Clin Transl Oncol (2015) 17:215–222 DOI 10.1007/s12094-014-1216-4
RESEARCH ARTICLE
Expression and clinical significance of IMP3 in microdissected premalignant and malignant pancreatic lesions B.-J. Wang • L. Wang • S.-Y. Yang Z.-J. Liu
•
Received: 24 June 2014 / Accepted: 8 August 2014 / Published online: 3 September 2014 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2014
Abstract Introduction Insulin-like growth factor 2 (IGF-2) mRNAbinding protein 3 (IMP3) is overexpressed in pancreatic cancer, while remaining undetectable in the normal pancreas, indicating its important role in pancreatic cancer pathogenesis. The role of IMP3 in pancreatic carcinogenesis has not been fully understood. The main goal of this study was to probe the expression profile of IMP3 in different stages of pancreatic ductal adenocarcinoma (PDAC) development, and evaluate their prognostic significance in PDAC patients. Materials and methods We used quantitative real-time RT-PCR combined manual microdissection to precisely detect IMP3 expression in 97 microdissected foci from 50 patients with PDAC. Nonparametric test, Log-rank test and Cox regression analysis were used to evaluate the clinical significance of DNMTs expression. Results Expression of IMP3 increased from normal duct to pancreatic intraductal neoplasia and to PDAC. IMP3 mRNA expression statistically correlated with TNM staging. Univariate analysis showed that high level of IMP3 expression, tumor differentiation, TNM staging and alcohol consumption were statistically significant risk factors. Multivariate analysis showed that high level of IMP3
B.-J. Wang L. Wang Department of Laboratory Medicine, The Second Affiliated Hospital, Nanjing Medical University, Nanjing 210011, People’s Republic of China S.-Y. Yang Z.-J. Liu (&) Department of General Surgery, Nanjing First Hospital Affiliated to Nanjing Medical University, Nanjing 210006, Jiangsu, People’s Republic of China e-mail: [email protected]
expression and tumor differentiation were statistically significant independent poor prognostic factors. Conclusions These results suggested that pancreatic carcinogenesis involves an increased IMP3 mRNA expression, and it may become valuable diagnostic and prognostic markers as well as potential therapeutic targets for pancreatic cancer. Keywords IMP3 Pancreatic intraductal neoplasia Pancreatic ductal adenocarcinoma Microdissection
Introduction Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant disease with a 5-year survival rate of 4 %. Accumulating evidence suggested that PDAC develops from histologically well-defined premalignant lesions, called pancreatic intraductal neoplasia (PanIN) [1, 2]. Therefore, an in-depth study of molecular and histopathological transformations during the progression from PanINs to PDAC may refine our understanding of pancreatic carcinogenesis. IMP3 is a member of a family of insulin-like growth factor 2 (IGF-2) mRNA-binding proteins (IMPs) including IMP1, IMP2 and IMP3 [3]. IMPs play important roles in RNA trafficking, stabilization, localization and cell migration, especially during early stages of both human and mouse embryogenesis [4]. IMP3 was first cloned from a pancreatic tumor cDNA screen and was originally designated as KH domain-containing protein overexpressed in cancer (KOC) [5]. It is known as an oncofetal protein because IMP3 is expressed during embryogenesis but not in most adult tissues [3, 4]. Importantly, IMP3 is reexpressed in several malignant tissues including pancreatic
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cancer [6–11]. The expression of IMP3 has also been associated with an unfavorable outcome in renal clear cell carcinoma [6, 12], ovarian clear cell carcinoma [13] and PDAC [14]. However, no previous studies have examined IMP3 mRNA expression in pancreatic cancer, especially in the premalignant lesions of PanINs. The correlation between the clinicopathological characteristics of PDAC and IMP3 mRNA expression level remains to be determined. Prior studies on detecting IMP3 expression mainly used immunohistochemistry (IHC) techniques [10, 14–16]. The IHC technique is a semi-quantitative method and the scoring criteria rely on the individual judgement. Therefore, it is necessary to develop more reliable and reproducible methods. Microdissection-based quantitative mRNA analysis can contribute to the precise detection of IMP3 expression in tissue specimens [17–19]. Tissue microdissection techniques can avoid the problem of cellular heterogeneity in favor of the molecular analysis of interesting cells without contamination by neighboring cells. Thus, the main goal of this study was to probe the expression profile of IMP3 in different stages of PDAC development (from normal to PanIN and PDAC) using quantitative real-time RT-PCR coupled with manual microdissection, showing the relationship between IMP3 expression and the development and of prognosis of PDAC.
Materials and methods Patients and tissue specimens Pancreatic tissue samples were obtained from 50 patients (average age 61.5 years; range 41–78 years) who underwent pancreaticoduodenectomy (Whipple resection) at Nanjing First Hospital Affiliated to Nanjing Medical University, China, between 2009 and 2012. Each of the patients gave informed consent and the study was approved by the Ethics Committees of Nanjing Medical University. 50 pairs of tumoral and nontumoral tissue samples from these patients were collected during surgery. Fresh tissue specimens obtained were embedded in OCT within 5 min upon collection, snap frozen in liquid nitrogen, and stored at -80 °C until analysis. All tissue samples were histologically examined by a pathologist to confirm the diagnosis. The clinicopathological characteristics of the 50 PDAC patients are summarized in Table 1. Histological grade of tumor differentiation was assigned according to the World Health Organization criteria [20]. Tumor–node–metastasis (TNM) staging was based on the sixth edition of the American Joint Committee on Cancer (AJCC) guidelines [21].
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Clin Transl Oncol (2015) 17:215–222 Table 1 Correlations of IMP3 mRNA expression with clinicopathological variables (n = 50) Variable
Case
IMP3 Mean
Median
25
0.067
0.070
25
0.051
0.042
\60
23
0.054
0.054
C60
27
0.063
0.059
Head
31
0.054
0.053
Body and tail
19
0.067
0.078
B2
15
0.056
0.047
[2
35
0.060
0.059
5
0.068
0.085
Gender Male Female Age (years)
0.211
0.606
Location of tumor
0.342
Size of tumor (cm)
0.695
Differentiation
0.225
Well Moderate
37
0.056
0.054
8
0.068
0.075
11 30
0.056 0.060
0.054 0.057
9
0.060
0.060
Poor TNM staging IA ? IB IIA ? IIB
0.002*
III ? IV Smoking
0.874
No
30
0.059
0.059
Yes
20
0.058
0.056
No
36
0.055
0.053
Yes
14
0.070
0.073
Alcohol consumption
0.261
Serum CA19-9 (kU/L)
0.788
B39
11
0.063
0.063
[39
39
0.058
0.054
B25
15
0.065
0.065
[25
35
0.056
0.053
33 17
0.057 0.062
0.054 0.066
Serum CA50 (kU/L)
0.664
Serum CEA (lg/L) B4.3 [4.3
p
0.798
* p \ 0.05
Manual microdissection Manual microdissection was performed according to the previously published studies [22, 23]. 5-lm-thick frozen sections were prepared for every five pieces from tissue blocks and stained with hematoxylin and eosin (H & E). The others were serially sectioned into 8-lm thick and immediately fixed on slides in 100 % ethanol and stored at -80 °C for further manual microdissection in \1 week.
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5-lm-thick sections were reviewed for histological examination. The PanINs lesions were classified into PanIN-1A, PanIN-1B, PanIN-2, and PanIN-3 according to an international nomenclature and grading scheme [24]. For statistical analysis, PanIN-1A and 1B were considered ‘‘low-grade’’ PanIN lesions (PanIN-L), whereas PanIN-2 and 3 were combined into ‘‘high-grade’’ PanIN (PanIN-H) according to the previously published studies [25, 26] (Fig. 1a–f). 8-lm-thick sections with the required tissue components were selected for microdissection based on the review of 5-lm-thick sections. The selected 8-lm-thick sections were briefly treated with H & E (all solutions were prepared with DEPC-treated water). Pancreatic tissue surrounding the required tissue components was first removed by a sterile injection needle (size 0.65 9 25 mm). Then, the target lesions were covered by drops of lysis buffer and were collected into 1.5-ml reaction tubes for further RNA extraction. It was estimated that averagely \20 % of the cells collected was surrounding nonductal cells. In total, 97 tissue samples, including 14 samples of normal ducts, 21 samples of low-grade PanINs, 12 samples of high-grade PanINs and 50 samples of PDACs, were microdissected. Total RNA was isolated using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Quantitative real-time RT-PCR 1 lg of total RNA was used for reverse transcription (RT) in a 20 ll final volume with iScript cDNA Synthesis Kit (Bio-Rad, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using TaqMan Gene Expression Assays (Life Technologies, Carlsbad, CA, USA) in a StepOne Plus Real-time PCR System (Life Technologies). The TaqMan Gene Expression Assay Mix used for IMP3 had the product number Hs00251000_s1 (Life Technologies). Human 18S rRNA (product number Hs099999901_s1, Life Technologies) was used to calibrate the original concentration of mRNA. The reactions were performed in a volume of 10 ll containing 4.5 ll diluted cDNA, 0.5 ll TaqMan Gene Expression Assay Mix (209), and 5 ll TaqMan Universal PCR Master Mix (29). The thermal cycling conditions comprised an initial denaturation step at 95 °C for 10 min and 40 cycles at 95 °C for 15 s and at 65 °C for 1 min. Each quantification PCR was performed in triplicate, and the number of target gene was normalized to 18S gene. Statistical analysis Statistical analysis was performed using the SPSS software (Version 11.0). Differences in the IMP3 mRNA expression levels between different groups were analyzed using the
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Mann–Whitney U test. Correlations between the IMP3 mRNA expression and various clinicopathological, epidemiological or serological variables were analyzed by the Mann–Whitney U or Kruskal–Wallis test. Receiver operating characteristic (ROC) curve analysis determined the IMP3 expression level cut-off value for survival analysis [27, 28]. Survival distributions were estimated with the Kaplan–Meier method, and the significance of differences between survival rates was calculated by the Log-rank test. Univariate and multivariate analysis was performed using the Cox regression analysis. For the multivariate analysis, we used 0.10 as the cut-off p value to select the analyzed factors from the univariate analysis result. p \ 0.05 was considered statistically significant.
Results IMP3 mRNA expression in normal ducts, PanINs and PDACs The expression level of IMP3 normalized to that of 18S is shown in Fig. 1g. IMP3 expression was not detected in most of the 14 samples of NP. The mean IMP3 expression was 0.0029 (95 % CI 0.0017–0.0042) in PanIN-L, 0.0298 (95 % CI 0.0179–0.0416) in PanIN-H, and 0.0589 (95 % CI 0.0487–0.0692) in PDAC. PanIN-Ls expressed significantly higher levels of IMP3 than NPs (PanIN-L versus NP, p \ 0.001). PanIN-Hs expressed significantly higher levels of IMP3 than PanIN-Ls (PanIN-H versus PanIN-L, p \ 0.001). PDACs expressed significantly higher levels of IMP3 than PanIN-Hs (PDAC versus PanIN-H, p \ 0.05). Correlations between IMP3 mRNA expression and clinicopathological variables of PDAC patients IMP3 mRNA expression of PDACs was found to be significantly correlated with TNM staging (p = 0.002). No significant correlations were identified between the levels of tumor IMP3 expression and other variables (Table 1). Effect of IMP3 mRNA expression on patient survival 50 PDAC patients were enrolled in the survival analysis. 39 patients died, while the remaining 11 patients were alive at the last follow-up (31 March 2014). To determine the IMP3 expression level cut-off value for survival analysis, the patients were divided into two groups based on the length of overall survival: short-term survivors (survival period \24 months) and long-term survivors (C24 months). The threshold value of 0.495 was chosen as the cut-off score for high- and low-level expression of IMP3, since 0.495 (within 95 % CI 0.049–0.069 of IMP3 expression) was on
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Fig. 1 Quantitative real-time RT-PCR analysis of IMP3 mRNA expression in microdissected pancreatic ductal epithelia with different tissue lesions. Representative images of NP (a), PanIN-1A (b), PanIN-1B (c), PanIN-2 (d), PanIN-3 (e), PDAC (f) in frozen sections. PanIN-1A and PanIN-1B composing ‘PanIN-L’; PanIN-2 and PanIN3 composing ‘PanIN-H’; H & E stain; original magnifications 9200. g Quantitative real-time RT-PCR analysis of IMP3 mRNA expression
in NPs, PanIN-Ls, PanIN-Hs, and PDACs. The bottom and the top edges of the box mark the lower bound and upper bound of the 95 % confidence interval (CI) for the mean, respectively; The center horizontal line is drawn at the sample mean; the center vertical lines drawn from the boxes extend to the minimum and the maximum; *p \ 0.05; #p \ 0.001
the ROC curve closest to (0.0, 1.0). This maximized both sensitivity and specificity for the survival outcome (Fig. 2a). The area under the ROC curve (AUC) was 0.809 (95 % CI 0.656–0.962, p = 0.002). The Kaplan–Meier
survival curves showed that patients with high level (C0.495) expression of IMP3 had longer survival than those with low-level (\0.495) expression (Fig. 2b, p \ 0.001).
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Fig. 2 Correlation between IMP3 mRNA expression and patient survival. a ROC curve for IMP3 expression and cut-off value selection for high- and low-level IMP3 expression. b Kaplan–Meier
survival curves in 50 patients with PDAC according to their IMP3 mRNA expression status. The p value was calculated by the Log-rank test
Analysis of prognostic factors for PDAC patients
whether or not high-level IMP3 expression was independent predictor of overall survival, a multivariate, Cox regression analysis was done in a forward or backward stepwise method by including only those factors that showed significant (threshold for inclusion, p \ 0.10) relations with patient survival. Multivariate analysis demonstrated that high-level IMP3 expression and tumor differentiation were significant independent risk factors for predicting the prognosis of PDAC patients (Table 3).
To evaluate the effects of IMP3 mRNA expression and clinicopathological variables on the prognosis of PDAC patients, both univariate and multivariate analyses were performed. Univariate analysis demonstrated that high-level expression of IMP3, tumor differentiation, TNM staging and alcohol consumption were statistically significant risk factors affecting the outcome of PDAC patients (Table 2). To assess
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Table 2 Univariate analysis of prognostic factors in PDAC patients (n = 50) Cases
Events
Mean survival (months)
HR (95 % CI)
Male
25
20
14.460
1
Female
25
19
15.954
0.915 (0.485–1.725)
\60
23
19
13.024
1
C60
27
20
16.908
0.775 (0.413–1.455)
Variable
Gender
p
0.783
Age (years)
0.428
Location of tumor
0.478
Head
31
23
15.641
1
Body and tail
19
16
13.754
1.261 (0.665–2.391)
Size of tumor (cm)
0.944
B2
15
12
15.232
1
[2
35
27
15.449
1.025 (0.519–2.025)
5
3
22.400
1
37
29
16.113
8.777 (2.097–36.741)
0.003*
8
7
4.500
2.258 (0.684–7.462)
0.182
IA ? IB
11
12
23.136
IIA ? IIB
30
25
14.441
4.604 (1.697–12.488)
0.003*
9
34
6.556
1.766 (0.779–4.006)
0.173
No
30
24
15.489
1
Yes
20
15
14.380
0.971 (0.508–1.856) 1
Differentiation Well Moderate Poor
0.004*
TNM staging
III ? IV
0.009* 1
Smoking
0.929
Alcohol consumption
0.016*
No
36
26
18.115
Yes
14
13
8.833
2.350 (1.169–4.724)
Serum CA19-9 (kU/L)
0.963
B39
11
9
14.364
1
[39
39
30
15.510
1.018 (0.482–2.147)
Serum CA50 (kU/L)
0.877
B25
15
12
13.800
1
[25
35
27
15.832
0.947 (0.478–1.876)
Serum CEA (lg/L)
0.886
B4.3
33
26
15.719
1
[4.3
17
13
15.569
1.050 (0.538–2.048)
Low
20
12
24.566
1
High
30
27
9.073
\0.001*
IMP3 4.218 (2.007–8.866)
HR hazard ratio, 95 % CI 95 % confidence interval * p \ 0.05
Discussion In this study, quantitative real-time RT-PCR was used to study the IMP3 mRNA expression in a large number of microdissected pancreatic tissue samples representing different stages of PDAC development. The results helped us to analyze the clinical significance of IMP3 in PDAC. The present study showed that IMP3 is expressed de novo in pancreatic cancer. IMP3 mRNA expression was
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not detected in most of the NP samples, which was consistent with previous reports [10, 14–16] in pancreatic tumor samples detected by IHC. IMP3 expression increases by small increment in the early stage of pancreatic carcinogenesis, while increases markedly in the late stage of progression towards pancreatic cancer. These results demonstrated that IMP3 is a neo-expression gene in pancreatic carcinogenesis, and its marked increase is correlated with the development and progression of PDAC. Our data showed that the higher IMP3 expression level correlated to a higher TNM stage, suggesting that IMP3 may play important roles in lymph node metastasis and distant metastasis. No significant correlations were identified between IMP3 mRNA expression and tumor differentiation stage. Microdissection was used to overcome the heterogeneous nature of the tissues in this study. Combining with a standardized real-time RT-PCR technique, it is possible to exactly detect gene expression in tissue specimens. Thus, we presumed that tumor differentiation is a very complex process and might be influenced by multiple factors in vivo, in addition to the role of IMP3. We also evaluated the effect of IMP3 mRNA expression status on the prognosis of PDAC patients and found a significant difference in patient survival between groups with high- and low-level of IMP3 expression. High level of IMP3 mRNA expression in PDACs was significantly associated with a poorer overall survival, which was compatible with a previous report in Canada populations [14], demonstrating that IMP3 overexpression in PDAC correlates with poor survival. These results suggested that IMP3 may be used as a marker in predicting the prognosis of patients with PDAC. Multivariate analysis demonstrated that IMP3 expression status was an independent poor prognostic factor of PDAC patients. Our results implied that realtime RT-PCR examination of IMP3 in microdissected cells from surgically resected specimens may be a sensitive, reliable and useful method of predicting postoperative survival of PDAC patients, and IMP3 mRNA expression status may be used as an independent predictor of PDAC patient survival. In summary, this is the first report to describe IMP3 mRNA expression in premalignant lesions and in ductal cancers of the pancreas. This study provided new evidence supporting the role of IMP3 in the progression of PDAC. Meanwhile, our data validated the prognostic value of IMP3 mRNA expression in PDAC. These results suggested that using quantitative real-time RT-PCR combined with microdissection techniques to accurately analyze IMP3 mRNA expression in frozen pancreatic specimens is a promising approach for the diagnosis of
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Table 3 Multivariate analysis of prognostic factors in PDAC patients (n = 50) Variable
Forward stepwise HR (95 % CI)
Differentiation
Backward stepwise p
HR (95 % CI)
0.022*
Well
1
Moderate
5.323 (1.212–23.371)
Poor
1.565 (0.461–5.318)
TNM staging
0.031* 1
0.027*
4.995 (1.117–22.326)
0.473
1.540 (0.448–5.286)
0.325
IA ? IB
–
IIA ? IIB III ? IV
– –
Alcohol consumption –
Yes
–
IMP3
0.035* 0.493 0.483
– 0.233 0.953
– –
0.060
No
p
0.320 0.939 0.064
1 1.984 (0.960–4.097) 0.001*
0.001*
Low
1
1
High
3.740 (1.741–8.034)
3.568 (1.627–7.822)
HR hazard ratio, 95 % CI 95 % confidence interval * p \ 0.05
early pancreatic cancer and for monitoring the progression of treatment. Acknowledgments The authors thank all the patients and their family members for their consent and cooperation, without them this study would not have been possible. Conflict of interest
None declared.
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RESEARCH ARTICLE
Expression and clinical significance of IMP3 in microdissected premalignant and malignant pancreatic lesions B.-J. Wang • L. Wang • S.-Y. Yang Z.-J. Liu
•
Received: 24 June 2014 / Accepted: 8 August 2014 / Published online: 3 September 2014 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2014
Abstract Introduction Insulin-like growth factor 2 (IGF-2) mRNAbinding protein 3 (IMP3) is overexpressed in pancreatic cancer, while remaining undetectable in the normal pancreas, indicating its important role in pancreatic cancer pathogenesis. The role of IMP3 in pancreatic carcinogenesis has not been fully understood. The main goal of this study was to probe the expression profile of IMP3 in different stages of pancreatic ductal adenocarcinoma (PDAC) development, and evaluate their prognostic significance in PDAC patients. Materials and methods We used quantitative real-time RT-PCR combined manual microdissection to precisely detect IMP3 expression in 97 microdissected foci from 50 patients with PDAC. Nonparametric test, Log-rank test and Cox regression analysis were used to evaluate the clinical significance of DNMTs expression. Results Expression of IMP3 increased from normal duct to pancreatic intraductal neoplasia and to PDAC. IMP3 mRNA expression statistically correlated with TNM staging. Univariate analysis showed that high level of IMP3 expression, tumor differentiation, TNM staging and alcohol consumption were statistically significant risk factors. Multivariate analysis showed that high level of IMP3
B.-J. Wang L. Wang Department of Laboratory Medicine, The Second Affiliated Hospital, Nanjing Medical University, Nanjing 210011, People’s Republic of China S.-Y. Yang Z.-J. Liu (&) Department of General Surgery, Nanjing First Hospital Affiliated to Nanjing Medical University, Nanjing 210006, Jiangsu, People’s Republic of China e-mail: [email protected]
expression and tumor differentiation were statistically significant independent poor prognostic factors. Conclusions These results suggested that pancreatic carcinogenesis involves an increased IMP3 mRNA expression, and it may become valuable diagnostic and prognostic markers as well as potential therapeutic targets for pancreatic cancer. Keywords IMP3 Pancreatic intraductal neoplasia Pancreatic ductal adenocarcinoma Microdissection
Introduction Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant disease with a 5-year survival rate of 4 %. Accumulating evidence suggested that PDAC develops from histologically well-defined premalignant lesions, called pancreatic intraductal neoplasia (PanIN) [1, 2]. Therefore, an in-depth study of molecular and histopathological transformations during the progression from PanINs to PDAC may refine our understanding of pancreatic carcinogenesis. IMP3 is a member of a family of insulin-like growth factor 2 (IGF-2) mRNA-binding proteins (IMPs) including IMP1, IMP2 and IMP3 [3]. IMPs play important roles in RNA trafficking, stabilization, localization and cell migration, especially during early stages of both human and mouse embryogenesis [4]. IMP3 was first cloned from a pancreatic tumor cDNA screen and was originally designated as KH domain-containing protein overexpressed in cancer (KOC) [5]. It is known as an oncofetal protein because IMP3 is expressed during embryogenesis but not in most adult tissues [3, 4]. Importantly, IMP3 is reexpressed in several malignant tissues including pancreatic
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cancer [6–11]. The expression of IMP3 has also been associated with an unfavorable outcome in renal clear cell carcinoma [6, 12], ovarian clear cell carcinoma [13] and PDAC [14]. However, no previous studies have examined IMP3 mRNA expression in pancreatic cancer, especially in the premalignant lesions of PanINs. The correlation between the clinicopathological characteristics of PDAC and IMP3 mRNA expression level remains to be determined. Prior studies on detecting IMP3 expression mainly used immunohistochemistry (IHC) techniques [10, 14–16]. The IHC technique is a semi-quantitative method and the scoring criteria rely on the individual judgement. Therefore, it is necessary to develop more reliable and reproducible methods. Microdissection-based quantitative mRNA analysis can contribute to the precise detection of IMP3 expression in tissue specimens [17–19]. Tissue microdissection techniques can avoid the problem of cellular heterogeneity in favor of the molecular analysis of interesting cells without contamination by neighboring cells. Thus, the main goal of this study was to probe the expression profile of IMP3 in different stages of PDAC development (from normal to PanIN and PDAC) using quantitative real-time RT-PCR coupled with manual microdissection, showing the relationship between IMP3 expression and the development and of prognosis of PDAC.
Materials and methods Patients and tissue specimens Pancreatic tissue samples were obtained from 50 patients (average age 61.5 years; range 41–78 years) who underwent pancreaticoduodenectomy (Whipple resection) at Nanjing First Hospital Affiliated to Nanjing Medical University, China, between 2009 and 2012. Each of the patients gave informed consent and the study was approved by the Ethics Committees of Nanjing Medical University. 50 pairs of tumoral and nontumoral tissue samples from these patients were collected during surgery. Fresh tissue specimens obtained were embedded in OCT within 5 min upon collection, snap frozen in liquid nitrogen, and stored at -80 °C until analysis. All tissue samples were histologically examined by a pathologist to confirm the diagnosis. The clinicopathological characteristics of the 50 PDAC patients are summarized in Table 1. Histological grade of tumor differentiation was assigned according to the World Health Organization criteria [20]. Tumor–node–metastasis (TNM) staging was based on the sixth edition of the American Joint Committee on Cancer (AJCC) guidelines [21].
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Clin Transl Oncol (2015) 17:215–222 Table 1 Correlations of IMP3 mRNA expression with clinicopathological variables (n = 50) Variable
Case
IMP3 Mean
Median
25
0.067
0.070
25
0.051
0.042
\60
23
0.054
0.054
C60
27
0.063
0.059
Head
31
0.054
0.053
Body and tail
19
0.067
0.078
B2
15
0.056
0.047
[2
35
0.060
0.059
5
0.068
0.085
Gender Male Female Age (years)
0.211
0.606
Location of tumor
0.342
Size of tumor (cm)
0.695
Differentiation
0.225
Well Moderate
37
0.056
0.054
8
0.068
0.075
11 30
0.056 0.060
0.054 0.057
9
0.060
0.060
Poor TNM staging IA ? IB IIA ? IIB
0.002*
III ? IV Smoking
0.874
No
30
0.059
0.059
Yes
20
0.058
0.056
No
36
0.055
0.053
Yes
14
0.070
0.073
Alcohol consumption
0.261
Serum CA19-9 (kU/L)
0.788
B39
11
0.063
0.063
[39
39
0.058
0.054
B25
15
0.065
0.065
[25
35
0.056
0.053
33 17
0.057 0.062
0.054 0.066
Serum CA50 (kU/L)
0.664
Serum CEA (lg/L) B4.3 [4.3
p
0.798
* p \ 0.05
Manual microdissection Manual microdissection was performed according to the previously published studies [22, 23]. 5-lm-thick frozen sections were prepared for every five pieces from tissue blocks and stained with hematoxylin and eosin (H & E). The others were serially sectioned into 8-lm thick and immediately fixed on slides in 100 % ethanol and stored at -80 °C for further manual microdissection in \1 week.
Clin Transl Oncol (2015) 17:215–222
5-lm-thick sections were reviewed for histological examination. The PanINs lesions were classified into PanIN-1A, PanIN-1B, PanIN-2, and PanIN-3 according to an international nomenclature and grading scheme [24]. For statistical analysis, PanIN-1A and 1B were considered ‘‘low-grade’’ PanIN lesions (PanIN-L), whereas PanIN-2 and 3 were combined into ‘‘high-grade’’ PanIN (PanIN-H) according to the previously published studies [25, 26] (Fig. 1a–f). 8-lm-thick sections with the required tissue components were selected for microdissection based on the review of 5-lm-thick sections. The selected 8-lm-thick sections were briefly treated with H & E (all solutions were prepared with DEPC-treated water). Pancreatic tissue surrounding the required tissue components was first removed by a sterile injection needle (size 0.65 9 25 mm). Then, the target lesions were covered by drops of lysis buffer and were collected into 1.5-ml reaction tubes for further RNA extraction. It was estimated that averagely \20 % of the cells collected was surrounding nonductal cells. In total, 97 tissue samples, including 14 samples of normal ducts, 21 samples of low-grade PanINs, 12 samples of high-grade PanINs and 50 samples of PDACs, were microdissected. Total RNA was isolated using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Quantitative real-time RT-PCR 1 lg of total RNA was used for reverse transcription (RT) in a 20 ll final volume with iScript cDNA Synthesis Kit (Bio-Rad, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using TaqMan Gene Expression Assays (Life Technologies, Carlsbad, CA, USA) in a StepOne Plus Real-time PCR System (Life Technologies). The TaqMan Gene Expression Assay Mix used for IMP3 had the product number Hs00251000_s1 (Life Technologies). Human 18S rRNA (product number Hs099999901_s1, Life Technologies) was used to calibrate the original concentration of mRNA. The reactions were performed in a volume of 10 ll containing 4.5 ll diluted cDNA, 0.5 ll TaqMan Gene Expression Assay Mix (209), and 5 ll TaqMan Universal PCR Master Mix (29). The thermal cycling conditions comprised an initial denaturation step at 95 °C for 10 min and 40 cycles at 95 °C for 15 s and at 65 °C for 1 min. Each quantification PCR was performed in triplicate, and the number of target gene was normalized to 18S gene. Statistical analysis Statistical analysis was performed using the SPSS software (Version 11.0). Differences in the IMP3 mRNA expression levels between different groups were analyzed using the
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Mann–Whitney U test. Correlations between the IMP3 mRNA expression and various clinicopathological, epidemiological or serological variables were analyzed by the Mann–Whitney U or Kruskal–Wallis test. Receiver operating characteristic (ROC) curve analysis determined the IMP3 expression level cut-off value for survival analysis [27, 28]. Survival distributions were estimated with the Kaplan–Meier method, and the significance of differences between survival rates was calculated by the Log-rank test. Univariate and multivariate analysis was performed using the Cox regression analysis. For the multivariate analysis, we used 0.10 as the cut-off p value to select the analyzed factors from the univariate analysis result. p \ 0.05 was considered statistically significant.
Results IMP3 mRNA expression in normal ducts, PanINs and PDACs The expression level of IMP3 normalized to that of 18S is shown in Fig. 1g. IMP3 expression was not detected in most of the 14 samples of NP. The mean IMP3 expression was 0.0029 (95 % CI 0.0017–0.0042) in PanIN-L, 0.0298 (95 % CI 0.0179–0.0416) in PanIN-H, and 0.0589 (95 % CI 0.0487–0.0692) in PDAC. PanIN-Ls expressed significantly higher levels of IMP3 than NPs (PanIN-L versus NP, p \ 0.001). PanIN-Hs expressed significantly higher levels of IMP3 than PanIN-Ls (PanIN-H versus PanIN-L, p \ 0.001). PDACs expressed significantly higher levels of IMP3 than PanIN-Hs (PDAC versus PanIN-H, p \ 0.05). Correlations between IMP3 mRNA expression and clinicopathological variables of PDAC patients IMP3 mRNA expression of PDACs was found to be significantly correlated with TNM staging (p = 0.002). No significant correlations were identified between the levels of tumor IMP3 expression and other variables (Table 1). Effect of IMP3 mRNA expression on patient survival 50 PDAC patients were enrolled in the survival analysis. 39 patients died, while the remaining 11 patients were alive at the last follow-up (31 March 2014). To determine the IMP3 expression level cut-off value for survival analysis, the patients were divided into two groups based on the length of overall survival: short-term survivors (survival period \24 months) and long-term survivors (C24 months). The threshold value of 0.495 was chosen as the cut-off score for high- and low-level expression of IMP3, since 0.495 (within 95 % CI 0.049–0.069 of IMP3 expression) was on
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Fig. 1 Quantitative real-time RT-PCR analysis of IMP3 mRNA expression in microdissected pancreatic ductal epithelia with different tissue lesions. Representative images of NP (a), PanIN-1A (b), PanIN-1B (c), PanIN-2 (d), PanIN-3 (e), PDAC (f) in frozen sections. PanIN-1A and PanIN-1B composing ‘PanIN-L’; PanIN-2 and PanIN3 composing ‘PanIN-H’; H & E stain; original magnifications 9200. g Quantitative real-time RT-PCR analysis of IMP3 mRNA expression
in NPs, PanIN-Ls, PanIN-Hs, and PDACs. The bottom and the top edges of the box mark the lower bound and upper bound of the 95 % confidence interval (CI) for the mean, respectively; The center horizontal line is drawn at the sample mean; the center vertical lines drawn from the boxes extend to the minimum and the maximum; *p \ 0.05; #p \ 0.001
the ROC curve closest to (0.0, 1.0). This maximized both sensitivity and specificity for the survival outcome (Fig. 2a). The area under the ROC curve (AUC) was 0.809 (95 % CI 0.656–0.962, p = 0.002). The Kaplan–Meier
survival curves showed that patients with high level (C0.495) expression of IMP3 had longer survival than those with low-level (\0.495) expression (Fig. 2b, p \ 0.001).
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Fig. 2 Correlation between IMP3 mRNA expression and patient survival. a ROC curve for IMP3 expression and cut-off value selection for high- and low-level IMP3 expression. b Kaplan–Meier
survival curves in 50 patients with PDAC according to their IMP3 mRNA expression status. The p value was calculated by the Log-rank test
Analysis of prognostic factors for PDAC patients
whether or not high-level IMP3 expression was independent predictor of overall survival, a multivariate, Cox regression analysis was done in a forward or backward stepwise method by including only those factors that showed significant (threshold for inclusion, p \ 0.10) relations with patient survival. Multivariate analysis demonstrated that high-level IMP3 expression and tumor differentiation were significant independent risk factors for predicting the prognosis of PDAC patients (Table 3).
To evaluate the effects of IMP3 mRNA expression and clinicopathological variables on the prognosis of PDAC patients, both univariate and multivariate analyses were performed. Univariate analysis demonstrated that high-level expression of IMP3, tumor differentiation, TNM staging and alcohol consumption were statistically significant risk factors affecting the outcome of PDAC patients (Table 2). To assess
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Table 2 Univariate analysis of prognostic factors in PDAC patients (n = 50) Cases
Events
Mean survival (months)
HR (95 % CI)
Male
25
20
14.460
1
Female
25
19
15.954
0.915 (0.485–1.725)
\60
23
19
13.024
1
C60
27
20
16.908
0.775 (0.413–1.455)
Variable
Gender
p
0.783
Age (years)
0.428
Location of tumor
0.478
Head
31
23
15.641
1
Body and tail
19
16
13.754
1.261 (0.665–2.391)
Size of tumor (cm)
0.944
B2
15
12
15.232
1
[2
35
27
15.449
1.025 (0.519–2.025)
5
3
22.400
1
37
29
16.113
8.777 (2.097–36.741)
0.003*
8
7
4.500
2.258 (0.684–7.462)
0.182
IA ? IB
11
12
23.136
IIA ? IIB
30
25
14.441
4.604 (1.697–12.488)
0.003*
9
34
6.556
1.766 (0.779–4.006)
0.173
No
30
24
15.489
1
Yes
20
15
14.380
0.971 (0.508–1.856) 1
Differentiation Well Moderate Poor
0.004*
TNM staging
III ? IV
0.009* 1
Smoking
0.929
Alcohol consumption
0.016*
No
36
26
18.115
Yes
14
13
8.833
2.350 (1.169–4.724)
Serum CA19-9 (kU/L)
0.963
B39
11
9
14.364
1
[39
39
30
15.510
1.018 (0.482–2.147)
Serum CA50 (kU/L)
0.877
B25
15
12
13.800
1
[25
35
27
15.832
0.947 (0.478–1.876)
Serum CEA (lg/L)
0.886
B4.3
33
26
15.719
1
[4.3
17
13
15.569
1.050 (0.538–2.048)
Low
20
12
24.566
1
High
30
27
9.073
\0.001*
IMP3 4.218 (2.007–8.866)
HR hazard ratio, 95 % CI 95 % confidence interval * p \ 0.05
Discussion In this study, quantitative real-time RT-PCR was used to study the IMP3 mRNA expression in a large number of microdissected pancreatic tissue samples representing different stages of PDAC development. The results helped us to analyze the clinical significance of IMP3 in PDAC. The present study showed that IMP3 is expressed de novo in pancreatic cancer. IMP3 mRNA expression was
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not detected in most of the NP samples, which was consistent with previous reports [10, 14–16] in pancreatic tumor samples detected by IHC. IMP3 expression increases by small increment in the early stage of pancreatic carcinogenesis, while increases markedly in the late stage of progression towards pancreatic cancer. These results demonstrated that IMP3 is a neo-expression gene in pancreatic carcinogenesis, and its marked increase is correlated with the development and progression of PDAC. Our data showed that the higher IMP3 expression level correlated to a higher TNM stage, suggesting that IMP3 may play important roles in lymph node metastasis and distant metastasis. No significant correlations were identified between IMP3 mRNA expression and tumor differentiation stage. Microdissection was used to overcome the heterogeneous nature of the tissues in this study. Combining with a standardized real-time RT-PCR technique, it is possible to exactly detect gene expression in tissue specimens. Thus, we presumed that tumor differentiation is a very complex process and might be influenced by multiple factors in vivo, in addition to the role of IMP3. We also evaluated the effect of IMP3 mRNA expression status on the prognosis of PDAC patients and found a significant difference in patient survival between groups with high- and low-level of IMP3 expression. High level of IMP3 mRNA expression in PDACs was significantly associated with a poorer overall survival, which was compatible with a previous report in Canada populations [14], demonstrating that IMP3 overexpression in PDAC correlates with poor survival. These results suggested that IMP3 may be used as a marker in predicting the prognosis of patients with PDAC. Multivariate analysis demonstrated that IMP3 expression status was an independent poor prognostic factor of PDAC patients. Our results implied that realtime RT-PCR examination of IMP3 in microdissected cells from surgically resected specimens may be a sensitive, reliable and useful method of predicting postoperative survival of PDAC patients, and IMP3 mRNA expression status may be used as an independent predictor of PDAC patient survival. In summary, this is the first report to describe IMP3 mRNA expression in premalignant lesions and in ductal cancers of the pancreas. This study provided new evidence supporting the role of IMP3 in the progression of PDAC. Meanwhile, our data validated the prognostic value of IMP3 mRNA expression in PDAC. These results suggested that using quantitative real-time RT-PCR combined with microdissection techniques to accurately analyze IMP3 mRNA expression in frozen pancreatic specimens is a promising approach for the diagnosis of
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Table 3 Multivariate analysis of prognostic factors in PDAC patients (n = 50) Variable
Forward stepwise HR (95 % CI)
Differentiation
Backward stepwise p
HR (95 % CI)
0.022*
Well
1
Moderate
5.323 (1.212–23.371)
Poor
1.565 (0.461–5.318)
TNM staging
0.031* 1
0.027*
4.995 (1.117–22.326)
0.473
1.540 (0.448–5.286)
0.325
IA ? IB
–
IIA ? IIB III ? IV
– –
Alcohol consumption –
Yes
–
IMP3
0.035* 0.493 0.483
– 0.233 0.953
– –
0.060
No
p
0.320 0.939 0.064
1 1.984 (0.960–4.097) 0.001*
0.001*
Low
1
1
High
3.740 (1.741–8.034)
3.568 (1.627–7.822)
HR hazard ratio, 95 % CI 95 % confidence interval * p \ 0.05
early pancreatic cancer and for monitoring the progression of treatment. Acknowledgments The authors thank all the patients and their family members for their consent and cooperation, without them this study would not have been possible. Conflict of interest
None declared.
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