Tumor Biol. DOI 10.1007/s13277-014-1880-6
RESEARCH ARTICLE
Combined evaluation of the expression of NUCKS and Ki-67 proteins as independent prognostic factors for patients with gastric adenocarcinoma Ming Yang & Xiaoxia Wang & Qi Zhao & Tianbo Liu & Guodong Yao & Wenhao Chen & Zhiwei Li & Xiaoyi Huang & Yanqiao Zhang
Received: 22 February 2014 / Accepted: 21 March 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS) has been recently documented in various malignancies. However, data regarding the expression and prognostic value of NUCKS in gastric adenocarcinoma are limited. Specimens from 159 gastric adenocarcinoma patients who underwent primary gastrectomy were collected. Immunohistochemical method was used to evaluate NUCKS and Ki-67 expression. The correlations between NUCKS and clinical significance were analyzed. Elevated NUCKS expression was significantly associated with TNM stage (P=0.034), depth of invasion (P=0.001), and expression of Ki-67 (P=0.035). Kaplan–Meier analysis indicated that NUCKS overexpression alone (P=0.045 for overall survival) or in combination with Ki-67 (P=0.007 for disease-free survival, P=0.002 for overall survival) was correlated with adverse prognosis of the patients. Multivariate analysis revealed that combined NUCKS and Ki-67 overexpression was an M. Yang : Q. Zhao : W. Chen : Z. Li : Y. Zhang (*) Department of Gastrointestinal Medical Oncology, The Affiliated Tumour Hospital of Harbin Medical University, Harbin, Heilongjiang, China e-mail: [email protected] X. Wang Department of Emergency, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China T. Liu Department of Gynecology, The Affiliated Tumour Hospital of Harbin Medical University, Harbin, Heilongjiang, China G. Yao Department of Pathology, The Affiliated Tumour Hospital of Harbin Medical University, Harbin, Heilongjiang, China X. Huang Department of Biotherapy, The Affiliated Tumour Hospital of Harbin Medical University, Harbin, Heilongjiang, China
independent prognostic factor for both disease-free survival (hazard ratio=1.623, P=0.02) and overall survival (hazard ratio=1.667, P=0.016) in gastric adenocarcinoma patients. The combination of NUCKS and Ki-67 overexpression in gastric adenocarcinoma further distinguished a subgroup of patients with poor prognosis. Keywords NUCKS . Ki-67 . Gastric adenocarcinoma . Immunohistochemistry . Prognosis
Introduction Nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS), a 27-kDa nuclear DNA binding protein, is widespread in vertebrates and expressed in almost all types of human cells [1]. NUCKS possesses several unique features and little-known functions. The NUCKS protein is soluble in 5 % perchloric acid, with the amino acid composition resembling that of the high-mobility group (HMG) proteins 1 and 2 [2], which constitute one of the most abundant and wellcharacterized classes of non-histone nuclear proteins [3]. The family of HMG proteins are presently attributed with various functions related to transcription modulation, DNA integration, and recombination [4]. In contrast to HMG proteins, NUCKS is phosphorylated on multiple sites in vivo, which is essential in sustaining chromatin structure and activity [5]. In addition, NUCKS is a substrate for second messenger-activated kinases in vitro, including cyclic AMP/ GMP-dependent protein, calcium/phospholipid-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II [6]. Meanwhile, NUCKS acts as a substrate for DNAactivated protein kinase, which is involved in DNA repair in vitro [7]. In addition, NUCKS is also phosphorylated by CK-2, Cdk1, Cdk2, Cdk4, and Cdk6, which are implicated in
Tumor Biol.
cell growth and proliferation and participated in the regulation of the cell cycle [8, 9]. Moreover, the ubiquity and abundance of NUCKS particularly increase in rapidly growing cells, as well as in various cancers [10–15], suggesting that its possible function as a cancer-related protein involved in facilitating and maintaining transcriptional activity [5]. Although occurrence of NUCKS was detected in human gastric normal tissues [16], little is known about its expression and function in gastric cancer, where no data has been reported regarding the expression level of NUCKS so far. Gastric cancer, one of the most aggressive malignancies with very poor prognosis, remains as the world’s second leading cause of cancer mortality in less-developed areas [17]. The characteristics of proliferation may reflect the aggressiveness of gastric tumors and their eventual prognosis. In addition, various biomarkers with the potential function of predicting the disease outcome are essential factors in cell proliferation. Thus, understanding the proliferative changes in gastric cancer, particularly the identification of novel biomarkers for cell proliferation, may aid in the improvement of prevention, diagnosis, and treatment. As a proliferation marker, Ki-67 is the most robust immunohistochemical (IHC) biomarker identified to date. The Ki-67 protein is a non-histone protein present in G1, S, G2, and M phases of the cell cycle, but is absent in G0 cells [18, 19]. A series of studies demonstrated the potential involvement of Ki-67 in the prognosis of a variety of cancers [20–23], including gastric cancer [24]. Together with the proliferation-promoting feature of NUCKS, parallel detection of the expression of NUCKS and investigation of the correlation between NUCKS and Ki-67 may yield additional diagnostic information in gastric adenocarcinoma. In this study, the expression of NUCKS and its correlation with the clinicopathologic data, as well as with Ki-67 expression, were assessed to better understand the potential role of NUCKS in gastric adenocarcinoma.
Materials and methods Patients and tissue samples One hundred fifty-nine formalin-fixed, paraffin-embedded specimens were collected from patients with gastric adenocarcinoma who received surgical resection in The Affiliated Tumor Hospital of Harbin Medical University from September 2007 and June 2008. All primary gastric adenocarcinoma patients who received surgery were included in this study. Patients with prior chemotherapy, radiotherapy, or immunotherapy were excluded. All tissue specimens and slides were confirmed via pathologic examination. This study was approved by Harbin Medical
University Ethics Committee, with written informed consent provided by all the patients. The clinicopathological characteristics of the 159 patients were summarized in details as shown in Table 1. Median age of the gastric adenocarcinoma patients was 58, ranging from 30 to 88 years. The staging of tumors was performed according to the American Joint Committee on Cancer (AJCC) criteria [25]. Gastric cancer cases were categorized as intestinal or diffuse according to the Lauren classification [26]. Immunohistochemistry Formalin-fixed, paraffin-embedded specimens of the tissue sections from patients with gastric adenocarcinoma were available for immunohistochemical analysis. Sections of 4 μm thickness were cut on a microtome. The slides were deparaffinized in xylene and rehydrated in a series of graded Table 1 Association of NUCKS protein expression with clinicopathological variables of gastric adenocarcinoma patients Variables
Age(years) ≤60 >60 Gender Male Female TNM stage I–II III–IV Depth of invasion T1+T2 T3+T4 Lymph node metastasis Negative Positive Distant metastasis M0 M1 Differentiation Well/ moderately Poorly Location Upper Middle Lower Lauren classification Intestinal Diffuse Ki-67 expression Low High a
χ2 test
b
Fisher’s exact test
No. of patients (n=159)
95 64 118 41 56 103 29 130 41 118 145 14 50 109 29 40 90 99 60 44 115
NUCKS expression Low (54.7 %) (n=87)
High (45.3 %) (n=72)
57 (60.0) 30 (46.9) 68 (57.6) 19 (46.3) 37 (66.1) 50 (48.5) 24 (82.8) 63 (48.5) 26 (63.4) 61 (51.7) 77 (53.1) 10 (71.4) 24 (48.0) 63 (57.8) 17 (58.6) 23 (57.5) 47 (52.2) 56 (56.6) 31 (51.7) 30 (68.2) 57 (49.6)
38 (40.0) 34 (53.1) 50 (42.4) 22 (53.7) 19 (33.9) 53 (51.5) 5 (17.2) 67 (51.5) 15 (36.6) 57 (48.3) 68 (46.9) 4 (28.6) 26 (52.0) 46 (42.2) 12 (41.4) 17 (42.5) 43 (47.8) 43 (43.4) 29 (48.3) 14 (31.8) 58 (50.4)
P valuea
0.103 0.211 0.034 0.001 0.194 0.263b 0.249 0.767 0.547 0.035
Tumor Biol.
alcohol using standard methods. A pressure cooker antigen retrieval was performed in citrate buffer (pH 6.0) for 5 min to enhance immunoreactivity. Three percent hydrogen peroxide (Zhong Shan Golden Bridge Biological Technology Inc., Beijing, China) was used to block nonspecific peroxidase reaction. After washing with phosphate-buffered saline (PBS, pH 7.4), the sections were incubated with a rabbit polyclonal antibodies against NUCKS (1:800 dilution; ab84710, Abcam, Cambridge, MA, USA) or rabbit polyclonal antibodies against Ki-67 (1:200 dilution; ab66155, Abcam, Cambridge, MA, USA) overnight at 4 °C. After washing three times with PBS, sections were incubated at 37 °C with rabbit secondary antibody (PV-6001, Zhong Shan Golden Bridge Biological Technology Inc., Beijing, China) for 20 min at room temperature. The visualization was achieved by incubation with 3,3′-diaminobenzidine tetrahydrochloride (Dako, Hamburg, Germany), and the slides were counterstained with Mayer hematoxylin. After hydrated in graded alcohol and cleared with xylene, the slides were mounted with PermountTM Mounting Medium (C-0073, Beijing Biosynthesis Biotechnology CO., China). Each experiment was independently done twice. Immunohistochemical staining evaluation All the tissue sections were coded and evaluated by two expert gastric pathologists independently who were blind to the patient identity or clinical status, a consensus on the final Fig. 1 Immunohistochemical staining of NUCKS and Ki-67 in gastric adenocarcinoma. a NUCKS high expression in gastric adenocarcinoma. b NUCKS low expression in gastric adenocarcinoma. c Ki-67 high expression in gastric adenocarcinoma. d Ki-67 low expression in gastric adenocarcinoma (original magnification×200)
result was obtained. For NUCKS, intranuclear staining was graded semi-quantitatively as described by Kikuchi et al [13]. Briefly, immunohistochemical results were interpreted according to the intensity and percentage of immunoreactive product in nuclei of tumor cells. The staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The extent of staining was scored as 1 (1–10 % of the tumor cells stained), 2 (11–40 %), 3 (41–70 %), or 4 (71–100 %). The sum of the intensity and extent scores, potentially ranging from 1 to 7, was used as the final staining score. As in the study by Kikuchi et al [13], for the purpose of statistical analysis we defined final staining score of ≥6 as high expression group and ≤5 as low expression group. For Ki-67, the patients were divided into two groups according to the Ki-67 staining frequency (low expression, 0.05). Figure 2 demonstrated that the 5-year survival of patients with high NUCKS expression showed a trend towards poorer prognosis than patients with low NUCKS expression for OS (P=0.045), but not for DFS (P=0.082). Patients with high Ki-67 expression showed a trend towards poorer prognosis than patients with low Ki-67 expression for both OS and DFS (P=0.006 and 0.009, respectively). When NUCKS and Ki-67 were combined, patients with both high NUCKS and Ki-67 expression had a significantly poorer prognosis than other patients (P=0.002 for OS, P=0.007 for DFS). In multivariate analysis for both DFS and OS prediction, advanced TNM stage (P
RESEARCH ARTICLE
Combined evaluation of the expression of NUCKS and Ki-67 proteins as independent prognostic factors for patients with gastric adenocarcinoma Ming Yang & Xiaoxia Wang & Qi Zhao & Tianbo Liu & Guodong Yao & Wenhao Chen & Zhiwei Li & Xiaoyi Huang & Yanqiao Zhang
Received: 22 February 2014 / Accepted: 21 March 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS) has been recently documented in various malignancies. However, data regarding the expression and prognostic value of NUCKS in gastric adenocarcinoma are limited. Specimens from 159 gastric adenocarcinoma patients who underwent primary gastrectomy were collected. Immunohistochemical method was used to evaluate NUCKS and Ki-67 expression. The correlations between NUCKS and clinical significance were analyzed. Elevated NUCKS expression was significantly associated with TNM stage (P=0.034), depth of invasion (P=0.001), and expression of Ki-67 (P=0.035). Kaplan–Meier analysis indicated that NUCKS overexpression alone (P=0.045 for overall survival) or in combination with Ki-67 (P=0.007 for disease-free survival, P=0.002 for overall survival) was correlated with adverse prognosis of the patients. Multivariate analysis revealed that combined NUCKS and Ki-67 overexpression was an M. Yang : Q. Zhao : W. Chen : Z. Li : Y. Zhang (*) Department of Gastrointestinal Medical Oncology, The Affiliated Tumour Hospital of Harbin Medical University, Harbin, Heilongjiang, China e-mail: [email protected] X. Wang Department of Emergency, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China T. Liu Department of Gynecology, The Affiliated Tumour Hospital of Harbin Medical University, Harbin, Heilongjiang, China G. Yao Department of Pathology, The Affiliated Tumour Hospital of Harbin Medical University, Harbin, Heilongjiang, China X. Huang Department of Biotherapy, The Affiliated Tumour Hospital of Harbin Medical University, Harbin, Heilongjiang, China
independent prognostic factor for both disease-free survival (hazard ratio=1.623, P=0.02) and overall survival (hazard ratio=1.667, P=0.016) in gastric adenocarcinoma patients. The combination of NUCKS and Ki-67 overexpression in gastric adenocarcinoma further distinguished a subgroup of patients with poor prognosis. Keywords NUCKS . Ki-67 . Gastric adenocarcinoma . Immunohistochemistry . Prognosis
Introduction Nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS), a 27-kDa nuclear DNA binding protein, is widespread in vertebrates and expressed in almost all types of human cells [1]. NUCKS possesses several unique features and little-known functions. The NUCKS protein is soluble in 5 % perchloric acid, with the amino acid composition resembling that of the high-mobility group (HMG) proteins 1 and 2 [2], which constitute one of the most abundant and wellcharacterized classes of non-histone nuclear proteins [3]. The family of HMG proteins are presently attributed with various functions related to transcription modulation, DNA integration, and recombination [4]. In contrast to HMG proteins, NUCKS is phosphorylated on multiple sites in vivo, which is essential in sustaining chromatin structure and activity [5]. In addition, NUCKS is a substrate for second messenger-activated kinases in vitro, including cyclic AMP/ GMP-dependent protein, calcium/phospholipid-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II [6]. Meanwhile, NUCKS acts as a substrate for DNAactivated protein kinase, which is involved in DNA repair in vitro [7]. In addition, NUCKS is also phosphorylated by CK-2, Cdk1, Cdk2, Cdk4, and Cdk6, which are implicated in
Tumor Biol.
cell growth and proliferation and participated in the regulation of the cell cycle [8, 9]. Moreover, the ubiquity and abundance of NUCKS particularly increase in rapidly growing cells, as well as in various cancers [10–15], suggesting that its possible function as a cancer-related protein involved in facilitating and maintaining transcriptional activity [5]. Although occurrence of NUCKS was detected in human gastric normal tissues [16], little is known about its expression and function in gastric cancer, where no data has been reported regarding the expression level of NUCKS so far. Gastric cancer, one of the most aggressive malignancies with very poor prognosis, remains as the world’s second leading cause of cancer mortality in less-developed areas [17]. The characteristics of proliferation may reflect the aggressiveness of gastric tumors and their eventual prognosis. In addition, various biomarkers with the potential function of predicting the disease outcome are essential factors in cell proliferation. Thus, understanding the proliferative changes in gastric cancer, particularly the identification of novel biomarkers for cell proliferation, may aid in the improvement of prevention, diagnosis, and treatment. As a proliferation marker, Ki-67 is the most robust immunohistochemical (IHC) biomarker identified to date. The Ki-67 protein is a non-histone protein present in G1, S, G2, and M phases of the cell cycle, but is absent in G0 cells [18, 19]. A series of studies demonstrated the potential involvement of Ki-67 in the prognosis of a variety of cancers [20–23], including gastric cancer [24]. Together with the proliferation-promoting feature of NUCKS, parallel detection of the expression of NUCKS and investigation of the correlation between NUCKS and Ki-67 may yield additional diagnostic information in gastric adenocarcinoma. In this study, the expression of NUCKS and its correlation with the clinicopathologic data, as well as with Ki-67 expression, were assessed to better understand the potential role of NUCKS in gastric adenocarcinoma.
Materials and methods Patients and tissue samples One hundred fifty-nine formalin-fixed, paraffin-embedded specimens were collected from patients with gastric adenocarcinoma who received surgical resection in The Affiliated Tumor Hospital of Harbin Medical University from September 2007 and June 2008. All primary gastric adenocarcinoma patients who received surgery were included in this study. Patients with prior chemotherapy, radiotherapy, or immunotherapy were excluded. All tissue specimens and slides were confirmed via pathologic examination. This study was approved by Harbin Medical
University Ethics Committee, with written informed consent provided by all the patients. The clinicopathological characteristics of the 159 patients were summarized in details as shown in Table 1. Median age of the gastric adenocarcinoma patients was 58, ranging from 30 to 88 years. The staging of tumors was performed according to the American Joint Committee on Cancer (AJCC) criteria [25]. Gastric cancer cases were categorized as intestinal or diffuse according to the Lauren classification [26]. Immunohistochemistry Formalin-fixed, paraffin-embedded specimens of the tissue sections from patients with gastric adenocarcinoma were available for immunohistochemical analysis. Sections of 4 μm thickness were cut on a microtome. The slides were deparaffinized in xylene and rehydrated in a series of graded Table 1 Association of NUCKS protein expression with clinicopathological variables of gastric adenocarcinoma patients Variables
Age(years) ≤60 >60 Gender Male Female TNM stage I–II III–IV Depth of invasion T1+T2 T3+T4 Lymph node metastasis Negative Positive Distant metastasis M0 M1 Differentiation Well/ moderately Poorly Location Upper Middle Lower Lauren classification Intestinal Diffuse Ki-67 expression Low High a
χ2 test
b
Fisher’s exact test
No. of patients (n=159)
95 64 118 41 56 103 29 130 41 118 145 14 50 109 29 40 90 99 60 44 115
NUCKS expression Low (54.7 %) (n=87)
High (45.3 %) (n=72)
57 (60.0) 30 (46.9) 68 (57.6) 19 (46.3) 37 (66.1) 50 (48.5) 24 (82.8) 63 (48.5) 26 (63.4) 61 (51.7) 77 (53.1) 10 (71.4) 24 (48.0) 63 (57.8) 17 (58.6) 23 (57.5) 47 (52.2) 56 (56.6) 31 (51.7) 30 (68.2) 57 (49.6)
38 (40.0) 34 (53.1) 50 (42.4) 22 (53.7) 19 (33.9) 53 (51.5) 5 (17.2) 67 (51.5) 15 (36.6) 57 (48.3) 68 (46.9) 4 (28.6) 26 (52.0) 46 (42.2) 12 (41.4) 17 (42.5) 43 (47.8) 43 (43.4) 29 (48.3) 14 (31.8) 58 (50.4)
P valuea
0.103 0.211 0.034 0.001 0.194 0.263b 0.249 0.767 0.547 0.035
Tumor Biol.
alcohol using standard methods. A pressure cooker antigen retrieval was performed in citrate buffer (pH 6.0) for 5 min to enhance immunoreactivity. Three percent hydrogen peroxide (Zhong Shan Golden Bridge Biological Technology Inc., Beijing, China) was used to block nonspecific peroxidase reaction. After washing with phosphate-buffered saline (PBS, pH 7.4), the sections were incubated with a rabbit polyclonal antibodies against NUCKS (1:800 dilution; ab84710, Abcam, Cambridge, MA, USA) or rabbit polyclonal antibodies against Ki-67 (1:200 dilution; ab66155, Abcam, Cambridge, MA, USA) overnight at 4 °C. After washing three times with PBS, sections were incubated at 37 °C with rabbit secondary antibody (PV-6001, Zhong Shan Golden Bridge Biological Technology Inc., Beijing, China) for 20 min at room temperature. The visualization was achieved by incubation with 3,3′-diaminobenzidine tetrahydrochloride (Dako, Hamburg, Germany), and the slides were counterstained with Mayer hematoxylin. After hydrated in graded alcohol and cleared with xylene, the slides were mounted with PermountTM Mounting Medium (C-0073, Beijing Biosynthesis Biotechnology CO., China). Each experiment was independently done twice. Immunohistochemical staining evaluation All the tissue sections were coded and evaluated by two expert gastric pathologists independently who were blind to the patient identity or clinical status, a consensus on the final Fig. 1 Immunohistochemical staining of NUCKS and Ki-67 in gastric adenocarcinoma. a NUCKS high expression in gastric adenocarcinoma. b NUCKS low expression in gastric adenocarcinoma. c Ki-67 high expression in gastric adenocarcinoma. d Ki-67 low expression in gastric adenocarcinoma (original magnification×200)
result was obtained. For NUCKS, intranuclear staining was graded semi-quantitatively as described by Kikuchi et al [13]. Briefly, immunohistochemical results were interpreted according to the intensity and percentage of immunoreactive product in nuclei of tumor cells. The staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The extent of staining was scored as 1 (1–10 % of the tumor cells stained), 2 (11–40 %), 3 (41–70 %), or 4 (71–100 %). The sum of the intensity and extent scores, potentially ranging from 1 to 7, was used as the final staining score. As in the study by Kikuchi et al [13], for the purpose of statistical analysis we defined final staining score of ≥6 as high expression group and ≤5 as low expression group. For Ki-67, the patients were divided into two groups according to the Ki-67 staining frequency (low expression, 0.05). Figure 2 demonstrated that the 5-year survival of patients with high NUCKS expression showed a trend towards poorer prognosis than patients with low NUCKS expression for OS (P=0.045), but not for DFS (P=0.082). Patients with high Ki-67 expression showed a trend towards poorer prognosis than patients with low Ki-67 expression for both OS and DFS (P=0.006 and 0.009, respectively). When NUCKS and Ki-67 were combined, patients with both high NUCKS and Ki-67 expression had a significantly poorer prognosis than other patients (P=0.002 for OS, P=0.007 for DFS). In multivariate analysis for both DFS and OS prediction, advanced TNM stage (P
