Open Access

Nm23/nucleoside diphosphate kinase-A as a potent prognostic marker in invasive pancreatic ductal carcinoma identified by proteomic analysis of laser micro-dissected formalin-fixed paraffin-embedded tissue

  • Tatsuyuki Takadate1,
  • Tohru Onogawa1Email author,
  • Kiyonaga Fujii2,
  • Fuyuhiko Motoi1,
  • Sayaka Mikami3,
  • Tetsuya Fukuda3,
  • Makoto Kihara4,
  • Takashi Suzuki5,
  • Taro Takemura6,
  • Takashi Minowa6,
  • Nobutaka Hanagata6,
  • Kengo Kinoshita7,
  • Takanori Morikawa1,
  • Keiichi Shirasaki1,
  • Toshiki Rikiyama1,
  • Yu Katayose1, 8,
  • Shinichi Egawa1,
  • Toshihide Nishimura9 and
  • Michiaki Unno1, 8
Clinical Proteomics20129:8

https://doi.org/10.1186/1559-0275-9-8

Received: 23 February 2012

Accepted: 12 June 2012

Published: 27 June 2012

Abstract

Background

Pancreatic cancer is among the most lethal malignancies worldwide. This study aimed to identify a novel prognostic biomarker, facilitating treatment selection, using mass spectrometry (MS)-based proteomic analysis with formalin-fixed paraffin-embedded (FFPE) tissue.

Results

The two groups with poor prognosis (n = 4) and with better prognosis (n = 4) had been carefully chosen among 96 resected cases of pancreatic cancer during 1998 to 2007 in Tohoku University Hospital. Although those 2 groups had adjusted background (UICC-Stage IIB, Grade2, R0, gemcitabine adjuvant), there was a significant difference in postoperative mean survival time (poor 21.0 months, better 58.1 months, P = 0.0067). Cancerous epithelial cells collected from FFPE tissue sections by laser micro-dissection (LMD) were processed for liquid chromatography-tandem mass spectrometry (LC-MS/MS). In total, 1099 unique proteins were identified and 6 proteins showed different expressions in the 2 groups by semi-quantitative comparison. Among these 6 proteins, we focused on Nm23/Nucleoside Diphosphate Kinase A (NDPK-A) and immunohistochemically confirmed its expression in the cohort of 96 cases. Kaplan-Meier analysis showed high Nm23/NDPK-A expression to correlate with significantly worse overall survival (P = 0.0103). Moreover, in the multivariate Cox regression model, Nm23/NDPK-A over-expression remained an independent predictor of poor survival with a hazard ratio of 1.97 (95% CI 1.16-3.56, P = 0.0110).

Conclusions

We identified 6 candidate prognostic markers for postoperative pancreatic cancer using FFPE tissues and immunohistochemically demonstrated high Nm23/NDPK-A expression to be a useful prognostic marker for pancreatic cancer.

Keywords

Proteomics Prognostic biomarker Formalin-fixed paraffin-embedded (FFPE) Laser micro-dissection (LMD) Liquid chromatography-tandem mass spectrometry (LC-MS/MS) Nm23/Nucleoside Diphosphate Kinase A (NDPK-A)

Background

Pancreatic cancer has the worst prognosis of any major malignancy, with a 5-year survival rate of less than 5% after diagnosis [1]. It is the fifth leading cause of cancer death and the incidence of pancreatic cancer is rising in Japan [2, 3]. The majority of exocrine pancreatic cancers are invasive ductal carcinomas. Radical surgical resection is the only chance for cure, but only 10% to 20% of patients are candidates for curative resection, [2] and even if curative resection is performed, the postoperative 5-year survival rate is only 15% to 25% because of high recurrence rates [4, 5]. In an effort to explain improved survival rates, several studies have analyzed determinants of long-term survival in postoperative pancreatic cancer patients [615]. However, the prognosis of pancreatic cancer patients has improved little in the past 20 years [2]. Thus, there is an urgent need to identify novel molecular targets for early diagnosis and selecting optimal treatments.

In recent decades, many exploratory studies have aimed to discover biomarkers for early detection, optimal treatment selection and predicting therapeutic outcomes, with the goal of improving prognosis [16]. Proteomics has become a major tool for the discovery of diagnostic and prognostic cancer biomarkers, but pancreatic cancer analysis is challenging due to histological heterogeneity within the tumor. However, laser micro-dissection (LMD) provides an ideal method of extracting tumor specimens and isolating targeted cells with defined morphologies. Formalin-fixed paraffin-embedded (FFPE) tissues, long collected and stored in hospitals worldwide, represent an enormous untapped information resource concerning disease progression as well as drug responses and toxicity. FFPE tissue is particularly advantageous for discovering biomarkers because of the wealth of accompanying clinical data [17, 18].

We performed retrospective proteomics by liquid chromatography–tandem mass spectrometry (LC-MS/MS) using FFPE tissues from resected invasive pancreatic ductal carcinomas and semi-quantitative group comparison. To identify novel prognostic biomarkers, we selected 2 groups with poor prognosis and better prognosis. We focused on differentially expressed proteins between the 2 groups and immunohistochemically confirmed the candidate proteins in a cohort of 96 cases of invasive pancreatic ductal carcinoma.

Methods

Patient characteristics and FFPE tissue samples

The study subjects were selected from among 156 histologically diagnosed invasive pancreatic ductal carcinoma cases undergoing pancreatectomy during the January 1998 through December 2007 period at our hospital. We selected 96 cases suitable for monitoring, with microscopic complete resection (R0), and no neoadjuvant chemotherapy (Figure 1). To identify novel biomarkers, we standardized known prognostic factors: 1) pathological stage, [68, 1012, 14] 2) histological differentiation, [10, 12, 14] 3) postoperative carbohydrate antigen 19–9 (CA19-9) level, [19] and 4) adjuvant chemotherapy [9, 10, 13, 15]. We excluded 3 cases because the observation period was too short, leaving 8 cases divided into 2 groups, poor prognosis (n = 4) and better prognosis (n = 4), for shotgun proteomic analysis (Figure 1). There was a significant difference in postoperative mean survival time (poor 21.0 ± 4.8 months, better 58.1 ± 13.0 months, P = 0.0067). First, we performed shotgun proteomic analysis using FFPE tissues from these 8 invasive pancreatic ductal carcinomas. FFPE tissues were utilized for this semi-quantitative proteomic study with approval from the Ethics Committee of Tohoku University (2006–119), and informed consent was obtained from individual patients.
Figure 1

Patient selection for proteomic analysis.a Histological residuum R0 shows microscopically negative margins. b Unio Internationalis Contra Cancrum/Union for International Cancer Control. UICC stage IIB: Cancer has spread to nearby lymph nodes and may also have spread to adjacent tissues and organs. c Japan Pancreas Society. JPS stage 3: Cancer has not extended into the portal vein, extra-pancreatic nerve plexus, or other organs in UICC stage IIB. d Formalin-Fixed Paraffin-Embedded

LMD and protein extraction

Tumor tissues were fixed in 4% paraformaldehyde and routinely processed for paraffin sectioning. Cancerous lesions were identified on serial sections stained with hematoxylin and eosin. For shotgun proteomic analysis, 10-μm sections prepared from the same tissue block were attached to DIRECTOR™ slides (Expression Pathology, MD, USA), de-paraffinized three times with xylene for 5 min, rehydrated with graded ethanol solutions and distilled water, and then stained with hematoxylin. Stained, uncovered slides were air-dried and approximately 30,000 cancerous cells (8 mm2) were collected into the cap of a 0.2 mL PCR tube using Leica LMD6000 (Leica Microsystems GmbH, Germany). Peptides were extracted using a Liquid Tissue™ MS Protein Prep kit (Expression Pathology) [20] according to the manufacturer’s instructions. Briefly, the cellular material, suspended in the Liquid Tissue buffer, was incubated at 95 °C for 90 min, then cooled on ice for 3 min. Trypsin was added (approximately 15–18 U) followed by incubation at 37 °C overnight. Dithiothreitol was added to a final concentration of 10 mM, and the samples were heated for 5 min at 95 °C. The Liquid Tissue digestate was stored at −20 °C until analysis.

LC-MS/MS shotgun analysis

Exploratory LC-MS/MS analysis of a digested sample was performed using reversed phase (RP)-LC interfaced with an LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific, Germany) using a nanoelectrospray device (AMR, Tokyo, Japan), as previously reported [21]. The RP-LC system (Paradigm MS4B, Michrom BioResources, CA, USA) consisted of a peptide Cap-Trap cartridge (2.0 × 0.5 mm inside diameter) and an analytical column (L-column Micro, 150 × 0.2 mm L-C18, 3 μm, 12 nm, Chemical Evaluation Research Institute, Tokyo, Japan) fitted with an emitter tip (FortisTip, OmniSeparo-TJ, Hyogo, Japan). Peptide-mixture samples processed from FFPE tissues were loaded onto the trap cartridge and washed with mobile phase A (98% H2O with 2% acetonitrile and 0.1% formic acid) for concentration and desalting. Subsequently, the samples were eluted over 70 min from the analytical column via the trap cartridge using a linear gradient of 5–40% mobile phase B (10% H2O with 90% acetonitrile and 0.1% formic acid) at a flow-rate of 1 μL/min. General MS conditions were as follows: electrospray voltage, 3.0 kV, no sheath and auxiliary gas flow; ion transfer tube temperature, 200 °C; collision energy, 35%; ion selection threshold, 1000 counts for MS/MS. MS/MS was performed on the top 3 ions in each MS scan using the dynamic exclusion principle, i.e. temporary (180 s) placement of a mass on an exclusion list after its MS/MS spectrum has been acquired.

Data analysis and protein identification

All MS/MS spectral data were searched against Homo sapiens entries in the Swiss-Prot database (Release 57.13, 20,349 entries) using MASCOT software (version 2.1.1, Matrix Science, UK). The peptide mass tolerance was 20 ppm, fragment mass tolerance 0.8 Da, and trypsin specificity was applied with a maximum of 2 missed cleavages. For variable peptide modifications, methionine oxidation, and N-formylation, including formyl (K), formyl (R), and formyl (N-terminus), were taken into account. To estimate the false positive rate for protein identification, a decoy database was created by reversing the protein sequences in the original database. Based on search results for the decoy database, the estimated false positive rate of peptide matches was 4.0% under P < 0.05 for protein score threshold conditions. Results were obtained from triplicate LC-MS/MS runs for each sample.

Spectral counting analysis of identified proteins

To compare protein expression across all tissue samples from the results of shotgun analysis, I used the label-free spectral counting method [18]. The number of peptide spectra with high confidence (Mascot ion score, P < 0.05) served as the spectral count value. All proteins with at least one peptide spectrum in a single LC-MS/MS analysis were considered for protein quantification using spectral counting. The averaged values based on triplicate analyses were estimated as the spectral count values of each protein for individual tissue samples, taking into consideration statistical significance.

Fold changes in expressed proteins on a base-2 logarithmic scale were calculated using the protein ratio from spectral counting (Rsc) [22]. Rsc > 1 or < −1 corresponds to fold changes > 2 or < 0.5. Differences in relative abundances of identified proteins were also assessed by spectral index (SpI), allowing group comparison [23]. SpI values ranged from −1 to +1, and those close to 0 indicated near-equal relative peptide abundance in the 2 groups. Candidate proteins of the 2 groups chosen based on Rsc > 1 or < −1 and SpI > 0.75 or < -0.75, showed statistical significance at P < 0.05 by non-parametric G-test [24].

Immunohistochemistry

Immunohistochemistry was performed using FFPE tissue sections stained with the horseradish peroxidase EnVision + System (DAKO, CA, USA). Antigen retrieval was performed by heating the sections in 10 mmol/L citrate buffer (pH 6.0) for 5 minutes. Mouse monoclonal nm23/nucleoside diphosphate kinase-A (Nm23/NDPK-A) antibody, clone37.6 (Abcam, MA, USA), was used as the primary antibody at a 1:100 dilution. A breast cancer tissue section served as the positive control [25]. For negative controls, mouse IgG2a isotype control (R&D Systems, MN, USA) was used as the primary antibody. The sections were lightly counterstained with hematoxylin. After completely reviewing all slides of immunostained sections for each carcinoma, three of the authors (T.T., T.O. and T.S.) independently and blindly classified carcinoma cases into 2 groups: those in which the percentage of carcinoma cells positive for Nm23/NDPK-A exceeded 10% were the positive group, while the negative group comprised those with fewer than 10% positive cells, as in previous studies [26, 27].

Statistical analysis

The χ 2 -test was used to compare categorical variables. In univariate analysis, survival rates were calculated by the Kaplan-Meier method and compared using the log-rank test. In multivariate analysis, independent prognostic factors were determined by the Cox proportional hazards model. P < 0.05 was considered statistically significant. JMP software version 9.0 was used for all analyses. Analyses were performed using only available data; missing information was assumed to be non-informative.

Results

The present study aimed to investigate molecular profiles of proteins relevant to pancreatic cancer and to confirm these proteins to be potential prognostic biomarkers. The workflow is illustrated in Figure 2.
Figure 2

Strategy for shotgun proteomic analysis and immunohistochemical confirmation.a Liquid chromatography-tandem mass spectrometry

Patient characteristics

First, we performed shotgun proteomic analysis using FFPE tissues from 8 invasive pancreatic ductal carcinomas. The better and poor prognostic groups each provided 4 independent primary tumor tissues. The 2 groups did not differ in age, gender, tumor location, pathological staging, lymph node metastasis, tumor differentiation, postoperative CA19-9, or adjuvant chemotherapy (Table 1).
Table 1

Patients characteristics in the poor and better prognostic group

Variable

Category

Poor prognostic group (n = 4)

Better prognostic group (n = 4)

P value

Postoperative survival months

Mean ± SDa

21.0 ± 4.80

58.1 ± 13.0

0.0067b

Age

Mean ± SD

66.75 ± 10.66

63.25 ± 12.95

0.6909c

Gender

Male

3(75)

3(75)

 
 

Female

1(25)

1(25)

 

Location

Head

4(100)

4(100)

 

T

T3

4(100)

4(100)

 

N

N1

4(100)

4(100)

 

M

M0

4(100)

4(100)

 

UICC stage

IIB

4(100)

4(100)

 

JPS stage

3

4(100)

4(100)

 

Differentiation

Moderate

4(100)

4(100)

 

Residuum

R0

4(100)

4(100)

 

Postoperative CA19-9

<37 U/ml

4(100)

4(100)

 

Adjuvant chemotherapy

Gemcitabine

4(100)

4(100)

 

a Standard deviation.

b The log-rank test was used.

c The t-test was used.

Protein identification and semi-quantitative comparison

In shotgun proteomic analysis, 845 proteins in the better prognostic group, 924 in the poor prognostic group and 1099 proteins in total were identified ( Additional file 1: Table S1). The identified proteins were semi-quantitatively compared using spectral counting analysis. We identified that ADP-ribosylation factor 4 (ARF4), Collagen alpha-3(VI) chain (CO6A3), DNA-binding protein A (DBPA), Malate dehydrogenase, cytoplasmic (MDHC), Nucleoside diphosphate kinase A (NDKA) and Probable transcription factor PML (PML) were differentially expressed in the 2 groups, including 4 and 2 over-expressed proteins in the poor and better prognostic groups, respectively (Table 2).
Table 2

Proteins identified differentially expressed between better and poor prognostic groups

Accession number

Entry name

Protein name

      

Better prognostic group

Poor prognostic group

Fold change (Rsca)

Spectral index (SpI)

G score

P-value

P15531

NDKA_HUMAN

Nucleoside diphosphate kinase A

1

6

1.74

0.82

3.88

0.049

P16989

DBPA_HUMAN

DNA-binding protein A

2

10

1.85

0.79

5.89

0.015

P18085

ARF4_HUMAN

ADP-ribosylation factor 4

5

25

2.13

0.79

15.1

<0.01

P12111

CO6A3_HUMAN

Collagen alpha-3(VI) chain

6

31

2.21

0.76

19.2

<0.01

P29590

PML_HUMAN

Probable transcription factor PML

10

1

−2.27

−0.89

7.85

<0.01

P40925

MDHC_HUMAN

Malate dehydrogenase, cytoplasmic

7

0

−2.67

−1

8.81

<0.01

a protein ratio from spectral counting.

Nm23/NDPK-A expression in invasive pancreatic ductal carcinoma

To confirm expressions of the 6 candidate proteins in tissue sections, we immunohistochemically analyzed the 8 cases with shotgun proteomic data. Among the 6 candidate proteins, we focused on NDKA (also called Nm23/NDPK-A), because the immunohistochemical results were most compatible with those of spectral counting analysis. Nm23/NDPK-A has previously been described as being associated with prognosis in non-pancreatic cancers. Nm23/NDPK-A was over-expressed in the poor prognostic group on semi-quantitative comparison. The immunostaining results for Nm23/NDPK-A are shown in Figure 3. As with semi-quantitative analysis, 3 of 4 cases were negative in the better prognostic group, while all 4 cases in the poor prognostic group were positive for Nm23/NDPK-A, which was detected in the carcinoma cell cytoplasm.
Figure 3

Immunohistochemistry for Nm23/NDPK-A in invasive pancreatic ductal carcinoma. A), B) hematoxylin-eosin staining, C) Nm23/NDPK-A immunoreactivity was detected in the cytoplasm of carcinoma cells. D) No Nm23/NDPK-A immunoreactivity was detected in carcinoma cells. Scale bar = 100 μm, original magnification, ×200

Relationships between Nm23/NDPK-A expression and clinicopathological features in 96 invasive pancreatic ductal carcinoma patients

To confirm the association between Nm23/NDPK-A and the prognosis of invasive pancreatic ductal carcinoma, we immunohistochemically analyzed a 96-case cohort (62 men, 34 women; mean age, 64 years). Baseline characteristics are presented in Table 3. Median survival of patients in this cohort was 19.1 months and 20.4% were alive at 5 years. Nm23/NDPK-A was expressed in the carcinoma cell cytoplasm in 73 of the 96 (76%) tumor specimens. Nm23/NDPK-A expression status was compared across survival-associated clinical parameters (Table 3). Nm23/NDPK-A staining patterns did not differ by age, gender, tumor location, histological differentiation, pathological stage, lymph node metastasis, National Comprehensive Cancer Network (NCCN) resectability status, pre- and postoperative CA19-9 levels, or adjuvant chemotherapy. It was suggested that Nm23/NDPK-A was a potentially independent prognostic factors of pancreatic cancer. Thus, we had investigated the correlation between the expression of Nm23/NDPK-A and prognosis.
Table 3

Patient characteristics as stratified by Nm23/NDPK-A status

Variable

Category

All patients (n = 96)

Nm23/NDPK-A positive (n = 73)

Nm23/NDPK-A negative (n = 23)

P value

Age

< 65

45 (47%)

32 (44%)

13 (57%)

0.2877

 

≥ 65

51 (53%)

41 (56%)

10 (43%)

 

Gender

Male

62 (65%)

45 (62%)

17 (74%)

0.2833

 

Female

34 (35%)

28 (38%)

6 (26%)

 

Location

Head

73 (76%)

56 (77%)

17 (74%)

0.7839

 

Body-Tail

23 (24%)

17 (23%)

6 (26%)

 

Histological differentiationa

Well

8 (8%)

6 (8%)

2 (9%)

0.9838

 

Moderate + Poor

77 (80%)

58 (80%)

19 (82%)

 

Tumor stage

T1 + T2

10 (10%)

7 (10%)

3 (13%)

0.6363

 

T3 + T4

86 (90%)

66 (90%)

20 (87%)

 

Nodal stage

N0

33 (34%)

25 (34%)

8 (35%)

0.9624

 

N1

63 (66%)

48 (66%)

15 (65%)

 

NCCNb criteria

Resectable

50 (52%)

38 (52%)

12 (52%)

0.9920

 

Borderline rsectable

46 (48%)

35 (48%)

11 (48%)

 

Preoperative CA19-9

< 37 U/mL

25 (26%)

17 (23%)

8 (35%)

0.2733

 

≥ 37 U/mL

71 (74%)

56 (77%)

15 (65%)

 

Postoperative CA19-9

< 37 U/mL

57 (59%)

41 (56%)

16 (70%)

0.2538

 

≥ 37 U/mL

39 (41%)

32 (44%)

7 (30%)

 

Adjuvant chemotherapy

Gemcitabine

58 (60%)

42 (58%)

16 (70%)

0.3035

 

Othersc

38 (40%)

31 (42%)

7 (30%)

 

a Other histological classifications including papillary, mucinous, adenosquamous and anaplastic carcinoma in 11 cases.

b National Comprehensive Cancer Network.

c Others include 5FU and surgery alone.

The χ 2 -test was used to compare categorical variables. None of the categories was significantly associated with Nm23/NDPK-A status.

Survival and the expression of Nm23/NDPK-A

Kaplan-Meier analysis revealed patients with carcinoma cells expressing Nm23/NDPK-A to have significantly worse overall survival (OS) and disease free survival (DFS) than those without Nm23/NDPK-A (Figure 4, OS; P = 0.0103, DFS; P = 0.0186). In univariate analysis, lymph node metastasis (P = 0.0039), NCCN criteria (P = 0.0339), postoperative CA19-9 level (P = 0.0082), and Nm23/NDPK-A expression (P = 0.0103) were significantly associated with worse OS (Table 4). With the multivariate Cox regression model, lymph node metastasis (Hazard Ratio [HR] 2.20, 95% Confidence Interval [CI] 1.33-3.80, P = 0.0020), postoperative CA19-9 level (HR 1.92, 95% CI 1.21-3.04, P = 0.0062), and Nm23/NDPK-A expression (HR 1.97, 95% CI 1.16-3.56, P = 0.0110) remained independent factors predicting poor survival (Table 4).
Figure 4

Survival analysis for Nm23/NDPK-A immunoreactivity by the Kaplan-Meier method. A) Nm23/NDPK-A positivity was significantly associated with poor overall survival (log-rank test, P = 0.0103). B) Nm23/NDPK-A positivity was significantly associated with poor disease free survival (log-rank test, P = 0.0186). The day of recurrence was unclear in 11 cases

Table 4

Univariate and Multivariate analyses of clinicopathological factors influencing overall survival

Variable

Category

No. of patients

P value by log-rank test

HRa

95% CIb

P value by Cox proportional hazards

Gender

Male

62

0.1288

   
 

Female

34

    

Age

< 65

45

0.8967

   
 

≥ 65

51

    

Location

Head

73

0.8589

   
 

Body-Tail

23

    

Histological differentiation

Well

8

0.2069

   
 

Moderate + Poor

77

    

Tumor stage

T1 + T2

10

0.1996

   
 

T3 + T4

86

    

Nodal stage

N0

63

0.0039

2.20

1.33-3.80

0.0020

 

N1

33

    

NCCN criteria

Resectable

50

0.0339

1.40

0.90-2.21

0.1419

 

Borderline resectable

46

    

Preoperative CA19-9

< 37 U/mL

25

0.3444

   
 

≥ 37 U/mL

71

    

Postoperative CA19-9

< 37 U/mL

57

0.0082

1.92

1.21-3.04

0.0062

 

≥ 37 U/mL

39

    

Adjuvant chemotherapy

Gemcitabine

58

0.4201

   
 

Others

38

    

Nm23/NDPK-A status

Negative

23

0.0103

1.97

1.16-3.56

0.0110

 

Positive

73

    

a Hazard ratio.

b Confidence Interval.

Univariate analysis was performed using the log-rank test. Multivariate analysis was performed using the Cox proportional hazard model for variables that were significant on univariate analysis.

Discussion

Recently, efforts have been made to identify novel biomarker proteins by gene microarray and proteomics methods. Pancreatic cancer is aggressive and has an extremely poor prognosis due to delayed diagnosis, early metastasis, and resistance to most cytotoxic agents. Thus, it is very important to find new diagnostic, prognostic and therapeutic biomarkers [16]. We explored novel independent prognostic biomarkers of pancreatic cancer by shotgun proteomics using LC-MS/MS, with clinically-documented micro-dissected FFPE tissues, spectral counting for semi-quantification and immunohistochemical confirmation.

FFPE, a routine process providing easily-stored archived tissues, is well-defined pathologically. Extensive FFPE tissue archives have been collected and stored at room temperature for decades in hospitals worldwide. FFPE tissue contains associated clinical and experimental information representing an extremely valuable, untapped reservoir of protein biomarkers [18, 20]. For decades, however, FFPE tissue has been used only for light microscopic diagnosis and staging of tumors [28]. A methodological limitation is that, due to various combinations of intra- and inter-molecular covalent cross-linking between proteins by formaldehyde, proteins are preserved but rendered insoluble [2830]. Recent developments in extraction methodologies have finally made analysis of FFPE tissue by MS possible, providing access to this vast and clinically important sample set for biomarker discovery [18, 20, 3133]. By spectral counting for semi-quantification, we identified 6 proteins differentially expressed in the 2 groups, including 4 and 2 over-expressed proteins in the poor and better prognostic groups, respectively. Though we confirmed all 6 candidate proteins, we focused on Nm23/NDPK-A because its immunohistochemical results were most compatible with those of spectral counting analysis. Nm23/NDPK-A has previously been described as being associated with prognosis in non-pancreatic cancers.

The NM23 gene family was initially identified as putative metastasis suppressors based on reduced expression in certain highly metastatic cell lines and tumors [34, 35]. In humans, 10 genes belong to the NM23 gene family (also known as NME genes). The two most abundantly expressed are NM23-H1 and NM23-H2 encoding the A and B subunits, respectively, of NDPK [36]. Nm23/NDPK-A has several biochemical functions [37]: 1) nucleoside diphosphate kinase, 2) phosphotransferase and histidine protein kinase, 3) 3’-5’-exonuclease, and 4) regulation of GTP-binding proteins. Inverse correlations between NM23-H1 expression and high tumor metastatic potential in several tumor types, e.g. hepatocellular carcinoma, melanomas, breast cancer, ovarian cancer and gastric cancer, have been demonstrated [3841] In other human carcinomas, such as colon cancer, pancreatic ductal carcinoma, neuroblastoma and non-Hodgkin’s lymphoma, high mRNA and protein levels of NM23-H1 have been detected in aggressive tumors [25, 4247]. Andolfo et al reported that NM23-H1 functioned as an inhibitor of tumor invasion in vitro, but had the opposite function in vivo[27]. Although the mechanisms underlying these differences are presently unknown, the action of Nm23/NDPK-A might be altered or regulated in different ways in various organs, under the influence of other genes and proteins.

Only a few studies have examined Nm23/NDPK-A expression in pancreatic cancer. Nakamori et al reported strong immunoreactivity for Nm23/NDPK-A and -B to be associated with lymph node metastasis and poor prognosis in 47 pancreatic cancer cases [25, 44]. We immunohistochemically analyzed Nm23/NDPK-A in 96 cases, a larger sample size than that of Nakamori et al. Our findings also indicated higher Nm23/NDPK-A expression levels to be associated with poor prognosis, but there was no significant correlation with lymph node metastasis. Furthermore, in our study, Nm23/NDPK-A expression was an independent prognostic factor for OS on multivariate analysis. Ohshio et al found no significant correlation between Nm23/NDPK-A expression and prognosis of resected cases [42]. This discrepancy in results between our study and that of Ohshio et al might reflect differences in sample case backgrounds. Nakamori et al and our group examined only resected pancreatic cancer samples whereas Ohshio et al investigated samples from both resected pancreatic cancers and metastases.

Although further study is needed to determine the precise role of Nm23/NDPK-A in malignant behavior of cells, Nm23/NDPK-A expression is potentially useful for assessing prognosis or selecting treatments in pancreatic cancer patients. Positivity for Nm23/NDPK-A in resected specimens might serve as an index providing information useful to physicians for the administration of adjuvant therapy and intensive follow-up of cancer patients likely to suffer a recurrence. By combining the expression of Nm23/NDPK-A, lymph node matastasis and postoperative CA19-9 level that were independent prognostic factors in this study, it might be possible to know the prognosis and make treatment more accurately. Herein, Nm23/NDPK-A expression was confirmed immunohistochemically, though Okabe-Kado et al reported that the serum Nm23/NDPK-A level may contribute to predicting prognosis of neuroblastoma patients [48]. It would be interesting to examine whether serum Nm23/NDPK-A levels are associated with poor prognosis of postoperative pancreatic cancer patients. Identification of Nm23/NDPK-A by immunocytochemistry using endoscopic ultrasound-guided fine needle aspiration or brushing cytology specimens might be also anticipated to be useful for diagnosis and selecting optimal treatments for preoperative patients. Moreover, if the precise role of Nm23/NDPK-A in malignant behavior of cells is elucidated, Nm23/NDPK-A might be therapeutic target.

Conclusions

We performed a proteomic analysis to identify novel prognostic biomarkers for postoperative pancreatic cancer using FFPE tissues. We semi-quantitatively compared expressed proteins between poor and better prognostic groups. The results were immunohistochemically confirmed. A high level of Nm23/NDPK-A expression correlated with poor OS and DFS. Measurement of Nm23/NDPK-A expression is potentially useful for obtaining prognostic and treatment information for pancreatic cancer patients. MS-based proteomic analysis with FFPE tissue offers new opportunities to identify biomarkers and therapeutic targets using archival samples with their corresponding pathological and clinical records.

Abbreviations

ARF4: 

ADP-ribosylation factor 4

CA19-9: 

Carbohydrate antigen 19–9

CI: 

Confidence interval

CO6A3: 

Collagen alpha-3(VI) chain

DBPA: 

DNA-binding protein A

DFS: 

Disease free survival

FFPE: 

Formalin-fixed paraffin-embedded

HR: 

Hazard ratio

JPS: 

Japan pancreas society

LC: 

Liquid chromatography

LMD: 

Laser micro dissection

MDHC: 

Malate dehydrogenase: cytoplasmic

MS: 

Mass spectrometry

MS/MS: 

Tandem mass spectrometry

NDKA: 

Nucleoside diphosphate kinase A

Nm23/NDPK-A: 

Nm23/nucleoside diphosphate kinase-A

NCCN: 

National comprehensive cancer network

OS: 

Overall survival

PML: 

Probable transcription factor PML

RP: 

Reverse phase

Rsc: 

Protein ratio from spectral counting

SpI: 

Spectral index

UICC: 

Unio internationalis contra cancrum/Union for International Cancer Control.

Declarations

Acknowledgments

We thank Emiko Shibuya and Keiko Inabe (Department of Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan), Megumi Maeda and Seiko Yamada (Leica Microsystems K.K., Tokyo, Japan), and Yasuhiko Bando (Biosys Technologies, Incorporated, Tokyo, Japan) for technical assistance, and Brent Bell for reading the manuscript.

Authors’ Affiliations

(1)
Division of Gastroenterological Surgery, Department of Surgery, Tohoku University Graduate School of Medicine
(2)
Department of Structural Biology, Hokkaido University Graduate School of Pharmaceutical Sciences
(3)
Biosys Technologies, Incorporated
(4)
Medical ProteoScope Company, Limited
(5)
Department of Pathology and Histotechnology, Tohoku University Graduate School of Medicine
(6)
Nanotechnology Innovation Station, National Institute for Materials Science
(7)
Department of Applied Information Sciences, Tohoku University Graduate School of Information Science
(8)
Division of Integrated Surgery and Oncology, Tohoku University Graduate School of Medicine
(9)
Department of Surgery I, Tokyo Medical University

References

  1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ: Cancer statistics, 2009. CA Cancer J Clin. 2009, 59 (4): 225-249. 10.3322/caac.20006View ArticlePubMedGoogle Scholar
  2. Matsuno S, Egawa S, Fukuyama S, Motoi F, Sunamura M, Isaji S, Imaizumi T, Okada S, Kato H, Suda K, Nakao A, Hiraoka T, Hosotani R, Takeda K: Pancreatic Cancer Registry in Japan: 20 years of experience. Pancreas. 2004, 28 (3): 219-230. 10.1097/00006676-200404000-00002View ArticlePubMedGoogle Scholar
  3. Hirata K, Egawa S, Kimura Y, Nobuoka T, Oshima H, Katsuramaki T, Mizuguchi T, Furuhata T: Current status of surgery for pancreatic cancer. Dig Surg. 2007, 24 (2): 137-147. 10.1159/000102067View ArticlePubMedGoogle Scholar
  4. Wagner M, Redaelli C, Lietz M, Seiler CA, Friess H, Buchler MW: Curative resection is the single most important factor determining outcome in patients with pancreatic adenocarcinoma. Br J Surg. 2004, 91 (5): 586-594. 10.1002/bjs.4484View ArticlePubMedGoogle Scholar
  5. Carpelan-Holmstrom M, Nordling S, Pukkala E, Sankila R, Luttges J, Kloppel G, Haglund C: Does anyone survive pancreatic ductal adenocarcinoma? A nationwide study re-evaluating the data of the Finnish Cancer Registry. Gut. 2005, 54 (3): 385-387. 10.1136/gut.2004.047191PubMed CentralView ArticlePubMedGoogle Scholar
  6. Schnelldorfer T, Ware AL, Sarr MG, Smyrk TC, Zhang L, Qin R, Gullerud RE, Donohue JH, Nagorney DM, Farnell MB: Long-term survival after pancreatoduodenectomy for pancreatic adenocarcinoma: is cure possible?. Ann Surg. 2008, 247 (3): 456-462. 10.1097/SLA.0b013e3181613142View ArticlePubMedGoogle Scholar
  7. Riediger H, Keck T, Wellner U: zur Hausen A, Adam U, Hopt UT, Makowiec F: The lymph node ratio is the strongest prognostic factor after resection of pancreatic cancer. J Gastrointest Surg. 2009, 13 (7): 1337-1344. 10.1007/s11605-009-0919-2View ArticlePubMedGoogle Scholar
  8. Magistrelli P, Antinori A, Crucitti A, La Greca A, Masetti R, Coppola R, Nuzzo G, Picciocchi A: Prognostic factors after surgical resection for pancreatic carcinoma. J Surg Oncol. 2000, 74 (1): 36-40. 10.1002/1096-9098(200005)74:1<36::AID-JSO9>3.0.CO;2-FView ArticlePubMedGoogle Scholar
  9. Lim JE, Chien MW, Earle CC: Prognostic factors following curative resection for pancreatic adenocarcinoma: a population-based, linked database analysis of 396 patients. Ann Surg. 2003, 237 (1): 74-85. 10.1097/00000658-200301000-00011PubMed CentralView ArticlePubMedGoogle Scholar
  10. Kim R, Tsao R, Tan A, Byrne M, Almhanna K, Lazaryan A, Elson P, Pelley RJ: A single institution review of adjuvant therapy outcomes for resectable pancreatic adenocarcinoma: outcome and prognostic indicators. J Gastrointest Surg. 2010, 14 (7): 1159-1169. 10.1007/s11605-010-1213-zView ArticlePubMedGoogle Scholar
  11. Fortner JG, Klimstra DS, Senie RT, Maclean BJ: Tumor size is the primary prognosticator for pancreatic cancer after regional pancreatectomy. Ann Surg. 1996, 223 (2): 147-153. 10.1097/00000658-199602000-00006PubMed CentralView ArticlePubMedGoogle Scholar
  12. Cleary SP, Gryfe R, Guindi M, Greig P, Smith L, Mackenzie R, Strasberg S, Hanna S, Taylor B, Langer B, Gallinger S: Prognostic factors in resected pancreatic adenocarcinoma: analysis of actual 5-year survivors. J Am Coll Surg. 2004, 198 (5): 722-731. 10.1016/j.jamcollsurg.2004.01.008View ArticlePubMedGoogle Scholar
  13. Burris HA: Moore MJ, Andersen J, Green MR, Rothenberg ML, Modiano MR, Cripps MC, Portenoy RK, Storniolo AM, Tarassoff P, Nelson R, Dorr FA, Stephens CD, Von Hoff DD: Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol. 1997, 15 (6): 2403-2413.PubMedGoogle Scholar
  14. Badger SA, Brant JL, Jones C, McClements J, Loughrey MB, Taylor MA, Diamond T, McKie LD: The role of surgery for pancreatic cancer: a 12-year review of patient outcome. Ulster Med J. 2010, 79 (2): 70-75.PubMed CentralPubMedGoogle Scholar
  15. Ahmad NA, Lewis JD, Ginsberg GG, Haller DG, Morris JB, Williams NN, Rosato EF, Kochman ML: Long term survival after pancreatic resection for pancreatic adenocarcinoma. Am J Gastroenterol. 2001, 96 (9): 2609-2615. 10.1111/j.1572-0241.2001.04123.xView ArticlePubMedGoogle Scholar
  16. Cecconi D, Palmieri M, Donadelli M: Proteomics in pancreatic cancer research. Proteomics. 2011, 11 (4): 816-828. 10.1002/pmic.201000401View ArticlePubMedGoogle Scholar
  17. Nishimura T, Nomura M, Tojo H, Hamasaki H, Fukuda T, Fujii K, Mikami S, Bando Y, Kato H: Proteomic analysis of laser-microdissected paraffin-embedded tissues: (2) MRM assay for stage-related proteins upon non-metastatic lung adenocarcinoma. J Proteomics. 2010, 73 (6): 1100-1110. 10.1016/j.jprot.2009.11.010View ArticlePubMedGoogle Scholar
  18. Kawamura T, Nomura M, Tojo H, Fujii K, Hamasaki H, Mikami S, Bando Y, Kato H, Nishimura T: Proteomic analysis of laser-microdissected paraffin-embedded tissues: (1) Stage-related protein candidates upon non-metastatic lung adenocarcinoma. J Proteomics. 2010, 73 (6): 1089-1099. 10.1016/j.jprot.2009.11.011View ArticlePubMedGoogle Scholar
  19. Motoi F, Rikiyama T, Katayose Y, Egawa S, Unno M: Retrospective evaluation of the influence of postoperative tumor marker status on survival and patterns of recurrence after surgery for pancreatic cancer based on RECIST guidelines. Ann Surg Oncol. 2011, 18 (2): 371-379. 10.1245/s10434-010-1311-xView ArticlePubMedGoogle Scholar
  20. Prieto DA, Hood BL, Darfler MM, Guiel TG, Lucas DA, Conrads TP, Veenstra TD, Krizman DB: Liquid Tissue: proteomic profiling of formalin-fixed tissues. Biotechniques. 2005, 38 (S6): S32-S35. 10.2144/05386SU06. 10.2144/05386SU06View ArticleGoogle Scholar
  21. Kawase H, Fujii K, Miyamoto M, Kubota KC, Hirano S, Kondo S, Inagaki F: Differential LC-MS-based proteomics of surgical human cholangiocarcinoma tissues. J Proteome Res. 2009, 8 (8): 4092-4103. 10.1021/pr900468kView ArticlePubMedGoogle Scholar
  22. Old WM, Meyer-Arendt K, Aveline-Wolf L, Pierce KG, Mendoza A, Sevinsky JR, Resing KA, Ahn NG: Comparison of label-free methods for quantifying human proteins by shotgun proteomics. Mol Cell Proteomics. 2005, 4 (10): 1487-1502. 10.1074/mcp.M500084-MCP200View ArticlePubMedGoogle Scholar
  23. Fu X, Gharib SA, Green PS, Aitken ML, Frazer DA, Park DR, Vaisar T, Heinecke JW: Spectral index for assessment of differential protein expression in shotgun proteomics. J Proteome Res. 2008, 7 (3): 845-854. 10.1021/pr070271+View ArticlePubMedGoogle Scholar
  24. Zhang B, VerBerkmoes NC, Langston MA, Uberbacher E, Hettich RL, Samatova NF: Detecting differential and correlated protein expression in label-free shotgun proteomics. J Proteome Res. 2006, 5 (11): 2909-2918. 10.1021/pr0600273View ArticlePubMedGoogle Scholar
  25. Nakamori S, Ishikawa O, Ohigashi H, Imaoka S, Sasaki Y, Kameyama M, Kabuto T, Furukawa H, Iwanakga T, Kimura N: Clinicopathological features and prognostic significance of nucleoside diphosphate kinase/nm23 gene product in human pancreatic exocrine neoplasms. Int J Pancreatol. 1993, 14 (2): 125-133.PubMedGoogle Scholar
  26. Li XR, Liu M, Zhang YJ, Wang JD, Zheng YQ, Li J, Ma B, Song X: CK5/6, EGFR, Ki-67, cyclin D1, and nm23-H1 protein expressions as predictors of pathological complete response to neoadjuvant chemotherapy in triple-negative breast cancer patients. Med Oncol. 2011, 28 (S1): S129-S134. 10.1007/s12032-010-9742-6View ArticlePubMedGoogle Scholar
  27. Andolfo I, De Martino D, Liguori L, Petrosino G, Troncone G, Tata N, Galasso A, Roma C, Chiancone F, Zarrilli S, Arrigoni G, Staibano S, Imbimbo C, Zollo M: Correlation of NM23-H1 cytoplasmic expression with metastatic stage in human prostate cancer tissue. Naunyn Schmiedebergs Arch Pharmacol. 2011, 384 (4–5): 489-498.View ArticlePubMedGoogle Scholar
  28. Shi SR, Cote RJ, Taylor CR: Antigen retrieval techniques: current perspectives. J Histochem Cytochem. 2001, 49 (8): 931-937. 10.1177/002215540104900801View ArticlePubMedGoogle Scholar
  29. Fox CH, Johnson FB, Whiting J, Roller PP: Formaldehyde fixation. J Histochem Cytochem. 1985, 33 (8): 845-853. 10.1177/33.8.3894502View ArticlePubMedGoogle Scholar
  30. Sutherland BW, Toews J, Kast J: Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions. J Mass Spectrom. 2008, 43 (6): 699-715. 10.1002/jms.1415View ArticlePubMedGoogle Scholar
  31. Hood BL, Darfler MM, Guiel TG, Furusato B, Lucas DA, Ringeisen BR, Sesterhenn IA, Conrads TP, Veenstra TD, Krizman DB: Proteomic analysis of formalin-fixed prostate cancer tissue. Mol Cell Proteomics. 2005, 4 (11): 1741-1753. 10.1074/mcp.M500102-MCP200View ArticlePubMedGoogle Scholar
  32. Hood BL, Conrads TP, Veenstra TD: Unravelling the proteome of formalin-fixed paraffin-embedded tissue. Brief Funct Genomic Proteomic. 2006, 5 (2): 169-175. 10.1093/bfgp/ell017View ArticlePubMedGoogle Scholar
  33. Hood BL, Conrads TP, Veenstra TD: Mass spectrometric analysis of formalin-fixed paraffin-embedded tissue: unlocking the proteome within. Proteomics. 2006, 6 (14): 4106-4114. 10.1002/pmic.200600016View ArticlePubMedGoogle Scholar
  34. Steeg PS, Bevilacqua G, Pozzatti R, Liotta LA, Sobel ME: Altered expression of NM23, a gene associated with low tumor metastatic potential, during adenovirus 2 Ela inhibition of experimental metastasis. Cancer Res. 1988, 48 (22): 6550-6554.PubMedGoogle Scholar
  35. Steeg PS, Bevilacqua G, Kopper L, Thorgeirsson UP, Talmadge JE, Liotta LA, Sobel ME: Evidence for a novel gene associated with low tumor metastatic potential. J Natl Cancer Inst. 1988, 80 (3): 200-204. 10.1093/jnci/80.3.200View ArticlePubMedGoogle Scholar
  36. Boissan M, De Wever O, Lizarraga F, Wendum D, Poincloux R, Chignard N, Desbois-Mouthon C, Dufour S, Nawrocki-Raby B, Birembaut P, Bracke M, Chavrier P, Gespach C, Lacombe ML: Implication of metastasis suppressor NM23-H1 in maintaining adherens junctions and limiting the invasive potential of human cancer cells. Cancer Res. 2010, 70 (19): 7710-7722. 10.1158/0008-5472.CAN-10-1887View ArticlePubMedGoogle Scholar
  37. Galasso A, Zollo M: The Nm23-H1-h-Prune complex in cellular physiology: a 'tip of the iceberg' protein network perspective. Mol Cell Biochem. 2009, 329 (1–2): 149-159.View ArticlePubMedGoogle Scholar
  38. Viel A: Dall'Agnese L, Canzonieri V, Sopracordevole F, Capozzi E, Carbone A, Visentin MC, Boiocchi M: Suppressive role of the metastasis-related nm23-H1 gene in human ovarian carcinomas: association of high messenger RNA expression with lack of lymph node metastasis. Cancer Res. 1995, 55 (12): 2645-2650.PubMedGoogle Scholar
  39. Iizuka N, Oka M, Noma T, Nakazawa A, Hirose K, Suzuki T: NM23-H1 and NM23-H2 messenger RNA abundance in human hepatocellular carcinoma. Cancer Res. 1995, 55 (3): 652-657.PubMedGoogle Scholar
  40. Hartsough MT, Steeg PS: Nm23/nucleoside diphosphate kinase in human cancers. J Bioenerg Biomembr. 2000, 32 (3): 301-308. 10.1023/A:1005597231776View ArticlePubMedGoogle Scholar
  41. Nakayama H, Yasui W, Yokozaki H, Tahara E: Reduced expression of nm23 is associated with metastasis of human gastric carcinomas. Jpn J Cancer Res. 1993, 84 (2): 184-190. 10.1111/j.1349-7006.1993.tb02853.xView ArticlePubMedGoogle Scholar
  42. Ohshio G, Imamura T, Okada N, Suwa H, Yamaki K, Imamura M, Ogasahara K, Tsukayama C, Yamabe H: Immunohistochemical expression of nm23 gene product, nucleotide diphosphate kinase, in pancreatic neoplasms. Int J Pancreatol. 1997, 22 (1): 59-66. 10.1007/BF02803906View ArticlePubMedGoogle Scholar
  43. Niitsu N, Okabe-Kado J, Kasukabe T, Yamamoto-Yamaguchi Y, Umeda M, Honma Y: Prognostic implications of the differentiation inhibitory factor nm23-H1 protein in the plasma of aggressive non-Hodgkin's lymphoma. Blood. 1999, 94 (10): 3541-3550.PubMedGoogle Scholar
  44. Nakamori S, Ishikawa O, Ohhigashi H, Kameyama M, Furukawa H, Sasaki Y, Inaji H, Higashiyama M, Imaoka S, Iwanaga T: Expression of nucleoside diphosphate kinase/nm23 gene product in human pancreatic cancer: an association with lymph node metastasis and tumor invasion. Clin Exp Metastasis. 1993, 11 (2): 151-158. 10.1007/BF00114973View ArticlePubMedGoogle Scholar
  45. Haut M, Steeg PS, Willson JK, Markowitz SD: Induction of nm23 gene expression in human colonic neoplasms and equal expression in colon tumors of high and low metastatic potential. J Natl Cancer Inst. 1991, 83 (10): 712-716. 10.1093/jnci/83.10.712View ArticlePubMedGoogle Scholar
  46. Hailat N, Keim DR, Melhem RF, Zhu XX, Eckerskorn C, Brodeur GM, Reynolds CP, Seeger RC, Lottspeich F, Strahler JR: High levels of p19/nm23 protein in neuroblastoma are associated with advanced stage disease and with N-myc gene amplification. J Clin Invest. 1991, 88 (1): 341-345. 10.1172/JCI115299PubMed CentralView ArticlePubMedGoogle Scholar
  47. Friess H, Guo XZ, Tempia-Caliera AA, Fukuda A, Martignoni ME, Zimmermann A, Korc M, Buchler MW: Differential expression of metastasis-associated genes in papilla of vater and pancreatic cancer correlates with disease stage. J Clin Oncol. 2001, 19 (9): 2422-2432.PubMedGoogle Scholar
  48. Okabe-Kado J, Kasukabe T, Honma Y, Hanada R, Nakagawara A, Kaneko Y: Clinical significance of serum NM23-H1 protein in neuroblastoma. Cancer Sci. 2005, 96 (10): 653-660. 10.1111/j.1349-7006.2005.00091.xView ArticlePubMedGoogle Scholar

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© Takadate et al.; licensee BioMed Central Ltd. 2012

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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