Med Oncol (2014) 31:131 DOI 10.1007/s12032-014-0131-4
ORIGINAL PAPER
Aberrant expression of nuclear KPNA2 is correlated with early recurrence and poor prognosis in patients with small hepatocellular carcinoma after hepatectomy Peng Jiang • Yunqiang Tang • Lu He Hui Tang • Min Liang • Cong Mai • Lijuan Hu • Jian Hong
•
Received: 28 June 2014 / Accepted: 9 July 2014 / Published online: 17 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Karyopherin a2 (KPNA2) functions as an adaptor that transports several proteins to the nucleus. Emerging evidence suggests that KPNA2 plays a crucial role in oncogenesis and early recurrence. In the present study, we evaluated the expression pattern of KPNA2 in 221 hepatocellular carcinoma (HCC) specimens and matching adjacent, non-tumorous tissues (NT) by immunohistochemical assays. We found that nuclear KPNA2 expression was significantly upregulated (30.3 %, 67/221) in HCC tissues; however, no nuclear expression of KPNA2 in NT tissues was observed. A correlation analysis demonstrated that nuclear KPNA2 expression was positively associated with serum AFP level, tumor differentiation, vascular invasion, BCLC stage and early recurrence (all p \ 0.05). Nuclear KPNA2 expression was associated with a poor prognosis in HCC patients. Univariate and multivariate analyses demonstrated that KPNA2 was an independent prognostic factor for both overall survival (p \ 0.001) and time to recurrence (p \ 0.001) in HCC patients. Furthermore, in a validation cohort, nuclear expression of KPNA2 was observed in 16 of 47 (34.0 %) small hepatocellular carcinoma patients. Importantly, the risk of recurrence associated with nuclear KPNA2 expression (9/16, 56.2 %) was significantly higher than the
P. Jiang Y. Tang (&) L. He H. Tang M. Liang C. Mai J. Hong Department of Hepatobiliary Oncology, Affiliated Tumor Hospital, Guangzhou Medical University, Guangzhou 510095, Guangdong, China e-mail: [email protected] L. Hu Department of Medical Oncology, Affiliated Tumor Hospital, Guangzhou Medical University, Guangzhou 510095, Guangdong, China
risk associated with an absence of nuclear KPNA2 expression (6/31, 19.3 %; p = 0.01). Our results demonstrate that nuclear KPNA2 expression is a poor prognostic biomarker for HCC, especially for early-stage HCC. Keywords Small hepatocellular carcinoma (sHCC) Hepatectomy KPNA2 Early recurrence
Introduction Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third cause of cancer-associated death worldwide, and its incidence and mortality increase every year [1]. Liver resection remains a standard treatment for patients with resectable HCC [2]. Despite great advances in surgical and medical management, the prognosis of HCC patients remains poor after curative surgical resection, even for small hepatocellular carcinoma (sHCC) patients, high incidences of tumor recurrence and metastasis are frequently observed [3]. Thus, it is crucial to identify patients, especially sHCC patients, with a high risk of recurrence, and to develop more effective and targeted treatment strategies to improve HCC disease outcome. Karyopherin a2 (KPNA2) is one of seven members of the Karyopherin family [4]; it consists of 529 amino acids and weighs approximately 58 kDa. It can deliver numerous cargo proteins to the nucleus [5–7]. The expression of KPNA2 is linked to various differentiation processes, such as spermatogenesis [8], myogenesis [9] and early embryogenesis [10]. Studies have also shown that KPNA2 is associated with viral infections, included Epstein-Barr virus [11], JC virus [12] and HPV [13] infections. In addition, emerging evidence suggests that KPNA2 plays an important role in cancer development. KPNA2
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Table 1 Correlation of KPNA2 protein expression with clinicopathological parameters Characteristics
n
KPNA2 Negative
p value Positive
Age (years) B50
93
65
28
85
63
22
B20
60
51
9
[20
160
104
56
[50 Serum AFP (lg/l)
0.531
[16], epithelial ovarian carcinoma [17] and bladder cancer [18]. KPNA2 might play a key role in cancer development and recurrence. In this study, we profiled the expression status and evaluated the prognostic significance of KPNA2 in 221 HCC samples. We also evaluated the correlation between KPNA2 expression and prognosis in 47 sHCC samples with no vessel invasion to investigate the prognostic predictive value of KPNA2 for early-stage patients.
0.002
Materials and methods
HBsAg Negative
26
21
5
Positive
192
124
68
B50
59
45
14
[50
162
111
51
A
204
143
61
B
15
12
3
158
115
43
Complete 22 Tumor differentiation
12
10
0.209
Patients and specimens
GGT (U/I) 0.263
Child–pugh score 0.416
Tumor capsule No/incomplete
I–II
161
122
39
60
34
26
No
185
138
47
Yes
36
18
18
No
82
61
21
Yes
139
95
44
III–IV
0.344
0.006
Vascular invasion 0.003
Liver cirrhosis 0.341
Tumor size (cm) B5
101
77
24
[5
119
78
41
Solitary
170
122
48
Multiple
51
34
17
BCLC stage 0/A
80
64
16
B
104
72
32
C
37
18
19
No
92
75
17
Yes
129
79
50
0.083
Ethical approval for the use human subjects was obtained from the research and ethics committee of the Affiliated Tumor Hospital of Guangzhou Medical University, and informed consent was obtained from each patient. Tumor specimens, including tumor and matched adjacent nontumorous tissues, were consecutively collected from 221 HCC patients and 47 sHCC patients during curative resection from June 2005 to June 2009, and from July 2009 to June 2012, respectively, at the Affiliated Tumor Hospital of Guangzhou Medical University. The cases selected were consecutive and based on the following criteria [19, 20]: Patient had undergone curative liver resection and had survived for at least 2 months after hepatectomy; pathological confirmation was HCC; no anticancer treatment was performed prior to hepatectomy; there was no previous malignant disease or a second primary tumor; and the patients had attended regular follow-up visits. In addition, the inclusion criteria for the sHCC patients were as follows: solitary tumor, B5 cm in diameter, and no lymph node metastasis or cancer embolus. Detailed clinicopathological parameters are listed in Table 1.
Tumor number 0.483
0.003
Early recurrence 0.001
Bold values (p \ 0.05) are statistically significant
overexpression has been observed in malignant tumor and is indication of a poor prognosis in patients with breast cancer [14], gastric cancer [15], non-small cell lung cancer
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Follow-up The HCC patients were monitored after surgery until April 30, 2014. They were followed every 3 months for the first year, every 6 months for the next 2 years and annually thereafter. The diagnostic examinations consisted of serum AFP measurement, MRI, CT, chest X-ray, abdominal ultrasonography and, when necessary, a bone scan to detect recurrence and/or metastasis. The time to recurrence (TTR) was computed from the date of resection to either the first detection of the recurrence of tumor or the last follow-up and was divided into early (B2 years) and late ([2 years) recurrence [21, 22]. Once evidence of HCC recurrence was confirmed, a second liver resection, radiofrequency ablation or trans-catheter arterial chemoembolization was
Med Oncol (2014) 31:131
suggested. Overall survival (OS) was defined as the interval between surgery and either death or the last follow-up.
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Results Nuclear KPNA2 expression in HCC samples
Immunohistochemistry (IHC) assay The resected surgical specimens fixed by formalin and embedded by paraffin were cut into 4-lm sections and mounted on glass slides. All sections were incubated at 62 °C for 2 h, deparaffinized in xylene and then rehydrated using a series of graded alcohols. The tissue slides were then treated with fresh 3 % hydrogen peroxide in methanol for 15 min to block endogenous peroxidase, and antigen retrieval was carried out in 0.01 M sodium citrate buffer (pH 6.0) using a microwave oven for 25 min. After the sections were colored for 30 min at room temperature, nonspecific staining was blocked by preincubation in 10 % normal goat serum for 1 h. The specimens were incubated with mouse monoclonal antibodies against KPNA2 (Santa Cruz; working dilution 1:100) overnight at 4 °C. Then, the tissue slides were incubated with HRP-labeled anti-mouse/ rabbit secondary antibody (DAKO) for 1 h at room temperature, and the reaction product was visualized with a non-biotin horseradish peroxidase detection system according to the manufacturer’s protocols (DAKO). Finally, specimens were counterstained with hematoxylin for 90 s. Two different pathologists who were specialized in liver cancer evaluated the IHC results of KPNA2 without the knowledge of clinical data. Cytoplasmic KPNA2 staining was found to be ubiquitous in both the tumor and the adjacent non-tumorous tissues. However, nuclear KPNA2 staining was only observed in the tumor specimens. Therefore, cytoplasmic staining was not considered during the data analysis. The extent of nuclear staining was assessed from 0 to 100 %. KPNA2 nuclear expression was classified as positive expression if it was observed in at least 5 % of the cancer cells; if the extent was \5 %, the sample was classified as having negative expression. Statistical analysis All of the data were analyzed by SPSS statistical software (version 22.0; SPSS, IBM). Survival curves were generated according to the Kaplan–Meier method, and differences between curves were analyzed using the log-rank test. Significant prognostic factors identified by univariate analysis were entered into multivariate analysis using the Cox proportional hazards model. The two-tailed chi-square test was used to analyze the association of KPNA2 nuclear expression with various clinicopathological parameters. The Student’s t test was used for comparisons. The results were considered statistically significant with p values \0.05.
The expression patterns of KPNA2 in 221 HCC (tumor and matched adjacent non-tumorous tissues) were examined by immunohistochemical (IHC) staining. We found that KPNA2 was predominantly immunoreactive in the nucleus in the tumor samples. We detected increased nuclear expression of KPNA2 in 67/221 (30.3 %) of the HCC tissue samples, whereas none of the adjacent non-tumorous tissues had positive staining (Fig. 1a). Our data also revealed that cytoplasmic KPNA2 expression in the matched adjacent non-tumorous tissues was higher than that in the tumor tissues, but this difference was not significant (p [ 0.05). Association of nuclear KPNA2 expression with HCC patient clinicopathological parameters To understand the clinicopathological significance of nuclear KPNA2 expression in HCC patients, we tested the correlation of nuclear KPNA2 expression status in the 221 HCC specimens with several standard clinicopathological parameters. The results of the statistical analysis showed that nuclear expression of KPNA2 was positively correlated with serum AFP, tumor differentiation, vascular invasion, BCLC stage and early recurrence (p \ 0.05, Table 1). No significant association was observed between unclear KPNA2 expression and other clinicopathological parameters, such as patient age, HBsAg, tumor size, tumor number and tumor capsule (p [ 0.05, Table 1). Prognostic value of KPNA2 In this study, the 1-, 2- and 3-year recurrence rates were 49.5, 61 and 68.9 %, respectively. The 1-, 2- and 3-year OS rates were 72.1, 58.2 and 49.3 %, respectively. Univariate and multivariate analyses were conducted to examine the impact of KPNA2 expression and other clinical pathological parameters on HCC patients. Univariate analysis revealed that tumor size, tumor number, vascular invasion, BCLC stage and nuclear KPNA2 expression were unfavorable predictors for TTR and OS (Table 2). In addition, GGT was associated with TTR (Table 2). The 1-, 2- and 3-year cumulative recurrence rates in the patients with positive nuclear KPNA2 expression were significantly higher than those in the patients with negative nuclear KPNA2 expression (67.5, 76.3 and 85.2 % vs. 42.2, 54.1 and 61.9 %, respectively; Fig. 1b). Similarly, the 1-, 2- and 3-year OS rates in the patients exhibiting positive nuclear KPNA2 expression were significantly lower than those in the patients exhibiting negative nuclear KPNA2 expression
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Fig. 1 IHC analysis of KPNA2 expression in 221 paired HCC samples. a Representative images showing KPNA2 staining in tumor (T) and adjacent non-tumorous (NT) tissue specimens. The lower panels are magnified images (95) of the corresponding upper panels. Scale bars 50 lm. A box plot graph to the right shows the quantitative evaluation of nuclear KPNA2 staining intensity. The significant differences between the two groups were analyzed by unpaired t test. **p \ 0.01. b, c Prognostic significance assessed by Kaplan–Meier survival estimates and log-rank tests. A comparison of OS and TTR as predicted by nuclear KPNA2 expression in HCC samples
Table 2 Univariate analysis of factors associated with survival and recurrence
Variables
OS
TTR
Hazard ratio (95 %CI)
p value
Hazard ratio (95 %CI)
p value
Age (years)
0.591 (0.390–0.896)
0.013
0.632 (0.439–0.910)
0.014
AFP (lg/l)
1.671 (1.295–2.157)
0.000
1.496 (1.217–1.838)
0.000
HBsAg
1.918 (0.889–4.137)
0.097
1.620 (0.895–2.930)
0.111
GGT (U/I)
1.594 (0.993–2.559)
0.053
1.721 (1.154–2.567)
0.008
Child–pugh score
1.378 (0.639–2.975)
0.414
1.085 (0.552–2.134)
0.813
Tumor size (cm)
1.985 (1.315–2.999)
0.001
1.665 (1.186–2.339)
0.003 0.007
Tumor number
1.803 (1.201–2.707)
0.004
1.646 (1.147–2.362)
Tumor capsule
0.904 (0.626–1.304)
0.589
1.030 (0.758–1.401)
0.849
Tumor differentiation
1.688 (1.112–2.562)
0.014
1.682 (1.182–2.394)
0.004
Vascular invasion
2.009 (1.278–3.159)
0.003
1.512 (1.000–2.289)
0.050 0.000
BCLC stage
2.375 (1.772–3.182)
0.000
1.985 (1.556–2.534)
Liver cirrhosis
0.986 (0.661–1.472)
0.946
1.030 (0.732–1.451)
0.864
Bold values (p \ 0.05) are statistically significant
KPNA2 expression
2.003 (1.343–2.987)
0.001
1.819 (1.292–2.561)
0.001
Table 3 Multivariate analysis of factors associated with survival and recurrence
Variables
OS Hazard ratio (95 %CI)
TTR p value
Hazard ratio (95 %CI)
p value
1.563 (1.032–2.367)
0.035
GGT (U/I)
NA
Tumor size (cm)
1.869 (1.230–2.840)
0.003
1.398 (0.978–1.997)
0.066
Tumor number
1.825 (1.209–2.756)
0.004
1.605 (1.110–2.320)
0.012
Tumor differentiation
1.130 (0.699–1.826)
0.617
1.289 (0.870–1.909)
0.205
NA not applicable
Vascular invasion
1.709 (1.029–2.838)
0.038
1.270 (0.810–1.993)
0.297
Bold values (p \ 0.05) are statistically significant
KPNA2
1.584 (1.042–2.409)
0.031
1.464 (1.023–2.096)
0.037
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Fig. 2 Nuclear KPNA2 expression is upregulated in tumor tissues. a IHC staining was used to evaluate the protein expression levels of KPNA2 in sHCC tissues. The ranges of nuclear KPNA2 expression (brown) are shown, with prominent staining in sHCC. Scale bars 50 lm. b Cumulative bar chart representing the correlation between nuclear immunoreactivity of KPNA2 and early recurrence in sHCC tissue samples. c, d Prognostic significance assessed by Kaplan–Meier survival estimates and log-rank tests. A comparison of OS and TTR as predicted by nuclear KPNA2 expression in sHCC samples
(56.7, 40.3 and 31.8 % vs. 78.9, 65.6 and 56.5 %, respectively; Fig. 1c). Further multivariate analysis demonstrated that KPNA2 was an independent prognostic factor for both the OS (p \ 0.05) and TTR (p \ 0.05) of HCC patients (Table 3). Nuclear expression of KPNA2 in patients with sHCC To further demonstrate the value of KPNA2 expression in predicting the survival of early-stage HCC patients, a validation cohort including 47 sHCC patients was employed. Positive nuclear KPNA2 expression was observed in 16 of 47 (34.0 %) sHCC specimens by IHC (Fig. 2a). Nuclear expression of KPNA2 was positively correlated with tumor differentiation (p = 0.02), vascular invasion (p = 0.004) and early recurrence (p = 0.019). We found that the risk of recurrence associated with nuclear
KPNA2 expression (9/16, 56.2 %) was significantly higher than the risk associated with an absence of nuclear KPNA2 expression (6/31, 19.3 %; p = 0.01; Fig. 2b). The survival analysis also revealed that the prognosis of sHCC patients with positive nuclear KPNA2 expression was significantly worse than for those with negative KPNA2 expression (Fig. 2c, d). The univariate and multivariate analyses further suggested that KPNA2 is a valuable predictor of recurrence potential in HCC patients, particularly in sHCC patients (p = 0.01).
Discussion The oncogenesis of HCC is linked to various molecular events, and the prognosis of HCC patients under conventional treatment strategies remains poor. Furthermore,
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prognosis predictor is absent, especially in sHCC patients, and prognostic predictors play an important role in the assessment of patients and in guiding clinical treatment [23]. Therefore, it is a great challenge for clinicians to discover sensitive molecular markers for HCC. Since the fundamental role of KPNA2 in nuclear protein transport was made clear [24], nucleo-cytoplasmic transport mechanisms have been the target of numerous studies. Increasing evidence suggests that the overexpression of KPNA2 may contribute to carcinogenesis and has potential as a novel therapeutic target. In this study, we evaluated the expression of KPNA2 in 221 HCC samples by IHC analysis. Our results demonstrated that, in adjacent non-carcinoma tissues, KPNA2 was only expressed in the cytoplasm; however, it was expressed in the nucleus in some HCC specimens. This finding indicates that nuclear KPNA2 expression might confer a selective advantage during the HCC tumorigenic processes. Furthermore, we evaluated the correlation between KPNA2 and HCC clinicopathological features, such as prognosis and recurrence. The data suggested that nuclear KPNA2 expression predicted poor prognosis. It was significantly associated with early recurrence and might facilitate HCC tumor invasion. The Kaplan–Meier analysis showed that the patients with positive nuclear KPNA2 expression exhibited shorter OS times and higher recurrence rates compared to patients with negative nuclear KPNA2 expression. The univariate and multivariate analyses further demonstrated that nuclear KPNA2 expression was a strong and independent predictor of reduced cancerspecific survival. With the improvement of the early diagnosis rate, the number of sHCC patients is increasing; however, sensitive molecular markers for predicting the prognosis of sHCC patients are still absent in clinical. Some well-accepted prognostic predictive parameters, such as tumor size, tumor number and cancer embolus, do not have prognostic predictive value for sHCC. Hence, it is important to identify a prognostic predicator for sHCC. In recent studies, aberrant nuclear KPNA2 expression has been observed in early lesions; this has been observed for non-muscle-invasive bladder cancer [18] and for peritumoral ductal carcinoma in situ in invasive breast cancer [25] samples. This suggests that KPNA2 participates in the early stage of carcinogenesis. Therefore, we studied a validation cohort of sHCC samples (solitary tumor, B5 cm in diameter, with no lymph node metastases or cancer emboli) to assess the predictive value of nuclear KPNA2 expression in sHCC patients. We evaluated the expression of nuclear KPNA2 in 47 sHCC samples by IHC analyses. Our results demonstrated that nuclear KPNA2 was overexpressed in sHCC samples. Furthermore, the correlation analysis between KPNA2 expression and early recurrence, and OS was conducted.
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We found that the association of nuclear KPNA2 expression with a higher risk of early recurrence and poor prognosis was statistically significant. The data suggested that nuclear KPNA2 expression was a predictor of early recurrence in sHCC patients. In conclusion, our study describes the expression pattern of nuclear KPNA2 in HCC. The above results suggest that nuclear KPNA2 is a valuable predictive biomarker for the prognosis of HCC patients. This was also true for sHCC patients, which is important and effective prognostic predicator for sHCC has not been previously identified. It is of great clinical value to continue to identify effective early markers for the diagnosis and prognosis of HCC. Acknowledgments We acknowledge National Natural Science Foundation of China (81201634); Basilic Special Financial of Affiliated Tumor Hospital Guangzhou Medical University (2011-yz-03); Ph.D. Startup Foundation of Guangzhou Medical University (2012C65). Conflict of interest
We declare that we have no conflict of interest.
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ORIGINAL PAPER
Aberrant expression of nuclear KPNA2 is correlated with early recurrence and poor prognosis in patients with small hepatocellular carcinoma after hepatectomy Peng Jiang • Yunqiang Tang • Lu He Hui Tang • Min Liang • Cong Mai • Lijuan Hu • Jian Hong
•
Received: 28 June 2014 / Accepted: 9 July 2014 / Published online: 17 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Karyopherin a2 (KPNA2) functions as an adaptor that transports several proteins to the nucleus. Emerging evidence suggests that KPNA2 plays a crucial role in oncogenesis and early recurrence. In the present study, we evaluated the expression pattern of KPNA2 in 221 hepatocellular carcinoma (HCC) specimens and matching adjacent, non-tumorous tissues (NT) by immunohistochemical assays. We found that nuclear KPNA2 expression was significantly upregulated (30.3 %, 67/221) in HCC tissues; however, no nuclear expression of KPNA2 in NT tissues was observed. A correlation analysis demonstrated that nuclear KPNA2 expression was positively associated with serum AFP level, tumor differentiation, vascular invasion, BCLC stage and early recurrence (all p \ 0.05). Nuclear KPNA2 expression was associated with a poor prognosis in HCC patients. Univariate and multivariate analyses demonstrated that KPNA2 was an independent prognostic factor for both overall survival (p \ 0.001) and time to recurrence (p \ 0.001) in HCC patients. Furthermore, in a validation cohort, nuclear expression of KPNA2 was observed in 16 of 47 (34.0 %) small hepatocellular carcinoma patients. Importantly, the risk of recurrence associated with nuclear KPNA2 expression (9/16, 56.2 %) was significantly higher than the
P. Jiang Y. Tang (&) L. He H. Tang M. Liang C. Mai J. Hong Department of Hepatobiliary Oncology, Affiliated Tumor Hospital, Guangzhou Medical University, Guangzhou 510095, Guangdong, China e-mail: [email protected] L. Hu Department of Medical Oncology, Affiliated Tumor Hospital, Guangzhou Medical University, Guangzhou 510095, Guangdong, China
risk associated with an absence of nuclear KPNA2 expression (6/31, 19.3 %; p = 0.01). Our results demonstrate that nuclear KPNA2 expression is a poor prognostic biomarker for HCC, especially for early-stage HCC. Keywords Small hepatocellular carcinoma (sHCC) Hepatectomy KPNA2 Early recurrence
Introduction Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third cause of cancer-associated death worldwide, and its incidence and mortality increase every year [1]. Liver resection remains a standard treatment for patients with resectable HCC [2]. Despite great advances in surgical and medical management, the prognosis of HCC patients remains poor after curative surgical resection, even for small hepatocellular carcinoma (sHCC) patients, high incidences of tumor recurrence and metastasis are frequently observed [3]. Thus, it is crucial to identify patients, especially sHCC patients, with a high risk of recurrence, and to develop more effective and targeted treatment strategies to improve HCC disease outcome. Karyopherin a2 (KPNA2) is one of seven members of the Karyopherin family [4]; it consists of 529 amino acids and weighs approximately 58 kDa. It can deliver numerous cargo proteins to the nucleus [5–7]. The expression of KPNA2 is linked to various differentiation processes, such as spermatogenesis [8], myogenesis [9] and early embryogenesis [10]. Studies have also shown that KPNA2 is associated with viral infections, included Epstein-Barr virus [11], JC virus [12] and HPV [13] infections. In addition, emerging evidence suggests that KPNA2 plays an important role in cancer development. KPNA2
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Table 1 Correlation of KPNA2 protein expression with clinicopathological parameters Characteristics
n
KPNA2 Negative
p value Positive
Age (years) B50
93
65
28
85
63
22
B20
60
51
9
[20
160
104
56
[50 Serum AFP (lg/l)
0.531
[16], epithelial ovarian carcinoma [17] and bladder cancer [18]. KPNA2 might play a key role in cancer development and recurrence. In this study, we profiled the expression status and evaluated the prognostic significance of KPNA2 in 221 HCC samples. We also evaluated the correlation between KPNA2 expression and prognosis in 47 sHCC samples with no vessel invasion to investigate the prognostic predictive value of KPNA2 for early-stage patients.
0.002
Materials and methods
HBsAg Negative
26
21
5
Positive
192
124
68
B50
59
45
14
[50
162
111
51
A
204
143
61
B
15
12
3
158
115
43
Complete 22 Tumor differentiation
12
10
0.209
Patients and specimens
GGT (U/I) 0.263
Child–pugh score 0.416
Tumor capsule No/incomplete
I–II
161
122
39
60
34
26
No
185
138
47
Yes
36
18
18
No
82
61
21
Yes
139
95
44
III–IV
0.344
0.006
Vascular invasion 0.003
Liver cirrhosis 0.341
Tumor size (cm) B5
101
77
24
[5
119
78
41
Solitary
170
122
48
Multiple
51
34
17
BCLC stage 0/A
80
64
16
B
104
72
32
C
37
18
19
No
92
75
17
Yes
129
79
50
0.083
Ethical approval for the use human subjects was obtained from the research and ethics committee of the Affiliated Tumor Hospital of Guangzhou Medical University, and informed consent was obtained from each patient. Tumor specimens, including tumor and matched adjacent nontumorous tissues, were consecutively collected from 221 HCC patients and 47 sHCC patients during curative resection from June 2005 to June 2009, and from July 2009 to June 2012, respectively, at the Affiliated Tumor Hospital of Guangzhou Medical University. The cases selected were consecutive and based on the following criteria [19, 20]: Patient had undergone curative liver resection and had survived for at least 2 months after hepatectomy; pathological confirmation was HCC; no anticancer treatment was performed prior to hepatectomy; there was no previous malignant disease or a second primary tumor; and the patients had attended regular follow-up visits. In addition, the inclusion criteria for the sHCC patients were as follows: solitary tumor, B5 cm in diameter, and no lymph node metastasis or cancer embolus. Detailed clinicopathological parameters are listed in Table 1.
Tumor number 0.483
0.003
Early recurrence 0.001
Bold values (p \ 0.05) are statistically significant
overexpression has been observed in malignant tumor and is indication of a poor prognosis in patients with breast cancer [14], gastric cancer [15], non-small cell lung cancer
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Follow-up The HCC patients were monitored after surgery until April 30, 2014. They were followed every 3 months for the first year, every 6 months for the next 2 years and annually thereafter. The diagnostic examinations consisted of serum AFP measurement, MRI, CT, chest X-ray, abdominal ultrasonography and, when necessary, a bone scan to detect recurrence and/or metastasis. The time to recurrence (TTR) was computed from the date of resection to either the first detection of the recurrence of tumor or the last follow-up and was divided into early (B2 years) and late ([2 years) recurrence [21, 22]. Once evidence of HCC recurrence was confirmed, a second liver resection, radiofrequency ablation or trans-catheter arterial chemoembolization was
Med Oncol (2014) 31:131
suggested. Overall survival (OS) was defined as the interval between surgery and either death or the last follow-up.
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Results Nuclear KPNA2 expression in HCC samples
Immunohistochemistry (IHC) assay The resected surgical specimens fixed by formalin and embedded by paraffin were cut into 4-lm sections and mounted on glass slides. All sections were incubated at 62 °C for 2 h, deparaffinized in xylene and then rehydrated using a series of graded alcohols. The tissue slides were then treated with fresh 3 % hydrogen peroxide in methanol for 15 min to block endogenous peroxidase, and antigen retrieval was carried out in 0.01 M sodium citrate buffer (pH 6.0) using a microwave oven for 25 min. After the sections were colored for 30 min at room temperature, nonspecific staining was blocked by preincubation in 10 % normal goat serum for 1 h. The specimens were incubated with mouse monoclonal antibodies against KPNA2 (Santa Cruz; working dilution 1:100) overnight at 4 °C. Then, the tissue slides were incubated with HRP-labeled anti-mouse/ rabbit secondary antibody (DAKO) for 1 h at room temperature, and the reaction product was visualized with a non-biotin horseradish peroxidase detection system according to the manufacturer’s protocols (DAKO). Finally, specimens were counterstained with hematoxylin for 90 s. Two different pathologists who were specialized in liver cancer evaluated the IHC results of KPNA2 without the knowledge of clinical data. Cytoplasmic KPNA2 staining was found to be ubiquitous in both the tumor and the adjacent non-tumorous tissues. However, nuclear KPNA2 staining was only observed in the tumor specimens. Therefore, cytoplasmic staining was not considered during the data analysis. The extent of nuclear staining was assessed from 0 to 100 %. KPNA2 nuclear expression was classified as positive expression if it was observed in at least 5 % of the cancer cells; if the extent was \5 %, the sample was classified as having negative expression. Statistical analysis All of the data were analyzed by SPSS statistical software (version 22.0; SPSS, IBM). Survival curves were generated according to the Kaplan–Meier method, and differences between curves were analyzed using the log-rank test. Significant prognostic factors identified by univariate analysis were entered into multivariate analysis using the Cox proportional hazards model. The two-tailed chi-square test was used to analyze the association of KPNA2 nuclear expression with various clinicopathological parameters. The Student’s t test was used for comparisons. The results were considered statistically significant with p values \0.05.
The expression patterns of KPNA2 in 221 HCC (tumor and matched adjacent non-tumorous tissues) were examined by immunohistochemical (IHC) staining. We found that KPNA2 was predominantly immunoreactive in the nucleus in the tumor samples. We detected increased nuclear expression of KPNA2 in 67/221 (30.3 %) of the HCC tissue samples, whereas none of the adjacent non-tumorous tissues had positive staining (Fig. 1a). Our data also revealed that cytoplasmic KPNA2 expression in the matched adjacent non-tumorous tissues was higher than that in the tumor tissues, but this difference was not significant (p [ 0.05). Association of nuclear KPNA2 expression with HCC patient clinicopathological parameters To understand the clinicopathological significance of nuclear KPNA2 expression in HCC patients, we tested the correlation of nuclear KPNA2 expression status in the 221 HCC specimens with several standard clinicopathological parameters. The results of the statistical analysis showed that nuclear expression of KPNA2 was positively correlated with serum AFP, tumor differentiation, vascular invasion, BCLC stage and early recurrence (p \ 0.05, Table 1). No significant association was observed between unclear KPNA2 expression and other clinicopathological parameters, such as patient age, HBsAg, tumor size, tumor number and tumor capsule (p [ 0.05, Table 1). Prognostic value of KPNA2 In this study, the 1-, 2- and 3-year recurrence rates were 49.5, 61 and 68.9 %, respectively. The 1-, 2- and 3-year OS rates were 72.1, 58.2 and 49.3 %, respectively. Univariate and multivariate analyses were conducted to examine the impact of KPNA2 expression and other clinical pathological parameters on HCC patients. Univariate analysis revealed that tumor size, tumor number, vascular invasion, BCLC stage and nuclear KPNA2 expression were unfavorable predictors for TTR and OS (Table 2). In addition, GGT was associated with TTR (Table 2). The 1-, 2- and 3-year cumulative recurrence rates in the patients with positive nuclear KPNA2 expression were significantly higher than those in the patients with negative nuclear KPNA2 expression (67.5, 76.3 and 85.2 % vs. 42.2, 54.1 and 61.9 %, respectively; Fig. 1b). Similarly, the 1-, 2- and 3-year OS rates in the patients exhibiting positive nuclear KPNA2 expression were significantly lower than those in the patients exhibiting negative nuclear KPNA2 expression
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Fig. 1 IHC analysis of KPNA2 expression in 221 paired HCC samples. a Representative images showing KPNA2 staining in tumor (T) and adjacent non-tumorous (NT) tissue specimens. The lower panels are magnified images (95) of the corresponding upper panels. Scale bars 50 lm. A box plot graph to the right shows the quantitative evaluation of nuclear KPNA2 staining intensity. The significant differences between the two groups were analyzed by unpaired t test. **p \ 0.01. b, c Prognostic significance assessed by Kaplan–Meier survival estimates and log-rank tests. A comparison of OS and TTR as predicted by nuclear KPNA2 expression in HCC samples
Table 2 Univariate analysis of factors associated with survival and recurrence
Variables
OS
TTR
Hazard ratio (95 %CI)
p value
Hazard ratio (95 %CI)
p value
Age (years)
0.591 (0.390–0.896)
0.013
0.632 (0.439–0.910)
0.014
AFP (lg/l)
1.671 (1.295–2.157)
0.000
1.496 (1.217–1.838)
0.000
HBsAg
1.918 (0.889–4.137)
0.097
1.620 (0.895–2.930)
0.111
GGT (U/I)
1.594 (0.993–2.559)
0.053
1.721 (1.154–2.567)
0.008
Child–pugh score
1.378 (0.639–2.975)
0.414
1.085 (0.552–2.134)
0.813
Tumor size (cm)
1.985 (1.315–2.999)
0.001
1.665 (1.186–2.339)
0.003 0.007
Tumor number
1.803 (1.201–2.707)
0.004
1.646 (1.147–2.362)
Tumor capsule
0.904 (0.626–1.304)
0.589
1.030 (0.758–1.401)
0.849
Tumor differentiation
1.688 (1.112–2.562)
0.014
1.682 (1.182–2.394)
0.004
Vascular invasion
2.009 (1.278–3.159)
0.003
1.512 (1.000–2.289)
0.050 0.000
BCLC stage
2.375 (1.772–3.182)
0.000
1.985 (1.556–2.534)
Liver cirrhosis
0.986 (0.661–1.472)
0.946
1.030 (0.732–1.451)
0.864
Bold values (p \ 0.05) are statistically significant
KPNA2 expression
2.003 (1.343–2.987)
0.001
1.819 (1.292–2.561)
0.001
Table 3 Multivariate analysis of factors associated with survival and recurrence
Variables
OS Hazard ratio (95 %CI)
TTR p value
Hazard ratio (95 %CI)
p value
1.563 (1.032–2.367)
0.035
GGT (U/I)
NA
Tumor size (cm)
1.869 (1.230–2.840)
0.003
1.398 (0.978–1.997)
0.066
Tumor number
1.825 (1.209–2.756)
0.004
1.605 (1.110–2.320)
0.012
Tumor differentiation
1.130 (0.699–1.826)
0.617
1.289 (0.870–1.909)
0.205
NA not applicable
Vascular invasion
1.709 (1.029–2.838)
0.038
1.270 (0.810–1.993)
0.297
Bold values (p \ 0.05) are statistically significant
KPNA2
1.584 (1.042–2.409)
0.031
1.464 (1.023–2.096)
0.037
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Fig. 2 Nuclear KPNA2 expression is upregulated in tumor tissues. a IHC staining was used to evaluate the protein expression levels of KPNA2 in sHCC tissues. The ranges of nuclear KPNA2 expression (brown) are shown, with prominent staining in sHCC. Scale bars 50 lm. b Cumulative bar chart representing the correlation between nuclear immunoreactivity of KPNA2 and early recurrence in sHCC tissue samples. c, d Prognostic significance assessed by Kaplan–Meier survival estimates and log-rank tests. A comparison of OS and TTR as predicted by nuclear KPNA2 expression in sHCC samples
(56.7, 40.3 and 31.8 % vs. 78.9, 65.6 and 56.5 %, respectively; Fig. 1c). Further multivariate analysis demonstrated that KPNA2 was an independent prognostic factor for both the OS (p \ 0.05) and TTR (p \ 0.05) of HCC patients (Table 3). Nuclear expression of KPNA2 in patients with sHCC To further demonstrate the value of KPNA2 expression in predicting the survival of early-stage HCC patients, a validation cohort including 47 sHCC patients was employed. Positive nuclear KPNA2 expression was observed in 16 of 47 (34.0 %) sHCC specimens by IHC (Fig. 2a). Nuclear expression of KPNA2 was positively correlated with tumor differentiation (p = 0.02), vascular invasion (p = 0.004) and early recurrence (p = 0.019). We found that the risk of recurrence associated with nuclear
KPNA2 expression (9/16, 56.2 %) was significantly higher than the risk associated with an absence of nuclear KPNA2 expression (6/31, 19.3 %; p = 0.01; Fig. 2b). The survival analysis also revealed that the prognosis of sHCC patients with positive nuclear KPNA2 expression was significantly worse than for those with negative KPNA2 expression (Fig. 2c, d). The univariate and multivariate analyses further suggested that KPNA2 is a valuable predictor of recurrence potential in HCC patients, particularly in sHCC patients (p = 0.01).
Discussion The oncogenesis of HCC is linked to various molecular events, and the prognosis of HCC patients under conventional treatment strategies remains poor. Furthermore,
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prognosis predictor is absent, especially in sHCC patients, and prognostic predictors play an important role in the assessment of patients and in guiding clinical treatment [23]. Therefore, it is a great challenge for clinicians to discover sensitive molecular markers for HCC. Since the fundamental role of KPNA2 in nuclear protein transport was made clear [24], nucleo-cytoplasmic transport mechanisms have been the target of numerous studies. Increasing evidence suggests that the overexpression of KPNA2 may contribute to carcinogenesis and has potential as a novel therapeutic target. In this study, we evaluated the expression of KPNA2 in 221 HCC samples by IHC analysis. Our results demonstrated that, in adjacent non-carcinoma tissues, KPNA2 was only expressed in the cytoplasm; however, it was expressed in the nucleus in some HCC specimens. This finding indicates that nuclear KPNA2 expression might confer a selective advantage during the HCC tumorigenic processes. Furthermore, we evaluated the correlation between KPNA2 and HCC clinicopathological features, such as prognosis and recurrence. The data suggested that nuclear KPNA2 expression predicted poor prognosis. It was significantly associated with early recurrence and might facilitate HCC tumor invasion. The Kaplan–Meier analysis showed that the patients with positive nuclear KPNA2 expression exhibited shorter OS times and higher recurrence rates compared to patients with negative nuclear KPNA2 expression. The univariate and multivariate analyses further demonstrated that nuclear KPNA2 expression was a strong and independent predictor of reduced cancerspecific survival. With the improvement of the early diagnosis rate, the number of sHCC patients is increasing; however, sensitive molecular markers for predicting the prognosis of sHCC patients are still absent in clinical. Some well-accepted prognostic predictive parameters, such as tumor size, tumor number and cancer embolus, do not have prognostic predictive value for sHCC. Hence, it is important to identify a prognostic predicator for sHCC. In recent studies, aberrant nuclear KPNA2 expression has been observed in early lesions; this has been observed for non-muscle-invasive bladder cancer [18] and for peritumoral ductal carcinoma in situ in invasive breast cancer [25] samples. This suggests that KPNA2 participates in the early stage of carcinogenesis. Therefore, we studied a validation cohort of sHCC samples (solitary tumor, B5 cm in diameter, with no lymph node metastases or cancer emboli) to assess the predictive value of nuclear KPNA2 expression in sHCC patients. We evaluated the expression of nuclear KPNA2 in 47 sHCC samples by IHC analyses. Our results demonstrated that nuclear KPNA2 was overexpressed in sHCC samples. Furthermore, the correlation analysis between KPNA2 expression and early recurrence, and OS was conducted.
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We found that the association of nuclear KPNA2 expression with a higher risk of early recurrence and poor prognosis was statistically significant. The data suggested that nuclear KPNA2 expression was a predictor of early recurrence in sHCC patients. In conclusion, our study describes the expression pattern of nuclear KPNA2 in HCC. The above results suggest that nuclear KPNA2 is a valuable predictive biomarker for the prognosis of HCC patients. This was also true for sHCC patients, which is important and effective prognostic predicator for sHCC has not been previously identified. It is of great clinical value to continue to identify effective early markers for the diagnosis and prognosis of HCC. Acknowledgments We acknowledge National Natural Science Foundation of China (81201634); Basilic Special Financial of Affiliated Tumor Hospital Guangzhou Medical University (2011-yz-03); Ph.D. Startup Foundation of Guangzhou Medical University (2012C65). Conflict of interest
We declare that we have no conflict of interest.
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