Expression of ROR1, pAkt, and pCREB in gastric adenocarcinoma

Expression of ROR1, pAkt, and pCREB in gastric adenocarcinoma

Annals of Diagnostic Pathology xxx (2015) xxx–xxx Contents lists available at ScienceDirect Annals of Diagnostic Pathology Original Contributions ...

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Annals of Diagnostic Pathology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Annals of Diagnostic Pathology

Original Contributions

Expression of ROR1, pAkt, and pCREB in gastric adenocarcinoma Hyeyoon Chang, MD, PhD a, Woon Yong Jung, MD, PhD b, Youngran Kang, MD, MS c, Hyunjoo Lee, MD, PhD d, Aeree Kim, MD, PhD a, Baek-hui Kim, MD, PhD a,⁎ a

Department of Pathology, Korea University Guro Hospital, Korea University College of Medicine, Seoul, Republic of Korea Department of Pathology, Catholic Kwandong University International St Mary's Hospital, Incheon, Republic of Korea Department of Pathology, Green Cross Laboratories, Yongin, Kyeonggi-Do, Republic of Korea d Department of Pathology, Kangbuk Samsung Hospital, Sungkyunkwan University College of Medicine, Seoul, Republic of Korea b c

a r t i c l e

i n f o

Available online xxxx Keywords: ROR1 Akt CREB Adenocarcinoma Stomach

a b s t r a c t The receptor tyrosine kinase–like orphan receptor 1 (ROR1) is a transmembrane protein of receptor tyrosine kinase family. High expression of ROR1 is reported in many types of malignancies and is thought to be involved in tumor growth, apoptosis, and epithelial-mesenchymal transition. In this study, we examined the expression of ROR1, pAkt, and pCREB in gastric adenocarcinoma and analyzed with clinicopathologic factors and tumor proliferation. Tissue microarray blocks containing 424 gastric adenocarcinomas were used for immunohistochemical staining. Ki-67 labeling index was used for tumor proliferation activity. High expression of ROR1 (63%), pAkt (36%), and pCREB (20%) was observed in gastric adenocarcinomas, and expression of these proteins was well intercorrelated. ROR1 and pCREB expression was associated with Ki-67 labeling index (P b .001). Expression of pAkt and pCREB group showed longer survival in univariate analysis (P = .007 and P b .001, respectively). This is the first study that analyzed ROR1 expression in gastric adenocarcinoma tissue samples. We revealed that gastric adenocarcinomas highly express ROR1 and related proteins and its prognostic significance. ROR1 in gastric adenocarcinoma could be possible candidate of therapeutic target, and more comprehensive study is required. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Gastric adenocarcinoma is one of the most common cancers worldwide and causing high cancer mortality [1]. Surgical treatment is the basic treatment for gastric adenocarcinoma, but various chemotherapies or target agents targeting human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), and c-Met have been developed [2,3]. However, the prognosis of advanced gastric cancer is not good yet, and discovery of new druggable therapeutic target in gastric adenocarcinoma is needed. The receptor tyrosine kinase–like orphan receptor 1 (ROR1) was first identified in a neuroblastoma cell line [4]. ROR family proteins are conserved during evolution, and their function in embryonic development was reported in variety of literature [5,6]. ROR1 consists of an immunoglobulin-like domain, a Frizzled domain, a Kringle domain, a tyrosine kinase domain, a serine/threonine-rich domain, and a prolinerich domain. ROR1 mediates variety of intracellular signaling including cell growth and epithelial-mesenchymal transition [7]. Although the expression of ROR1 is low in mature human tissues, some subsequent studies have revealed that ROR1 is expressed in cancer cells. In some blood malignancies including chronic lymphocytic leukemia, chronic ⁎ Corresponding author at: Department of Pathology, Korea University Guro Hospital, Korea University College of Medicine, Gurodongro 148, Guro-gu, Seoul 152-703, Republic of Korea. Tel.: +82 2 2626 3255; fax: +82 2 2626 1486. E-mail address: [email protected] (B. Kim).

myelogenous leukemia, and diffuse large B-cell lymphoma, expression of ROR1 was observed [8,9]. High expression of ROR1 is also observed in many solid malignancies such as colon, lung, pancreatic, prostate, and breast cancer tissues [10,11]. In gastric cancer cell lines, ROR1 expression was observed and associated with tumorigenesis [12]. In the area of cancer research, its role has been drawing attention because it plays an important role in major cell signal pathway such as PI3K/Akt/cAMP response element-binding protein (CREB) nuclear transduction and EGFR-induced signaling pathway and serves as a receptor for Wnt5a [7,13]. High expression of ROR1 was reported to activate Akt phosphorylation and CREB, enhancing tumor growth and proliferation in human breast cancer cells [10]. In this study, we observed the immunohistochemical expression of ROR1 and related proteins phosphor-Akt (pAkt) and phosphor-CREB (pCREB) in gastric adenocarcinoma tissues and analyzed correlation between these protein expression, clinicopathologic factors, and tumor proliferative activity measured by Ki-67 labeling index. 2. Materials and methods 2.1. Patients and clinicopathologic data We retrospectively collected formalin-fixed, paraffin-embedded tissue samples from 424 gastric cancer patients who had undergone gastrectomy at Korea University Guro Hospital from 2002 to 2005. No patient has received neoadjuvant chemotherapy.

http://dx.doi.org/10.1016/j.anndiagpath.2015.06.010 1092-9134/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Chang H, et al, Expression of ROR1, pAkt, and pCREB in gastric adenocarcinoma, Ann Diagn Pathol (2015), http:// dx.doi.org/10.1016/j.anndiagpath.2015.06.010

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H. Chang et al. / Annals of Diagnostic Pathology xxx (2015) xxx–xxx

Clinical data including age, sex, distant metastasis, and diseasespecific patient survival were identified by chart review. All pathology slides were reviewed, and pathologic data including pathologic tumor stage, histologic grade, Lauren classification, lymphatic invasion, and perineural invasion were evaluated [14,15]. Early gastric cancer is defined as invasive gastric cancer confined to the mucosa and/or submucosa irrespective of lymph node metastasis. The other cases were included in the advanced gastric cancer. This study was approved by Institutional Review Board of Korea University Guro Hospital (KUGH12225-002).

areas of positive tumor cells were recorded. We considered a case as high expression when 50% or more tumor cells showed moderate or strong staining intensity and otherwise as low expression (Fig. 1). Staining of Ki-67 showed a nuclear staining pattern. Nuclear staining intensity was graded in a 4-tier scale (0, negative; 1, weak; 2, moderate; 3, strong). Tumor cell with moderate or strong nuclear staining was counted as positive, and positive tumor cell fraction was recorded (Ki67 labeling index). All immunohistochemical stained slides were scored by 2 gastrointestinal pathologists, Kim BH and Chang HY. At least 2 tumor fields at a high magnification (×400) were examined.

2.2. Tissue microarray block construction and immunohistochemical staining

2.4. Statistical analysis

Representative areas of the tumor and nontumor portions were marked on the slide under a microscope and their corresponding area on the paraffin blocks were used for the tissue microarray (TMA) block. The tumor selected for inclusion in the TMA was exclusively from the main gastric mass. Each 2-mm diameter core tissue from donor blocks was implanted to the recipient blocks. The 4-μm-thick tissue sections of TMA blocks were mounted on electrostatic slides and used for staining. Immunohistochemical staining was done as previously described [16]. Antibodies used in this study are as follows: anti-ROR1 antibody (1:25, rabbit polyclonal; Abcam, Cambridge, MA), anti-pAkt (phosphor-Ser473) antibody (1:50, rabbit polyclonal; Abcam), antipCREB (phosphor-Ser133) antibody (1:100, rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), and Ki-67 (1:700, clone MIB-1; Dako, Glostrup, Denmark).

Relationship between clinicopathologic factors and immunohistochemical staining results was evaluated using χ 2 test, Fisher exact test, and independent t test. Disease-specific survival data were examined using the Kaplan-Meier plot. Log-rank test was used to verify the statistical significance of differences between survival curves. We performed multivariate analysis on factors that were statistically significantly associated with survival in the univariate analysis by Cox proportional hazards regression model to find the statistically significant independent factors. P b .05 was considered to be statistically significant. All statistical analyses were carried out using SPSS statistical software (version 12; SPSS, Chicago, IL). 3. Result 3.1. Patient characteristics

2.3. Analysis of immunohistochemical staining Immunohistochemical staining of ROR1, pAkt, and pCREB showed cytoplasmic and membranous pattern, cytoplasmic pattern, and nuclear pattern, respectively. The intensity of immunoreactivity was graded in a 4-tier scale (0, negative; 1, weak; 2, moderate; 3, strong), and percent

Among 424 patients, early gastric cancer patients were 163 (38%), and advanced gastric cancer patients were 261 (62%). Patient followup period ranged from 1 to 131 months (mean, 82.3 months; median, 96.1 months). Mean age of patients were 58.1 years (median, 61 years; range 23-84 years), and the male-to-female ratio was 2.07.

Fig. 1. Representative microscopic images of immunohistochemical staining (×200). Upper panel shows ROR1 staining (A-D), middle panel shows pCREB staining (E-H), and lower panel shows pAkt staining (I-L). Four-tiered grading of negative (A, E, and I), weak (B, F, and J), moderate (C, G, and K), and strong (D, H, and L) was used in analysis, and cases showing moderate or strong staining in 50% or more tumor cells were regarded as high expression.

Please cite this article as: Chang H, et al, Expression of ROR1, pAkt, and pCREB in gastric adenocarcinoma, Ann Diagn Pathol (2015), http:// dx.doi.org/10.1016/j.anndiagpath.2015.06.010

H. Chang et al. / Annals of Diagnostic Pathology xxx (2015) xxx–xxx

3.2. Immunohistochemical staining results

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Table 2 Intercorrelation between ROR1, pCREB, and pAkt.

High expression of ROR1 was observed in 269 patients (63%). ROR1 high expression was associated with lower pT stage (P = .020) and absence of perineural invasion (P = 0.002). High expression of pAkt was observed in 151 patients (36%) and associated with lower pT stage (P = .042). High expression of pCREB was observed in 86 patients (20%) and associated with lower pT stage (P b .001), pN stage (P b .001), absence of lymphatic invasion (P = .003), and absence of perineural invasion. (P = .001). Correlation between protein expression and patient age, distant metastasis, and histologic grade was not observed (Table 1). Intercorrelation between ROR1, pCREB, and pAkt showed statistical significance. Expression of ROR1 was correlated with pCREB (P = .011) and pAkt (P = .001) expression. Phospho-Akt expression showed association with pCREB (P = .024) (Table 2). 3.3. Expression of ROR1, pAkt, pCREB, and Ki-67 labeling index To compare proliferation activity between high expression group and low expression group, independent t test was used. Expression of ROR1, pAkt, and pCREB was compared with Ki-67 labeling index. ROR1 high expression group showed high Ki-67 labeling index (P b .001). Mean of Ki-67 labeling index was slightly higher in pAkt high expression group without statistical significance (P = .134). High expression group of pCREB also showed high Ki-67 labeling index (P b .001) (Fig. 2).

Clinicopathologic characteristics

ROR1 High

pCREB High expression Low expression pAkt High expression Low expression

pAkt Low

P

High

Low

39 112

47 226

.011⁎ 64 205

22 133

111 158

40 115

.001⁎

P .024⁎

⁎ Statistically significant p values are indicated in boldface type.

survival time in Kaplan-Meier plot, but statistical significance was not found (P = .189). High expression of pAkt and pCREB was associated with longer patient survival (P = .007 and P b .001, respectively) (Fig. 3). Multivariate analysis was performed by the Cox proportional hazards model. Parameters included in the analysis were age, histologic grade, invasion depth, lymph node metastasis, lymphatic invasion, pAkt expression, and pCREB expression. Invasion depth (P b .001), lymph node metastasis (P = .001), lymphatic invasion (P = .002), and old age (P = .010) were independent poor prognostic factors. In this analysis, expression of pAkt was not independent prognostic factor but showed marginal significance (P = .116). Expression of pCREB (P = .443) and histologic grade (P = .349) did not show statistical significance in multivariate analysis (Table 3).

3.4. Patient survival and expression of ROR1, pAkt, and pCREB Poor disease-specific survival was associated with age older than 60 years (P = .032), higher histologic grade (P b .001), diffuse-type Lauren classification (P b .001), higher pathologic T stage (P b .001), higher pathologic N stage (P b .001), distant metastasis (P b .001), and lymphatic invasion (P b .001). High expression of ROR1 showed longer

Table 1 Associations between protein immunoexpression level and clinicopathologic factors. Clinicopathologic characteristics Age (y) ≤60 N60 pT stage pT1 pT2 pT3 pT4 pN stage pN0 pN1 pN2 pN3 Distant metastasis Present Absent Lymphatic invasion Present Absent Perineural invasion Present Absent Histologic grade WD MD PD

ROR1

pAkt

pCREB

High Low P

High Low P

High Low P

.537 132 137

76 79

117 35 39 78

46 21 23 65

139 40 30 60

75 12 19 49

8 260

4 151

.020⁎

.185 79 72

129 144

71 20 17 43

92 36 45 100

88 16 18 29

126 36 31 80

5 146

7 265

.053

59 210

55 100

42 106 121

24 48 83

.002⁎

57 11 6 12

106 45 56 131

65 5 9 7

149 47 40 102

0 85

12 326

18 68

126 212

12 74

102 236

14 37 35

52 117 169

.437

.426 54 101

164 174

.064

.535

90 179

.042⁎

.376 44 42

96 177

36 115

78 195

18 55 78

48 99 126

.174

.180

b.001⁎

.065

.276 48 103

b.001⁎

.271

.003⁎

.001⁎

.272

Abbreviations: WD, well differentiated; MD, moderately differentiated; PD, poorly differentiated. ⁎ Statistically significant p values are indicated in boldface type.

4. Discussion ROR1 expression was reported in many types of cancers [10,11]. Some of these studies reported that ROR1 expression in breast and ovarian cancers is related to the higher tumor grade and aggressive behaviors [10,17]. However, another study reports that there is no different expression of ROR1 in progressive disease and nonprogressive disease of chronic lymphocytic leukemia [18]. The relationship between ROR1 expression and cancer prognosis is still controversial. There are no published data about ROR1 expression in gastric cancer tissue and its clinical relevance to patient outcome. It was reported that ROR1 is expressed in gastric cancer cell lines, and silencing of ROR1 in these cell lines showed impaired proliferation [12]. In this study, we evaluated the expression of ROR1 in gastric adenocarcinoma tissue samples. Of the 424 gastric cancer samples, 269 samples (63.4%) showed high expression of ROR1. High expression of ROR1 was associated with lower pT stage, and absence of perineural invasion, which are pathologic parameters associated with good clinical outcome. In the survival analysis, ROR1 high expression group showed longer survival curve in Kaplan-Meier plot but failed to show statistical significance. We hypothesized that the signaling pathway PI3K/Akt/CREB nuclear transduction induced by ROR1 would play a major role in the carcinogenesis of gastric cancer. So, we also conducted immunohistochemical staining of pAkt and pCREB on gastric cancer tissues and analyzed expression of these proteins. High expression of pAkt and pCREB was observed in 151 (35.6%) and 86 (20.3%) samples, respectively. There are some possible explanations about the difference between expression levels of ROR1, pAkt, and pCREB. The first is that antibody sensitivities could be not same between these proteins. The other explanation is that ROR1 is not only receptor related to Akt, and CREB is not only downward molecule in the Akt signaling [19]. Although there was considerable difference of high expression rate between ROR1, pAkt, and pCREB, intercorrelation of high expression between ROR1 and pCREB, between ROR1 and pAkt, and between pCREB and pAkt showed significant association, which corresponds with our hypothesis. PhosphoAkt expression showed correlation with lower pT stage, and pCREB expression showed correlation with lower pT stage, lower pN stage, absence of lymphatic invasion, and absence of perineural invasion.

Please cite this article as: Chang H, et al, Expression of ROR1, pAkt, and pCREB in gastric adenocarcinoma, Ann Diagn Pathol (2015), http:// dx.doi.org/10.1016/j.anndiagpath.2015.06.010

4

H. Chang et al. / Annals of Diagnostic Pathology xxx (2015) xxx–xxx

Fig. 2. Box plots showing Ki-67 labeling indexes in low and high expression groups of ROR1 (left), pAkt (middle), and pCREB (right).

Univariate survival analysis showed significant longer survival in patients with high expression of pAkt and pCREB, but in multivariate analysis, pAkt and pCREB expression was not independent prognostic factor. As in the previous literature of breast cancer tissue, we could find significant correlation between expressions of ROR1, phosphorylated Akt, and phosphorylated CREB [10]. In some types of cancer cells, it is known that ROR1 stimulates phosphorylation of Akt and phosphorylated Akt stimulates phosphorylation of CREB [10,20]. CREB is a transcription factor that overexpressed in some cancers including acute myeloid leukemia and lung cancer and regulates some genes related to oncogenesis [21]. Our staining results that high expression of ROR1, pAkt, and pCREB is significantly intercorrelated and is associated with good prognostic factors suggest that ROR1 plays a role through phosphorylation of Akt and CREB in gastric adenocarcinoma.

We noted that activated CREB could induce proliferation of cancer cells, and this may be related to the frequent expression of ROR1 in cancer cells [22]. To evaluate the relationship between ROR1/Akt/CREB pathway and tumor proliferation, we analyzed this protein expression and Ki-67 labeling index. High expression of ROR1 and pCREB was significantly correlated with high Ki-67 index. High expression group of pAkt showed tendency of association with high Ki-67 group but lacked statistical significance. This result suggests that the role of ROR1, pAkt, and pCREB in gastric adenocarcinoma may be related to the proliferation of tumor cells. In some gastrointestinal tumors including neuroendocrine tumors and gastrointestinal stromal tumors and breast cancers, high proliferation of tumor cells is related to the poor patient outcome. However, there are some controversies that high proliferation index in gastric

Fig. 3. Kaplan-Meier plots of univariate disease-specific survival analysis. Histologic grade (A), pathologic stage (B), lymphatic invasion (C), ROR1 expression (D), pCREB expression (E), and pAkt expression (F).

Please cite this article as: Chang H, et al, Expression of ROR1, pAkt, and pCREB in gastric adenocarcinoma, Ann Diagn Pathol (2015), http:// dx.doi.org/10.1016/j.anndiagpath.2015.06.010

H. Chang et al. / Annals of Diagnostic Pathology xxx (2015) xxx–xxx

Acknowledgment

Table 3 Multivariate analyses of variables in relation to disease-specific survival. Variables Age Invasion depth Lymph node metastasis Lymphatic invasion pAkt expression pCREB expression Histologic grade

Parameter N60 y vs ≤60 y pT2-4 vs pT1 Present vs absent Present vs absent High vs low High vs low PD vs WD and MD

Hazard ratio (95% CI)

P

1.607 (1.120-2.305) 4.409 (2.085-9.320) 2.585 (1.485-4.499) 1.806 (1.237-2.636) 0.724 (0.485-1.083) 0.787 (0.427-1.451) 1.195 (0.823-1.737)

.010⁎ b.001⁎ .001⁎ .002⁎ .116 .443 .349

Abbreviation: CI, confidence interval. ⁎ Statistically significant p values are indicated in boldface type.

adenocarcinoma represents poor prognosis [23]. Many other factors of tumor biology such as apoptosis, epithelial-mesenchymal transition, and telomerase activity are now known to affect patient survival. The role of ROR1 expression in malignant tumors is not thoroughly known yet, but recent studies are revealing variety of mechanisms of ROR1 in carcinogenesis and cancer progression. ROR1 has been shown to induce tumor cell growth mediated by Akt phosphorylation and CREB in breast cancer cell lines, to take important role in regulation of apoptosis in HeLa cells, and to be related to expression of genes involved in epithelialmesenchymal transition [10,24]. We revealed the relationship between ROR1 expression and phosphorylation of Akt and CREB, which could have resulted in high proliferation of tumor cells in gastric adenocarcinomas; we did not test other proteins involved in tumor progression. Our results that ROR1, pAkt, and pCREB expression is associated with tumor proliferation and good prognosis may suggest variety of roles of ROR1 in gastric adenocarcinoma progression. Further study is required to establish gastric adenocarcinoma–specific mechanism of ROR1 expression. ROR1, a transmembrane protein with frizzled domain and tyrosine kinase domain, is highly expressed in many types of cancers. Some investigators are challenging ROR1 as a possible target of cancer therapy. Monoclonal antibodies to ROR1 and frizzled domain showing cytotoxicity have been demonstrated in cell lines. Inhibition of tyrosine kinase or immunotargeting of ROR1 molecule also could be prospective in the treatment of cancers [8,9]. In this study, we firstly revealed that ROR1 is highly expressed in gastric adenocarcinoma and ROR1 could be a possible target for gastric cancer therapy. In conclusion, we observed that ROR1 was highly expressed in gastric adenocarcinoma tissue and was associated with pAkt, pCREB expression, and high proliferation index of tumor cells. Although they were not independent prognostic factors, pAkt and pCREB expression showed longer patient survival in univariate analysis and were correlated with good prognostic factors along with ROR1 expression and could be a possible marker of prognosis and future therapeutic target in gastric adenocarcinoma.

Conflict of interest There is nothing to declare.

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Please cite this article as: Chang H, et al, Expression of ROR1, pAkt, and pCREB in gastric adenocarcinoma, Ann Diagn Pathol (2015), http:// dx.doi.org/10.1016/j.anndiagpath.2015.06.010