefully and adjuvant therapy for them should be considered.
Funding This work was supported by a grant-in-aid for scientific research from the ministry of National Defense.
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In the present study we examined FAS expression in UTUC by immunohistochemistry and retrospectively evaluated the impact of FAS expression on pathological features and clinical outcome in 113 patients who underwent surgical resection. High FAS expression was significantly associated with tumour grade and poor RFS and ERFS in univariate analysis. Multivariate analysis of various clinicopathological parameters revealed that high FAS expression was an independent predictor of tumour recurrence. To the best of our knowledge, this is the first study examining FAS expression and evaluating the prognostic value of FAS expression in patients with UTUC. In contrast to normal tissue, which has a low level of FAS (1,2), various malignant tumours, especially a biologically aggressive subset, express increased levels of FAS (1 – 3,5). FAS was first identified as an oncogenic antigen in 1994 by Kuhajda et al. (14) who found it in breast cancer patients whose prognosis was extremely poor. As for urological malignancies, high FAS expression in radical prostatectomy specimens has been suggested to be an independent predictor of advanced pathological stage as well as higher Gleason score in prostate cancer (6). High FAS expression has been found in 82% of lethal castration-resistant prostate cancer cases examined at autopsy and appears to increase the risk of prostate cancer death 4.45-fold (15,16). In renal cell carcinoma, high FAS expression was associated with tumour aggressiveness (advanced pathological stage, nodal involvement, distant metastasis, higher tumour grade and microvascular invasion) and was an independent predictor of shortened CSS (5). High FAS expression in bladder UC has been reported to be significantly associated with higher pathological T stage and higher tumour grade (11), and in that study the high-score group for combined FAS and Ki-67 expression was significantly associated with poor CSS in univariate analysis. Moreover, high FAS expression in non-muscle-invasive bladder cancer has been reported to be associated with higher tumour grade and recurrence-free survival at 5 years (12). In our study, increased FAS expression was significantly associated with tumour grade in UTUC as well as other urological malignancies. Our results also showed that high FAS expression was significantly associated with poor RFS and ERFS. Of note, increased FAS expression was found predominantly in deeper lesions of the tumour, suggesting that FAS expression plays important roles in invasive and metastatic progression of UTUC. Surgical resection is the mainstay treatment for organ-confined UTUC, and 5-year survival after it has been reported to be 76.1% (9,10). A significant proportion of patients who undergo surgical resection eventually develop local recurrence and/or metastases, however, and the prognosis of patients with metastatic UTUC is poor (8). Local recurrence and metastasis are the major causes of mortality in patients with UTUC (8,17), so their accurate precise prediction is quite important. Patients with high FAS in surgical specimens should be followed carefully and might need adjuvant therapy.
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Conflict of interest statement None declared.
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