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High cytoplasmic HuR expression is associated with advanced pT stage, high grade and increased microvessel density in urothelial bladder carcinoma
Annals of Diagnostic Pathology 33 (2018) 40–44
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High cytoplasmic HuR expression is associated with advanced pT stage, high grade and increased microvessel density in urothelial bladder carcinoma
T
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Łukasz Piotr Fus , Paweł Pihowicz, Łukasz Koperski, Janina Maja Marczewska, Barbara Górnicka Department of Pathology, Medical University of Warsaw, Pawińskiego 7, 02-106 Warsaw, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords: Bladder cancer HuR Stage Grade Microvessel density
Purpose: HuR (human antigen R) protein is a RNA binding protein that stabilizes the mRNA and controls the translation of genes involved in cell proliferation, differentiation, and carcinogenesis. Overexpression of HuR was reported in a variety of cancers, however its clinical significance in urothelial bladder cancer (UBC) is still unknown. Our aim is to investigate the association between HuR expression and selected histopathological factors, such as tumor grade, pT stage, regional lymph nodes status and microvessel density (MVD). Methods: We studied expression of HuR protein in 119 patients with UBC in stages pTis and pTa–pT4 using immunohistochemistry (IHC). Tumor MVD was evaluated immunohistochemically using anti-CD31 antibody. Results: We observed no association between nuclear HuR immunoreactivity and tumor grade, stage or MVD. We found a significant association between cytoplasmic HuR positivity and high tumor grade, pT stage and MVD (p < 0,001). We also observed significantly higher MVD values in cases with positive cytoplasmic HuR expression (p < 0,001). No association between HuR immunoreactivity and lymph nodes status was found. Conclusions: Our results may suggest that HuR is involved in the process of acquiring malignant histopathological features and ability to invade the muscularis propria by UBC cells. Considering frequent difficulties in diagnosing UBC in specimens obtained from transurethral tumor resection and the risk of understaging, cytoplasmic HuR expression would suggest an advanced disease and necessitate serial sectioning of the specimen in search of muscle invasion. Association between HuR expression and MVD could suggest HuR involvement in the process of angiogenesis in UBC.
1. Introduction Bladder cancer is the most common urinary tract cancer and the fourth-most common malignancy in men in developed countries. In Europe and North America over 90% of bladder cancers are urothelial bladder carcinomas (UBC) [1]. The most important morphological features affecting prognosis are cancer grade and stage. The depth of tumor invasion – especially the invasion of muscularis propria – is of particular significance as it directly affects patient's treatment [2]. HuR (human antigen R) is a RNA binding protein which regulates the turnover of mRNA and controls the process of translation [3]. In physiologic conditions the intracellular location of HuR is mainly nuclear, but upon activation it is translocated to the cell cytoplasm where it binds to AU-rich-element containing 3′ untranslated region and stabilizes mRNA transcripts prolonging their half-life [4]. HuR can also directly regulate translation of several factors involved in cell proliferation, differentiation, apoptosis as well as angiogenesis and carcinogenesis [5]. It was shown that HuR stabilizes mRNA transcripts
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Corresponding author. E-mail address: [email protected] (Ł.P. Fus).
https://doi.org/10.1016/j.anndiagpath.2017.12.002
1092-9134/ © 2017 Elsevier Inc. All rights reserved.
coding cyclins A2, B1, D1 and E1, cytochrome C, COX-2, VEGF, interleukins 6 and 8, glucose transporter GLUT1, TGF-b, c-Myc, p53 and p21 [5-10]. Overexpression of HuR was described in a large number of malignant neoplasms and even though its high expression levels frequently correlated with disease progression and poor prognosis, some studies suggest that the role of HuR in carcinogenesis remains ambiguous [11-18]. The role of HuR in the pathogenesis of UBC is poorly understood and, to the best of our knowledge, there is only one published paper which investigates the presence of HuR protein in UBC cells. In their study Miyata et al. analyzed specimens obtained during transurethral resection of tumor (TURT) biopsy which consisted mainly of nonmuscle invasive bladder cancers (NMIBC, stage pTa and pT1) and relatively few cases of advanced, muscle-invasive bladder cancer (MIBC) staged pT2 and higher [19]. The goal of our study is to evaluate the presence and distribution of HuR protein in UBC cells in both nonmuscle invasive and muscle-invasive disease, and to investigate the association between HuR expression and tumor pT stage, histological
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grade, regional lymph nodes status and intratumoral microvessel density (MVD).
Table 1 Association of nuclear and cytoplasmic HuR positivity with clinicopathological variables in 119 patients with UBC.
2. Material and methods
Variable
Total
HuR nuclear positive, n (%)
HuR nuclear negative, n (%)
HuR cytoplasmic positive, n (%)
HuR cytoplasmic negative, n (%)
All cases pT stage pTis pTa pT1 pT2 pT3 pT4 p value Grade Low High p value Nodal status pN+ pN− n/a p value Sex F M p value Age (years) < 70 ≥ 70 p value MVD (vessels/ 0,43mm2) > 37 ≤ 37 p value
119
100
19
62
57
18 20 19 22 19 21
16 16 18 15 17 18 n.s.a
25 94
23 26 70
2.1. Patients To conduct our study we collected histopathological samples from 119 patients diagnosed with urothelial bladder carcinoma between 2005 and 2010. Samples were stored in the archives of our Department. The specimens represented 18 cases of pTis, 20 cases of pTa UBC, 19 cases of pT1 UBC, 22 cases of pT2 UBC, 19 cases of pT3 UBC and 22 cases of pT4 UBC. For log-linear regression analysis we categorized tumor staged pTis, pTa and pT1 as low (non-muscle invasive) stage and tumors pT2-pT4 as high (muscle invasive) stage. Samples of pTis, pTa and pT1 cancers were obtained during TURT biopsy. If the patient underwent more than one TURT, only the specimen from the first biopsy was evaluated. In more advanced UBC cases (pT2-pT4) tissue samples were taken from radical cystectomy specimens. The status of regional lymph nodes was evaluated only in cases staged pT2–pT4 in which radical cystectomy with lymphadenectomy was performed (49 cases). The mean patient's age was 69,8 ± 8,9 ( ± SD) years and ranged from 48 to 86. For statistical analysis we categorized cases according to patients age as patients < 70 years-old (53 cases) and ≥ 70 years-old (66 cases). 92 patients were males, 27 were females. 25 UBCs were diagnosed as low-grade (LG) and 94 as high-grade (HG) tumors. Among 20 cases of pTa UBCs tumors 18 were described as LG (90%) and 2 cases as HG (10%). 12 cases of pT1 cancers were HG (63,2%) and 7 cases were LG (36,8%). All cases diagnosed as pTis and pT2-pT4 cancers were described as HG. Among 49 cases in which regional lymph node status could be evaluated, 23 patients had lymph node metastases. 2.2. Immunohistochemistry Resected specimens were fixed in 10% buffered formalin and embedded in paraffin blocks. Immunohistochemical examinations were carried out on 3-μm tissue sections using anti-HuR mouse monoclonal antibody (3A2, Thermo Fisher). Sections were deparrafinized in xylene and rehydrated in graded ethanol solutions. Heat-induced antigen retrieval was performed with 0,01 M citrate buffer (pH 6.0) in a 700 W microwave (2 × 5 min, with 5-minute pause). Endogenous peroxidase was blocked with incubation in 3% hydrogen peroxide for 5 min. After washing with PBS, slides with sections were incubated with 2,5% horse serum for 20 min to block the non-specific antigen binding sites. Then sections were incubated for 45 min at room temperature with primary anti-HuR antibody (1:1000). After washing the slides with PBS, ImmPRESS HRP Anti-Mouse Polymer Detection Kit (Vector Labs) was applied for 30 min. The peroxidase reaction was visualized using ImmPACT DAB Peroxidase Substrate (Vector Labs). Sections were then counterstained with Mayer's hematoxylin, dehydrated, cleared in xylen and coverslipped using mounting solution. A negative control with UBC sections processed without the primary antibody was set up for every batch of slides to exclude nonspecific binding of the secondary antibody. As a positive control we observed expression of HuR in stromal cells and adjacent non-neoplastic epithelium. The cytoplasmic expression was observed predominantly in the tumor cells. No cytoplasmic immunoreactivity was seen in stromal cells and a very weak cytoplasmic reaction was seen in only few single normal urothelium cells.
(88,9) (80) (94,7) (68,2) (89,5) (85,7)
2 4 1 7 2 3
(11,1) (20) (5,2) (31,8) (10,5) (14,3)
5 (27,3) 3 (15) 5 (26,3) 20 (90,9) 13 (68,4) 16 (76,2) p < 0,001b
13 17 14 2 6 5
(72,7) (85) (73,7) (9,1) (31,6) (23,8)
21 (84) 79 (84) n.s.a
4 15
(16) (16)
3 (12) 59 (62,8) p < 0,001b
22 35
(88) (37,2)
20 19
3 7
(13) (26,9)
17 23
(73,9) (88,5)
6 3
(26,1) (11,5)
(87) (73,1)
n.s.a
n.s.a
27 92
22 (81,5) 78 (84,8) n.s.a
5 14
(18,5) (15,2)
18 44 n.s.b
(66,7) (47,8)
9 48
(33,3) (52,2)
53 66
46 (86,8) 54 (81,8) n.s.a
7 12
(13,2) (18,2)
30 32 n.s.b
(56,6) (48,5)
23 34
(43,4) (51,5)
50 69
41 (82) 59 (85,5) n.s.b
9 10
(18) (14,5)
39 (78) 23 (33,3) p < 0,001b
11 46
(22) (66,7)
n/a - data not available. a Chi-square test with Yates's correction. b Pearson's chi-square test.
percentage of positive cells. The intensity was scored as follows: 0 (negative staining), 1 (weak staining), 2 (moderate staining), 3 (strong staining). The percentage of positive cells was scored: 0 (no positive cells), 1 (< 10% positive cells), 2 (10–50% positive cells), 3 (51–80% positive cells), 4 (> 80% positive cells). For the immunoreactivity score (IRS) both values were multiplied, resulting in values: 0, 1, 2, 3, 4, 6, 8, 9 or 12. For statistical analysis, we categorized final scores as negative (IRS 0–6) or positive (IRS 8–12). 2.4. Microvessel density evaluation For microvessel density evaluation, CD31 (platelet/endothelial cell adhesion molecule-1) immunostaining was performed. CD31 is a panendothelial marker of epithelial cells and is considered to be a marker of choice for paraffin sections [21]. Staining was performed using automatic stainer (BOND-MAX, Leica) and a set of dedicated IHC reagents according to the manufacturer's instructions and protocols. In brief: after deparaffinization, antigen-retrieval procedure and blocking of endogenous peroxidase, 3-μm sections were incubated for 15 min with Ready-to-Use Primary CD31 antibody (Leica). Subsequently, tissues were incubated with HRP-polymer secondary antibody for 8 min, DAB chromogen for 10 min and counterstained with Mayer's hematoxylin. To measure MVD we used the vascular hot-spot technique described by Weidner et al. and modified by other authors [22]. At first we identified three regions of high vascular density within the tumor at low magnification (40 ×), and then evaluated each hot-spot at high magnification (200 ×). Each evaluated visual field covered 0,43 mm2. Any brown
2.3. Immunoreactivity scoring The expression of HuR in cancer cells was evaluated with immunoreactivity scoring performed as described previously by Wang et al. and Denkert et al. [15,20]. The nuclear and/or cytoplasmic expression of HuR was assesed by the intensity of staining and the 41
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Fig. 1. HuR immunoreactivity in normal urothelium and UBC. a, normal urothelium – urothelial cells as well as a number of stromal cells showing only nuclear immunoreactivity. b, low grade UBC in pTa stage – moderate nuclear reactivity detectable in > 80% of cancer cells (HuR nucl IRS = 8) with no cytoplasmic reactivity (HuR cyto IRS = 0). c, high grade UBC in pT1 stage (focus of invasion not shown here) – strong nuclear reactivity detectable in > 80% of cancer cells (HuR nucl IRS = 12) with no cytoplasmic reactivity (HuR cyto IRS = 0). d, high grade UBC in pT2 stage – moderate nuclear reactivity detectable in 51–80% of cancer cells (HuR nucl IRS = 6) with moderate cytoplasmic reactivity in 51–80% of cancer cells (HuR cyto IRS = 6). e, high grade UBC in pT3 stage – strong nuclear reactivity detectable in > 80% of cancer cells (HuR nucl IRS = 12) with strong cytoplasmic reactivity in > 80% of cancer cells (HuR cyto IRS = 12). f, high grade UBC in pT3 stage – moderate nuclear reactivity detectable in 51–80% of cancer cells (HuR nucl IRS = 6) with moderate cytoplasmic reactivity in > 80% of cancer cells (HuR cyto IRS = 8). Magnification – 200 ×, scale bars x × 100 μm.
relationships between clinicopathological factors and their influence on tumor stage or grade we performed log-linear regression analysis. For all tests the significance threshold was set at p ≤ 0,05. All analyses were performed using STATISTICA 12 (Dell Software).
Table 2 Association between nuclear and cytoplasmic HuR expression in 119 patients with UBC.
HuR nuclear positive, n HuR nuclear negative, n Total
a
HuR cytoplasmic positive, n
HuR cytoplasmic negative, n
Total
51
49
100
11
8
19
62
57 p valuea n.s.
3. Results HuR protein is a nuclear shuttling protein and its translocation from nucleus to cytoplasm reflects its increased activity. Therefore we separately evaluated HuR nuclear and cytoplasmic HuR expression in UBC cells. As shown in Table 1 and Fig. 1 nuclear expression was observed in 100 out of 119 cases (84%). In 62 cases (52,1%) a cytoplasmic expression was present. In 51 cases (42,6%) we found both positive nuclear and cytoplasmic expression. There was no correlation between nuclear and cytoplasmic immunoreactivity (Table 2). Mean MVD value in the analyzed cases was 37,42 ( ± 15,86) vessels/0,43mm2, and ranged from 13,33 to 92. We observed no significant association between nuclear HuR expression and clinicopathological factors such as patient's age, sex, tumor T stage, grade, regional lymph node status and microvessel density. Cytoplasmic HuR expression was significantly associated with high tumor T stage, high grade and high MVD of the tumor (p < 0,001). Cytoplasmic HuR expression increased with higher T stage: it was present in the cytoplasm in 20 (90,91%), 13 (68,42%) and 16 (76,19%) cases of tumors in pT2, pT3 and pT4 respectively while only 5 cases (27,3%) of pTis, 3 cases (15%) of pTa and 5 cases (26,3%) of pT1 tumors showed cytoplasmic expression. In HG tumors cytoplasmic expression was observed in 59 (62,8%) cases, and was significantly more frequent than in LG tumors, which showed positive cytoplasmic
Chi-square test with Yates's correction.
stained endothelial cell that was clearly separate from adjacent blood vessels, tumor cells and connective tissue elements was considered to be a single, countable vessel. Areas with extensive necrosis, inflammatory infiltrate or fibrosis were not included in the vessel count. The final MVD score was the average of the microvessels counts obtained from all three fields and given as continuous variable (average number of vessels/0,43 mm2). For statistical analysis we classified cases according to MVD values as poorly vascularized tumors (MVD ≤ 37 vessels/ 0,43mm2, 69 cases) and as well vascularized tumors (MVD > 37 vessels/0,43mm2, 50 cases).
2.5. Statistical analysis The association between HuR expression and histopathological parameters was assessed by chi-square tests (contingency tables) for categorical variables as wells as Mann-Whitney U Test for comparison of the mean values of continuous variables. To further evaluate the 42
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Fig. 2. Comparison of mean MVD values in groups classified according to nuclear and cytoplasmic HuR immunoreactivity. Tumors with positive cytoplasmic HuR expression had significantly higher mean MVD value than tumors showing no cytoplasmic immunoreactivity (44,84 SD ± 16,39 vs 29,35 SD ± 10,52, p < 0.001). Nuclear HuR expression status did not affect mean MVD value.
4. Discussion
reaction in 3 cases (12%). Well vascularized tumors showed positive cytoplasmic HuR expression in 39 cases (78%) in contrast to tumors with low MVD values which showed cytoplasmic HuR expression in only 23 cases (33,3%). Tumors with positive cytoplasmic HuR expression had significantly higher mean MVD value than those with no immunoreactivity (44,84 SD ± 16,39 vs 29,35 SD ± 10,52, p < 0,001) (Fig. 2). Other examined clinicopathological factors showed no correlation with cytoplasmic HuR immunoreactivity. To further evaluate the relationship between variables outlined in the frequency tables and tumor stage or grade assessment log-linear regression analysis was performed. All data was in the form of categorical variables. Two models were constructed: M1 (age, sex, HuR nuclear, HuR cytoplasmic, MVD, tumor stage) and M2 (age, sex, HuR nuclear, HuR cytoplasmic, MVD, tumor grade). The analysis demonstrated that in both models the main effects and only selected two-way interactions were included. Finally the determined models of best fit were as follows: M1 (chi-square = 23,115, df (degress of freedom) = 55, AIC (Akaike Information Criterion) = − 86,885, two-way interactions: tumor stage + HuR cytoplasmic and tumor stage + MVD) and M2 (chi-square = 32,129, df = 54, AIC = −75,871, two-way interactions: tumor grade + HuR cytoplasmic and tumor grade + MVD). To compare the effect sizes of interactions between the variables, odds ratios (OR) were computed. OR for HuR cytoplasmic expression and MVD were equal to 12,76 and 38,09 respectively in model M1 and 12,36 and 12,0 respectively in model M2. This demonstrated that in case of positive cytoplasmic HuR expression in UBC cells the risk of muscle invasive disease and high grade tumor is approximately 12times higher than in HuR-negative patients. In UBC with high MVD (> 37 vessels/0,43mm2) the risk of muscle invasive disease is approx 38-times higher and the risk of high grade tumors is 12-times higher than in poorly vascularized tumors. The log-linear regression analysis confirmed that cytoplasmic HuR expression and MVD are independent factors associated with high risk of muscle invasion and high-grade UBC. In normal urothelium HuR predominantly presented nuclear expression (Fig. 1). Nuclear immunoreactivity was also observed in stromal cells, mainly in fibroblasts, myocytes and macrophages. No cytoplasmic immunoreactivity was seen in stromal cells and a very weak cytoplasmic reaction was observed in only few single normal urothelium cells.
Cytoplasmic overexpression of HuR has been demonstrated in several cancers and is associated with disease progression and poor prognosis in patients with colon cancer, lung cancer, renal cell carcinoma and ovarian carcinoma [11,12,14,15]. However in some malignancies, such as breast cancer, positive cytoplasmic HuR expression can be associated with better prognosis [17]. There are also contradicting reports regarding the influence of HuR expression on histopathological factors such as tumor stage and grade in breast cancer and other malignancies [12,15,18,20]. In our study, we demonstrated that a cytoplasmic (but not nuclear) expression of HuR protein in UBC is associated with the depth of invasion (pT stage) and tumor grade (in both cases p < 0,001). Interestingly, the biggest change in cytoplasmic expression occurs between NMIBC (pTis, pTa and pT1) and MIBC (pT2-pT4). These observations may suggest that HuR is involved in the process of acquiring malignant histopathological features and ability to invade the muscularis propria by the cancer cells. We also found that HuR cytoplasmic immunoreactivity is closely associated with high intratumoral microvessel density, which could suggest that HuR protein is involved in the regulation of angiogenesis in UBC. We did not observe any association between the nuclear expression of HuR and any of the evaluated clinicopathological parameters. These results are in accord with the previous report from Miyata et al. that described correlation between cytoplasmic HuR expression and tumor stage, grade, MVD, presence of metastases and poor cancer-specific survival in non-muscle invasive urothelial bladder carcinoma [19]. The present study also supports the report form Liang et al. who investigated the expression of HuR in upper urinary tract urothelial carcinoma and showed strong association between cytoplasmic HuR immunoreactivity and tumor pT stage, high histological grade and poor prognosis [16]. The demonstrated association between cytoplasmic expression of HuR protein and known prognostic histopathological parameters such as tumor stage and grade suggests that the presence of HuR in urothelial bladder cancer cells' cytoplasm may be used as a prognostic or predictive factor. Considering frequent difficulties in diagnosing bladder cancer in specimens obtained from TURT, a positive cytoplasmic HuR immunostaining in cancer cells would suggest an invasion of muscularis propria and necessitate serial sectioning the specimen for further microscopic evaluation in search of a focus of muscle invasion. At the 43
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same time, lack of cytoplasmic HuR expression would mean that the risk of muscularis propria invasion is low. Status of cytoplasmic HuR expression may be useful in deciding which patients should undergo restaging transurethral resection (TUR). Restaging TUR is usually performed two to six weeks after the initial TURT when initial resection is incomplete, especially if the specimen is lacking muscularis propria or if the tumor is high grade pT1 lesion. Studies show that approx. 30% of apparent pT1 tumors will be understaged and restaging TUR will reveal stage pT2 or greater [23]. Assessing cytoplasmic HuR expression in initial TURT specimens would give additional information about the risk of muscularic propria invasion that may be useful in selecting patients in which restaging resection should be given high priority. Further large-scale studies with patient follow-up are necessary to establish whether immunohistochemical evaluation of HuR cytoplasmic expression can be used as a prognostic or predictive factor in UBC patients. The observed association between cytoplasmic HuR immunoreactivity and high microvessel density suggests that the translocation of HuR protein to cytoplasm can influence the process of angiogenesis in bladder cancer. HuR was demonstrated to regulate the stability of mRNA of many growth factors, cytokines, enzymes and other proteins, including proangiogenic factors such as VEGF, COX-2, MMP-9, IL-6 and IL-8 [24-28]. Further studies are needed to determine the exact role of HuR protein in the regulation of angiogenesis and its influence on urothelial bladder cancer pathogenesis in general. Recently Guo et al. reported the use of the antihelmintic drug and HuR protein inhibitor pyrvinium pamoate in urothelial bladder carcinoma [29]. Administration of pyrvinium pamoate with standard chemotherapeutics increased its cytotoxicity in UBC cell lines and inhibited the growth of UBC xenografts in mice. In their paper Guo et al. demonstrated that HuR inhibition leads to enhanced degradation of mRNA transcripts of DNA repairing genes which results in an increased number of DNA errors in cancer cells. Based on those observations as well as our findings we believe that HuR protein plays an important role in development and progression of UBC and that it may be a potential target for molecular anti-tumor therapy.
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High cytoplasmic HuR expression is associated with advanced pT stage, high grade and increased microvessel density in urothelial bladder carcinoma
T
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Łukasz Piotr Fus , Paweł Pihowicz, Łukasz Koperski, Janina Maja Marczewska, Barbara Górnicka Department of Pathology, Medical University of Warsaw, Pawińskiego 7, 02-106 Warsaw, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords: Bladder cancer HuR Stage Grade Microvessel density
Purpose: HuR (human antigen R) protein is a RNA binding protein that stabilizes the mRNA and controls the translation of genes involved in cell proliferation, differentiation, and carcinogenesis. Overexpression of HuR was reported in a variety of cancers, however its clinical significance in urothelial bladder cancer (UBC) is still unknown. Our aim is to investigate the association between HuR expression and selected histopathological factors, such as tumor grade, pT stage, regional lymph nodes status and microvessel density (MVD). Methods: We studied expression of HuR protein in 119 patients with UBC in stages pTis and pTa–pT4 using immunohistochemistry (IHC). Tumor MVD was evaluated immunohistochemically using anti-CD31 antibody. Results: We observed no association between nuclear HuR immunoreactivity and tumor grade, stage or MVD. We found a significant association between cytoplasmic HuR positivity and high tumor grade, pT stage and MVD (p < 0,001). We also observed significantly higher MVD values in cases with positive cytoplasmic HuR expression (p < 0,001). No association between HuR immunoreactivity and lymph nodes status was found. Conclusions: Our results may suggest that HuR is involved in the process of acquiring malignant histopathological features and ability to invade the muscularis propria by UBC cells. Considering frequent difficulties in diagnosing UBC in specimens obtained from transurethral tumor resection and the risk of understaging, cytoplasmic HuR expression would suggest an advanced disease and necessitate serial sectioning of the specimen in search of muscle invasion. Association between HuR expression and MVD could suggest HuR involvement in the process of angiogenesis in UBC.
1. Introduction Bladder cancer is the most common urinary tract cancer and the fourth-most common malignancy in men in developed countries. In Europe and North America over 90% of bladder cancers are urothelial bladder carcinomas (UBC) [1]. The most important morphological features affecting prognosis are cancer grade and stage. The depth of tumor invasion – especially the invasion of muscularis propria – is of particular significance as it directly affects patient's treatment [2]. HuR (human antigen R) is a RNA binding protein which regulates the turnover of mRNA and controls the process of translation [3]. In physiologic conditions the intracellular location of HuR is mainly nuclear, but upon activation it is translocated to the cell cytoplasm where it binds to AU-rich-element containing 3′ untranslated region and stabilizes mRNA transcripts prolonging their half-life [4]. HuR can also directly regulate translation of several factors involved in cell proliferation, differentiation, apoptosis as well as angiogenesis and carcinogenesis [5]. It was shown that HuR stabilizes mRNA transcripts
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Corresponding author. E-mail address: [email protected] (Ł.P. Fus).
https://doi.org/10.1016/j.anndiagpath.2017.12.002
1092-9134/ © 2017 Elsevier Inc. All rights reserved.
coding cyclins A2, B1, D1 and E1, cytochrome C, COX-2, VEGF, interleukins 6 and 8, glucose transporter GLUT1, TGF-b, c-Myc, p53 and p21 [5-10]. Overexpression of HuR was described in a large number of malignant neoplasms and even though its high expression levels frequently correlated with disease progression and poor prognosis, some studies suggest that the role of HuR in carcinogenesis remains ambiguous [11-18]. The role of HuR in the pathogenesis of UBC is poorly understood and, to the best of our knowledge, there is only one published paper which investigates the presence of HuR protein in UBC cells. In their study Miyata et al. analyzed specimens obtained during transurethral resection of tumor (TURT) biopsy which consisted mainly of nonmuscle invasive bladder cancers (NMIBC, stage pTa and pT1) and relatively few cases of advanced, muscle-invasive bladder cancer (MIBC) staged pT2 and higher [19]. The goal of our study is to evaluate the presence and distribution of HuR protein in UBC cells in both nonmuscle invasive and muscle-invasive disease, and to investigate the association between HuR expression and tumor pT stage, histological
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grade, regional lymph nodes status and intratumoral microvessel density (MVD).
Table 1 Association of nuclear and cytoplasmic HuR positivity with clinicopathological variables in 119 patients with UBC.
2. Material and methods
Variable
Total
HuR nuclear positive, n (%)
HuR nuclear negative, n (%)
HuR cytoplasmic positive, n (%)
HuR cytoplasmic negative, n (%)
All cases pT stage pTis pTa pT1 pT2 pT3 pT4 p value Grade Low High p value Nodal status pN+ pN− n/a p value Sex F M p value Age (years) < 70 ≥ 70 p value MVD (vessels/ 0,43mm2) > 37 ≤ 37 p value
119
100
19
62
57
18 20 19 22 19 21
16 16 18 15 17 18 n.s.a
25 94
23 26 70
2.1. Patients To conduct our study we collected histopathological samples from 119 patients diagnosed with urothelial bladder carcinoma between 2005 and 2010. Samples were stored in the archives of our Department. The specimens represented 18 cases of pTis, 20 cases of pTa UBC, 19 cases of pT1 UBC, 22 cases of pT2 UBC, 19 cases of pT3 UBC and 22 cases of pT4 UBC. For log-linear regression analysis we categorized tumor staged pTis, pTa and pT1 as low (non-muscle invasive) stage and tumors pT2-pT4 as high (muscle invasive) stage. Samples of pTis, pTa and pT1 cancers were obtained during TURT biopsy. If the patient underwent more than one TURT, only the specimen from the first biopsy was evaluated. In more advanced UBC cases (pT2-pT4) tissue samples were taken from radical cystectomy specimens. The status of regional lymph nodes was evaluated only in cases staged pT2–pT4 in which radical cystectomy with lymphadenectomy was performed (49 cases). The mean patient's age was 69,8 ± 8,9 ( ± SD) years and ranged from 48 to 86. For statistical analysis we categorized cases according to patients age as patients < 70 years-old (53 cases) and ≥ 70 years-old (66 cases). 92 patients were males, 27 were females. 25 UBCs were diagnosed as low-grade (LG) and 94 as high-grade (HG) tumors. Among 20 cases of pTa UBCs tumors 18 were described as LG (90%) and 2 cases as HG (10%). 12 cases of pT1 cancers were HG (63,2%) and 7 cases were LG (36,8%). All cases diagnosed as pTis and pT2-pT4 cancers were described as HG. Among 49 cases in which regional lymph node status could be evaluated, 23 patients had lymph node metastases. 2.2. Immunohistochemistry Resected specimens were fixed in 10% buffered formalin and embedded in paraffin blocks. Immunohistochemical examinations were carried out on 3-μm tissue sections using anti-HuR mouse monoclonal antibody (3A2, Thermo Fisher). Sections were deparrafinized in xylene and rehydrated in graded ethanol solutions. Heat-induced antigen retrieval was performed with 0,01 M citrate buffer (pH 6.0) in a 700 W microwave (2 × 5 min, with 5-minute pause). Endogenous peroxidase was blocked with incubation in 3% hydrogen peroxide for 5 min. After washing with PBS, slides with sections were incubated with 2,5% horse serum for 20 min to block the non-specific antigen binding sites. Then sections were incubated for 45 min at room temperature with primary anti-HuR antibody (1:1000). After washing the slides with PBS, ImmPRESS HRP Anti-Mouse Polymer Detection Kit (Vector Labs) was applied for 30 min. The peroxidase reaction was visualized using ImmPACT DAB Peroxidase Substrate (Vector Labs). Sections were then counterstained with Mayer's hematoxylin, dehydrated, cleared in xylen and coverslipped using mounting solution. A negative control with UBC sections processed without the primary antibody was set up for every batch of slides to exclude nonspecific binding of the secondary antibody. As a positive control we observed expression of HuR in stromal cells and adjacent non-neoplastic epithelium. The cytoplasmic expression was observed predominantly in the tumor cells. No cytoplasmic immunoreactivity was seen in stromal cells and a very weak cytoplasmic reaction was seen in only few single normal urothelium cells.
(88,9) (80) (94,7) (68,2) (89,5) (85,7)
2 4 1 7 2 3
(11,1) (20) (5,2) (31,8) (10,5) (14,3)
5 (27,3) 3 (15) 5 (26,3) 20 (90,9) 13 (68,4) 16 (76,2) p < 0,001b
13 17 14 2 6 5
(72,7) (85) (73,7) (9,1) (31,6) (23,8)
21 (84) 79 (84) n.s.a
4 15
(16) (16)
3 (12) 59 (62,8) p < 0,001b
22 35
(88) (37,2)
20 19
3 7
(13) (26,9)
17 23
(73,9) (88,5)
6 3
(26,1) (11,5)
(87) (73,1)
n.s.a
n.s.a
27 92
22 (81,5) 78 (84,8) n.s.a
5 14
(18,5) (15,2)
18 44 n.s.b
(66,7) (47,8)
9 48
(33,3) (52,2)
53 66
46 (86,8) 54 (81,8) n.s.a
7 12
(13,2) (18,2)
30 32 n.s.b
(56,6) (48,5)
23 34
(43,4) (51,5)
50 69
41 (82) 59 (85,5) n.s.b
9 10
(18) (14,5)
39 (78) 23 (33,3) p < 0,001b
11 46
(22) (66,7)
n/a - data not available. a Chi-square test with Yates's correction. b Pearson's chi-square test.
percentage of positive cells. The intensity was scored as follows: 0 (negative staining), 1 (weak staining), 2 (moderate staining), 3 (strong staining). The percentage of positive cells was scored: 0 (no positive cells), 1 (< 10% positive cells), 2 (10–50% positive cells), 3 (51–80% positive cells), 4 (> 80% positive cells). For the immunoreactivity score (IRS) both values were multiplied, resulting in values: 0, 1, 2, 3, 4, 6, 8, 9 or 12. For statistical analysis, we categorized final scores as negative (IRS 0–6) or positive (IRS 8–12). 2.4. Microvessel density evaluation For microvessel density evaluation, CD31 (platelet/endothelial cell adhesion molecule-1) immunostaining was performed. CD31 is a panendothelial marker of epithelial cells and is considered to be a marker of choice for paraffin sections [21]. Staining was performed using automatic stainer (BOND-MAX, Leica) and a set of dedicated IHC reagents according to the manufacturer's instructions and protocols. In brief: after deparaffinization, antigen-retrieval procedure and blocking of endogenous peroxidase, 3-μm sections were incubated for 15 min with Ready-to-Use Primary CD31 antibody (Leica). Subsequently, tissues were incubated with HRP-polymer secondary antibody for 8 min, DAB chromogen for 10 min and counterstained with Mayer's hematoxylin. To measure MVD we used the vascular hot-spot technique described by Weidner et al. and modified by other authors [22]. At first we identified three regions of high vascular density within the tumor at low magnification (40 ×), and then evaluated each hot-spot at high magnification (200 ×). Each evaluated visual field covered 0,43 mm2. Any brown
2.3. Immunoreactivity scoring The expression of HuR in cancer cells was evaluated with immunoreactivity scoring performed as described previously by Wang et al. and Denkert et al. [15,20]. The nuclear and/or cytoplasmic expression of HuR was assesed by the intensity of staining and the 41
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Fig. 1. HuR immunoreactivity in normal urothelium and UBC. a, normal urothelium – urothelial cells as well as a number of stromal cells showing only nuclear immunoreactivity. b, low grade UBC in pTa stage – moderate nuclear reactivity detectable in > 80% of cancer cells (HuR nucl IRS = 8) with no cytoplasmic reactivity (HuR cyto IRS = 0). c, high grade UBC in pT1 stage (focus of invasion not shown here) – strong nuclear reactivity detectable in > 80% of cancer cells (HuR nucl IRS = 12) with no cytoplasmic reactivity (HuR cyto IRS = 0). d, high grade UBC in pT2 stage – moderate nuclear reactivity detectable in 51–80% of cancer cells (HuR nucl IRS = 6) with moderate cytoplasmic reactivity in 51–80% of cancer cells (HuR cyto IRS = 6). e, high grade UBC in pT3 stage – strong nuclear reactivity detectable in > 80% of cancer cells (HuR nucl IRS = 12) with strong cytoplasmic reactivity in > 80% of cancer cells (HuR cyto IRS = 12). f, high grade UBC in pT3 stage – moderate nuclear reactivity detectable in 51–80% of cancer cells (HuR nucl IRS = 6) with moderate cytoplasmic reactivity in > 80% of cancer cells (HuR cyto IRS = 8). Magnification – 200 ×, scale bars x × 100 μm.
relationships between clinicopathological factors and their influence on tumor stage or grade we performed log-linear regression analysis. For all tests the significance threshold was set at p ≤ 0,05. All analyses were performed using STATISTICA 12 (Dell Software).
Table 2 Association between nuclear and cytoplasmic HuR expression in 119 patients with UBC.
HuR nuclear positive, n HuR nuclear negative, n Total
a
HuR cytoplasmic positive, n
HuR cytoplasmic negative, n
Total
51
49
100
11
8
19
62
57 p valuea n.s.
3. Results HuR protein is a nuclear shuttling protein and its translocation from nucleus to cytoplasm reflects its increased activity. Therefore we separately evaluated HuR nuclear and cytoplasmic HuR expression in UBC cells. As shown in Table 1 and Fig. 1 nuclear expression was observed in 100 out of 119 cases (84%). In 62 cases (52,1%) a cytoplasmic expression was present. In 51 cases (42,6%) we found both positive nuclear and cytoplasmic expression. There was no correlation between nuclear and cytoplasmic immunoreactivity (Table 2). Mean MVD value in the analyzed cases was 37,42 ( ± 15,86) vessels/0,43mm2, and ranged from 13,33 to 92. We observed no significant association between nuclear HuR expression and clinicopathological factors such as patient's age, sex, tumor T stage, grade, regional lymph node status and microvessel density. Cytoplasmic HuR expression was significantly associated with high tumor T stage, high grade and high MVD of the tumor (p < 0,001). Cytoplasmic HuR expression increased with higher T stage: it was present in the cytoplasm in 20 (90,91%), 13 (68,42%) and 16 (76,19%) cases of tumors in pT2, pT3 and pT4 respectively while only 5 cases (27,3%) of pTis, 3 cases (15%) of pTa and 5 cases (26,3%) of pT1 tumors showed cytoplasmic expression. In HG tumors cytoplasmic expression was observed in 59 (62,8%) cases, and was significantly more frequent than in LG tumors, which showed positive cytoplasmic
Chi-square test with Yates's correction.
stained endothelial cell that was clearly separate from adjacent blood vessels, tumor cells and connective tissue elements was considered to be a single, countable vessel. Areas with extensive necrosis, inflammatory infiltrate or fibrosis were not included in the vessel count. The final MVD score was the average of the microvessels counts obtained from all three fields and given as continuous variable (average number of vessels/0,43 mm2). For statistical analysis we classified cases according to MVD values as poorly vascularized tumors (MVD ≤ 37 vessels/ 0,43mm2, 69 cases) and as well vascularized tumors (MVD > 37 vessels/0,43mm2, 50 cases).
2.5. Statistical analysis The association between HuR expression and histopathological parameters was assessed by chi-square tests (contingency tables) for categorical variables as wells as Mann-Whitney U Test for comparison of the mean values of continuous variables. To further evaluate the 42
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Fig. 2. Comparison of mean MVD values in groups classified according to nuclear and cytoplasmic HuR immunoreactivity. Tumors with positive cytoplasmic HuR expression had significantly higher mean MVD value than tumors showing no cytoplasmic immunoreactivity (44,84 SD ± 16,39 vs 29,35 SD ± 10,52, p < 0.001). Nuclear HuR expression status did not affect mean MVD value.
4. Discussion
reaction in 3 cases (12%). Well vascularized tumors showed positive cytoplasmic HuR expression in 39 cases (78%) in contrast to tumors with low MVD values which showed cytoplasmic HuR expression in only 23 cases (33,3%). Tumors with positive cytoplasmic HuR expression had significantly higher mean MVD value than those with no immunoreactivity (44,84 SD ± 16,39 vs 29,35 SD ± 10,52, p < 0,001) (Fig. 2). Other examined clinicopathological factors showed no correlation with cytoplasmic HuR immunoreactivity. To further evaluate the relationship between variables outlined in the frequency tables and tumor stage or grade assessment log-linear regression analysis was performed. All data was in the form of categorical variables. Two models were constructed: M1 (age, sex, HuR nuclear, HuR cytoplasmic, MVD, tumor stage) and M2 (age, sex, HuR nuclear, HuR cytoplasmic, MVD, tumor grade). The analysis demonstrated that in both models the main effects and only selected two-way interactions were included. Finally the determined models of best fit were as follows: M1 (chi-square = 23,115, df (degress of freedom) = 55, AIC (Akaike Information Criterion) = − 86,885, two-way interactions: tumor stage + HuR cytoplasmic and tumor stage + MVD) and M2 (chi-square = 32,129, df = 54, AIC = −75,871, two-way interactions: tumor grade + HuR cytoplasmic and tumor grade + MVD). To compare the effect sizes of interactions between the variables, odds ratios (OR) were computed. OR for HuR cytoplasmic expression and MVD were equal to 12,76 and 38,09 respectively in model M1 and 12,36 and 12,0 respectively in model M2. This demonstrated that in case of positive cytoplasmic HuR expression in UBC cells the risk of muscle invasive disease and high grade tumor is approximately 12times higher than in HuR-negative patients. In UBC with high MVD (> 37 vessels/0,43mm2) the risk of muscle invasive disease is approx 38-times higher and the risk of high grade tumors is 12-times higher than in poorly vascularized tumors. The log-linear regression analysis confirmed that cytoplasmic HuR expression and MVD are independent factors associated with high risk of muscle invasion and high-grade UBC. In normal urothelium HuR predominantly presented nuclear expression (Fig. 1). Nuclear immunoreactivity was also observed in stromal cells, mainly in fibroblasts, myocytes and macrophages. No cytoplasmic immunoreactivity was seen in stromal cells and a very weak cytoplasmic reaction was observed in only few single normal urothelium cells.
Cytoplasmic overexpression of HuR has been demonstrated in several cancers and is associated with disease progression and poor prognosis in patients with colon cancer, lung cancer, renal cell carcinoma and ovarian carcinoma [11,12,14,15]. However in some malignancies, such as breast cancer, positive cytoplasmic HuR expression can be associated with better prognosis [17]. There are also contradicting reports regarding the influence of HuR expression on histopathological factors such as tumor stage and grade in breast cancer and other malignancies [12,15,18,20]. In our study, we demonstrated that a cytoplasmic (but not nuclear) expression of HuR protein in UBC is associated with the depth of invasion (pT stage) and tumor grade (in both cases p < 0,001). Interestingly, the biggest change in cytoplasmic expression occurs between NMIBC (pTis, pTa and pT1) and MIBC (pT2-pT4). These observations may suggest that HuR is involved in the process of acquiring malignant histopathological features and ability to invade the muscularis propria by the cancer cells. We also found that HuR cytoplasmic immunoreactivity is closely associated with high intratumoral microvessel density, which could suggest that HuR protein is involved in the regulation of angiogenesis in UBC. We did not observe any association between the nuclear expression of HuR and any of the evaluated clinicopathological parameters. These results are in accord with the previous report from Miyata et al. that described correlation between cytoplasmic HuR expression and tumor stage, grade, MVD, presence of metastases and poor cancer-specific survival in non-muscle invasive urothelial bladder carcinoma [19]. The present study also supports the report form Liang et al. who investigated the expression of HuR in upper urinary tract urothelial carcinoma and showed strong association between cytoplasmic HuR immunoreactivity and tumor pT stage, high histological grade and poor prognosis [16]. The demonstrated association between cytoplasmic expression of HuR protein and known prognostic histopathological parameters such as tumor stage and grade suggests that the presence of HuR in urothelial bladder cancer cells' cytoplasm may be used as a prognostic or predictive factor. Considering frequent difficulties in diagnosing bladder cancer in specimens obtained from TURT, a positive cytoplasmic HuR immunostaining in cancer cells would suggest an invasion of muscularis propria and necessitate serial sectioning the specimen for further microscopic evaluation in search of a focus of muscle invasion. At the 43
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same time, lack of cytoplasmic HuR expression would mean that the risk of muscularis propria invasion is low. Status of cytoplasmic HuR expression may be useful in deciding which patients should undergo restaging transurethral resection (TUR). Restaging TUR is usually performed two to six weeks after the initial TURT when initial resection is incomplete, especially if the specimen is lacking muscularis propria or if the tumor is high grade pT1 lesion. Studies show that approx. 30% of apparent pT1 tumors will be understaged and restaging TUR will reveal stage pT2 or greater [23]. Assessing cytoplasmic HuR expression in initial TURT specimens would give additional information about the risk of muscularic propria invasion that may be useful in selecting patients in which restaging resection should be given high priority. Further large-scale studies with patient follow-up are necessary to establish whether immunohistochemical evaluation of HuR cytoplasmic expression can be used as a prognostic or predictive factor in UBC patients. The observed association between cytoplasmic HuR immunoreactivity and high microvessel density suggests that the translocation of HuR protein to cytoplasm can influence the process of angiogenesis in bladder cancer. HuR was demonstrated to regulate the stability of mRNA of many growth factors, cytokines, enzymes and other proteins, including proangiogenic factors such as VEGF, COX-2, MMP-9, IL-6 and IL-8 [24-28]. Further studies are needed to determine the exact role of HuR protein in the regulation of angiogenesis and its influence on urothelial bladder cancer pathogenesis in general. Recently Guo et al. reported the use of the antihelmintic drug and HuR protein inhibitor pyrvinium pamoate in urothelial bladder carcinoma [29]. Administration of pyrvinium pamoate with standard chemotherapeutics increased its cytotoxicity in UBC cell lines and inhibited the growth of UBC xenografts in mice. In their paper Guo et al. demonstrated that HuR inhibition leads to enhanced degradation of mRNA transcripts of DNA repairing genes which results in an increased number of DNA errors in cancer cells. Based on those observations as well as our findings we believe that HuR protein plays an important role in development and progression of UBC and that it may be a potential target for molecular anti-tumor therapy.
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