High Expression of Plasminogen Activator Inhibitor-2 (PAI-2) is a Predictor of Improved Survival in Patients with Pancreatic Adenocarcinoma

High Expression of Plasminogen ActivatorInhibitor-2 (PAI-2) is a Predictor of ImprovedSurvival in Patients with PancreaticAdenocarcinomaRoss Smith,1 AiQun Xue,1 Anthony Gill,2 Christopher Scarlett,1 Alexander Saxby,1

Adele Clarkson,2 Thomas Hugh1

1University of Sydney, Department of Surgery, Royal North Shore Hospital, St. Leonards, NSW, Australia2Department of Anatomical Pathology, Royal North Shore Hospital, St. Leonards, NSW, Australia

Abstract

Objective: Recent findings suggest that the urokinase-type plasminogen activator (uPA), its

receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and -2 (PAI-2) play key roles in cancer

invasion.

Summary Background Data: The prognostic value of components of this system is well estab-

lished in breast cancer. However, little is known of its involvement in pancreatic cancer (PC).

Methods: Quantitative real-time polymerase chain reaction (Q-RT-PCR) was used on tissue-

banked specimens and immunohistochemistry (IHC) on paraffin specimens was used to measure

expression of uPA, uPAR, PAI-1, and PAI-2 proteins in 46 PC and 12 cystadenoma specimens.

Results were related to survival using Cox�s proportional hazards testing.

Results: Increased expression of uPA, uPAR, and PAI-1 in PC tissue were independently asso-

ciated with a higher Union Internationale Contre le Cancer [International Union Against Cancer

(UICC)] tumor stage (P < 0.001) and were intercorrelated (P < 0.001). Overexpression of uPAR

indicated reduced survival (P = 0.03). Conversely, PAI-2 messenger ribonucleic acid (mRNA)

overexpression, which occurred in 21 of 46 tumors, negatively correlated with tumor size

(P = 0.008) and survival (P < 0.007) but not with uPA, uPAR, or tumor stage. There was good

agreement between PAI-2 mRNA value and IHC score (P < 0.001). Using Cox�s stepwise anal-

ysis, PAI-2 mRNA value (HR = 0.24; P = 0.001) and UICC tumor stage (HR = 2.014; P = 0.001)

independently predicted survival. An IHC score for PAI-2 of 3+ or 4+ also independently predicted

improved survival (HR = 2.72; P = 0.025).

Conclusions: The uPA/uPAR/PAI-1 system is activated in advanced pancreatic cancer and may

account for the tumor�s aggressive behavior, whereas PAI-2 expression appears to be indepen-

dent of uPA/uPAR/PAI-1 and is associated with improved prognosis. Because of its intercorre-

lation with mRNA expression, PAI-2 IHC may be used as an indicator of survival.

Pancreatic cancer (PC) continues to be associated

with a very poor prognosis, which has not changed

over recent decades. Despite a general decrease in

mortality rates with surgical resection, the median 1-year

survival is 18 months, while 5-year survival is only

achieved in 15%–20% of patients.1,2 One reason for this

poor prognosis is that PC has a high metastatic potentialCorrespondence to: Ross Smith, e-mail: [email protected]

� 2007 by the Societe Internationale de Chirurgie World J Surg (2007) 31: 493–502

Published Online: 5 January 2007 DOI: 10.1007/s00268-006-0289-9

and a high risk of recurrence.3 There are few clinical

parameters that predict outcome with acceptable sensi-

tivity and specificity.4 Extensive research to find reliable

molecular markers that are both specific and sensitive is

vital for better prognostic stratification of PC patients. This

would allow for better selection of patients to surgery5 and

combined treatment modalities, respectively.

Cancer cells express the phenotypic traits that pro-

mote local invasion and distant metastasis.6 Proteolysis

of extracellular matrix (ECM) and basement mem-

branes is a prerequisite for tumor cell dissemination via

capillaries and lymph channels.7 Of particular signifi-

cance is the urokinase-type plasminogen activator

(uPA) system8 and the matrix metalloproteinase (MMP)

system.9 The uPA system is composed of the serine

protease uPA, its membrane-bound receptor (uPAR),

as well as its inhibitors, plasminogen activator inhibitor

type 1 (PAI-1) and type 2 (PAI-2).10 The inactive uPA

(pro-uPA) binds to its cell-surface receptor (uPAR),

then the active receptor-bound uPA in turn activates

the passage of plasminogen to plasmin, a strong pro-

teolytic enzym.11 Plasmin degrades several ECM

components and also activates a few pro-MMPs, thus

creating a localized microenvironment of matrix degra-

dation.12,13 Excessive proteolysis due to uPA–uPAR

binding and activation can be modulated by its inhibi-

tors, PAI-1 and PAI-2.14 During cancer invasion and

metastasis, however, this degradation system becomes

disordered, allowing cancer cells to cross their normal

tissue boundaries. Abnormalities of the components of

the uPA system are heightened in a number of solid

tumor types arising from the breast, ovary, prostate,

lung, endometrium, and colorectal mucosa.15–21 High

tumor-tissue concentrations of uPA and PAI-1 have

been shown to be predictors of poor prognosis in pa-

tients with breast22 and endometrial cancer,23 while a

high level of uPAR has been associated with good

prognosis in ovarian cancer.24 In contrast, a high level

of PAI-2 is associated with improved prognosis in pa-

tients with breast and non-small-cell lung cancer

(NSLC)25,26 but is associated with aggressive disease

in some studies of ovarian27 and colorectal cancers.28

Studies using immunohistochemistry (IHC),29 North-

ern blot,30 and in situ hybridization31 have indicated in-

creased expression of the components of the uPA system

and imply an association with survival. We have

previously shown that uPA messenger ribonucleic acid

(mRNA) is significantly overexpressed in PC tissue using

quantitative real-time polymerase chain reaction (Q-RT-

PCR) and IHC.32 There is little evidence, however, of the

role in the uPA system in PC progression.

This study used Q-RT-PCR and IHC to measure

expression of uPA, uPAR, PAI-1, and PAI-2 in human

pancreatic adenocarcinoma and benign mucinous cys-

tadenoma tumor tissue and correlate the expression

values with tumor grade, Union Internationale Contre le

Cancer [International Union Against Cancer (UICC)]

stage classification, and patient survival.

PATIENTS AND METHODS

Patients and Clinical Data

Informed consent was obtained from 58 patients sus-

pected to have pancreatic carcinoma (32 men and 26

women; median age: 68 years, range: 32–85 years) at

Royal North Shore Hospital between 2001 and 2005.

Tumor and adjacent pancreatic tissue samples were

harvested from 51 of these patients: 39 were from patients

with primary pancreatic adenocarcinoma (stages I–III), 12

were from patients who had mucinous cystadenomas who

underwent a pancreatic resection, and seven were from

patients who had pancreatic adenocarcinoma with

metastases (stage IV) who underwent a laparotomy and

biopsy of a metastatic site and did not have adjacent

pancreatic tissue sampled. This protocol was approved by

the Northern Sydney Health Human Research Ethics

Committee (Sydney, NSW, Australia). The tissue samples

were collected and stored in a tumor bank as previously

described.33

All pathological specimens were examined by one

pathologist (AG). Samples with a diagnosis of pancreatic

adenocarcinoma were graded as well differentiated

(grade 1), moderately differentiated (grade 2), or poorly

differentiated (grade 3). Tumor size was defined by the

tumor�s maximum dimension. All patients were staged

according to the UICC tumor–node–metastasis (TNM)

classification for PC.34 The tumors were also classified by

the Japan Pancreas Society (JPS) cancer staging system

using more stringent details of tumor extent, with factors

such as extension to the common bile duct and duode-

num, neural plexus invasion, and extrapancreatic exten-

sion indicating stage IV.32 Median follow-up after surgery

was 36 months (range: 20–55 months) postresection.

Total RNA Extraction and ReverseTranscriptase Reaction

Total RNA was extracted from frozen tissue using a

commercially available protocol (Qiagen, Hilden, Ger-

494 Smith et al.: High Expression of PAI-2 Predicts Improved Survival

many). We reverse transcribed 1.5 lg of total RNA to

generate first-strand cDNA (total volume 20 ll) using

random hexamer primers (Sigma, St. Louis, MO, USA)

and Superscript III RNase H- (Invitrogen, Grand Island,

NE, USA). The reverse-transcription reaction cycle was

performed at 25�C for 12 minutes, 42�C for 50 minutes,

then 15 minutes of heating at 70�C.

Quantitative Real-Time Polymerase ChainReaction (Q-RT-PCR)

Specific primers used for Q-RT-PCR analysis were as

follows: for uPA 5¢-TTG CTC ACC ACA ACG ACA TT-3¢(forward), 5¢-ATT TTC AGC TGC TCC GGA TA-3¢ (re-verse); for uPAR, 5¢-AGG CCC CAT GAA TCA ATG T-

3¢ (forward), 5¢-GCA GGA GAC ATC AAT GTG GTT-3¢(reverse); for PAI-1, 5¢-TGA TGG CTC AGA CCA ACA

AG-3¢ (forward), 5¢-ATA AGG GGC AGC AAT GAA CA-

3¢ (reverse); and for PAI-2, 5¢-CTC AGA ACC CCA

GGC AGT AG-3¢ (forward), 5¢-ACA GCA TTC ACC

AGG ACC AT-3¢ (reverse). Primers were designed with

the assistance of the PRIMER 3 software.35 To avoid

amplification of contaminating genomic DNA, the prim-

ers spanned different exons. PCR was performed using

the iCycler RT-PCR apparatus (BioRad, CA, USA)

employing SYBR Green I fluorescence (Sigma)

according to the manufacturer�s instructions (40 cycles).

The mRNA expression levels of uPA, uPAR, PAI-1, and

PAI-2 were defined as an N-fold difference in target-

gene expression relative to the ‘‘housekeeping gene’’

18S ribosomal RNA (rRNA) and its adjacent pancreatic

tissue value using the equation 1 of Pfaffl.36 Also we

used equation 4 of Pfaffl37 to make a quick estimation of

the relative ratio.

Cutoffs for uPA, uPAR, PAI-1, and PAI-2 mRNALevels

The cutoff level was arbitrarily decided upon because

of the ability to demonstrate a survival effect, which was

less significant at smaller or greater values. The coded

binary cutoffs for uPA, uPAR, PAI-1, and PAI-2 when

comparing cancer to adjacent tissue were 6-, 6-, 2-, and

2 -fold increases, respectively, in messenger RNA

(mRNA) expression.

Immunohistochemistry (IHC)

Protein expression of uPA (n = 15), uPAR (n = 14),

and PAI-2 (n = 45) in paraffin-embedded tumor-tissue

sections was analyzed by IHC. All specific antibodies

used in this study were from America Diagnostica

(Greenwich, CT, USA): anti-uPA (MAb, 3689, IgG1);

anti-uPAR (MAb, 3936, IgG2a); anti-PAI-2 (MAb, 3750,

IgG2a).

For uPA and PAI-2, deparaffinization and heat-induced

epitope retrieval was performed in Universal Decloaker

10X solution (UD1000MX, Biocare Medical, Walnut

Creek, CA, USA) at 97�C for 50 minutes. Sections were

then incubated with monoclonal antibodies at a dilution of

1:50 for 1 hour at 25�C. For uPA, after washing with 0.005

M Tris–phosphate buffered saline (PBS), slides were

incubated with the Dako rabbit EnVision Plus horseradish

peroxidase (HRP) detection system (DAKO4011, Dako

Corporation, Carpintaria, CA, USA) according to the

manufacturer�s instructions. For PAI-2, after washing in

0.005 M Tris–PBS, slides were incubated with the MACH

4 rabbit HRP detection system (M4U534G, Biocare

Medical) used according to the manufacturer�s instruc-

tions. For uPAR, enzyme-based antigen retrieval was

employed. Slides were incubated for 20 minutes at 37�Cwith 0.05 g porcine trypsin (T-7409, Sigma) and 0.05 g

CaCl2 in 50 ml 0.005 M Tris–PBS wash buffer, pH 7.4.

Sections were then incubated with monoclonal anti-uPAR

antibody at a dilution of 1:50 for 1 hour at 25�C. Afterwashing with 0.005 M Tris–PBS, slides were incubated

with the Dako rabbit EnVision Plus HRP detection system

(DAKO4011, Dako Corporation) according to the manu-

facturer�s instructions.

For both uPA and uPAR, a colonic adenocarcinoma

was used as a positive control, with adjacent normal co-

lonic tissue in the same tissue section used as a negative

control (Fig. 1A). For PAI-2, a squamous cell carcinoma

arising in buccal mucosa was used as a negative control,

with adjacent normal squamous mucosa in the same

tissue section used as a positive control (Fig. 1F). Stain

results were only accepted when the external positive

and negative controls from each batch stained appropri-

ately. The reactions were graded for each tumor using a

semiquantitative scale: 0 = no staining; 1 = weak staining

of some tumor cells; 2 = moderate positive staining of

most (<75%) of tumor cells; 3 = strong staining of most

(>75%) of tumor cells; 4 = intense staining of all tumor

cells. Immunohistochemical scoring was performed by a

single pathologist (AG) who was blinded to the Q-RT-

PCR and other data.

Statistical Analysis

The uPA, uPAR, PAI-1, and PAI-2 mRNA values were

compared with clinical, histological, and biological fea-

Smith et al.: High Expression of PAI-2 Predicts Improved Survival 495

tures using parametric and nonparametric tests where

appropriate (SPSS Software, v13.0, Chicago, IL, USA).

Comparison of uPA, uPAR, PAI-1, and PAI-2 mRNA

expression between pancreatic adenocarcinoma and

adjacent pancreatic tissue was performed using the one-

sample t-test and the binomial test procedure was used to

compare the frequency of abnormal results in cancer

specimens and their controls. Spearman�s rank order

correlation was applied to determine the correlation be-

tween groups defined by pathology or mRNA expression

profiling and IHC score. Overall survival estimates were

calculated using the Kaplan–Meier method and com-

pared between groups using the log-rank test. The Cox

proportional hazards model was used to calculate the

hazard ratio (HR) and its 95% confidence interval (95%

CI) in the univariate and multivariate analysis. All P val-

ues presented were two sided, with a P value of less than

0.05 considered statistically significant.

Figure 1. Urokinase-typeplasminogenactivator (uPA) system proteinexpression using immunohistochem-istry (IHC). A. A uPA external controlsection showing negative stainingin benign colonic mucosa(arrowhead) and positive stainingin colonic adenocarcinoma (arrow);B. Negative/1+ uPA expression inpancreatic cancer (PC); C. 3+positive uPA expression in PC;D. Negative/1+ uPA receptor (uPAR)expression in PC; E. Expression of 3+positive uPAR in PC; F. Plasminogenactivator inhibitor type 2 (PAI-2)external control section showingpositive staining of normal buccalmucosa (arrow) with negativestaining in squamous cell carcinoma(arrowhead); G. Negative PAI-2expression in PC; H. Expression of3+ positive PAI-2 in PC. Note that PAI-2 expression is exquisitely confined tothe tumor cells (arrow).


RESULTS

Messenger ribonucleic acid (mRNA) Levels ofuPA, uPAR, PAI-1, and PAI-2 in Malignant and

Benign Pancreatic Tumors

In cancer tissue, uPA, uPAR, PAI-1, and PAI-2 mRNA

expression was significantly increased when compared

with adjacent uninvolved pancreatic tissue (Table 1). In

addition, uPA and uPAR were significantly increased in

carcinomas when compared with pancreatic benign

mucinous cystadenoma tissue (Table 1).

Immunohistochemistry

Both uPA and uPAR showed diffuse cytoplasmic

staining in many of the tumors (Fig. 1A–E), and uPA

staining was generally more intense than uPAR staining.

Some weak staining for both uPA and uPAR was noted

in the stroma outside the tumor cell boundaries. This

phenomenon was most prominent with uPAR. This

stromal staining was most intense adjacent to the car-

cinoma and faded with distance from the tumor. Very

intense positive staining for both uPA and uPAR was

confined to the cytoplasm of tumor cells and not seen in

the intervening stroma. Incidental positivity for both uPA

and uPAR was noted in nonneoplastic pancreatic acinar

cells and islets of Langerhans. Benign pancreatic ducts

showed weak (1+) or negative staining for both uPA and

uPAR. IHC for PAI-2 showed much more intense and

specific staining than was seen for uPA or uPAR

(Fig. 2F–H). The staining pattern was both cytoplasmic

and nuclear but showed strong cytoplasmic accentua-

tion. Staining was exquisitely confined to the tumor cells

and was absent from the surrounding stroma. Benign

pancreatic ducts did not stain for PAI-2.

Correlation of uPA, uPAR, PAI-1, and PAI-2mRNA Values with Clinicopathologic

Parameters

Positive correlation was observed between uPA,

uPAR, and UICC tumor stage. A strong negative corre-

lation was observed between PAI-2 mRNA and tumor

size, while PAI-1 mRNA and tumor size were positively

correlated (Table 2). The mRNA levels of uPA, uPAR,

and PAI-1 were significantly intercorrelated, while the

trend for a negative correlation with PAI-2 mRNA failed to

reach significance (Table 2).

Overall Survival

A low expression of uPA, uPAR, and PAI-1 mRNA

tended to predicted higher survival probability after sur-

gery, but this was only statistically significant for uPAR.

Conversely, the patients displaying high expression of

PAI-2 had a significantly higher survival probability com-

pared with low expression of PAI-2 (Fig. 1D). Similar to

PAI-1 mRNA, patients with positive IHC score had a

significantly higher survival probability after surgery

compared with a low or negative score (Fig. 3).

Factors Influencing Survival

A significant univariate prediction of survival was dem-

onstrated for uPAR and PAI-2 mRNA values, PAI-2 IHC

score, and UICC tumor stage but not for uPA and PAI-1

mRNA, age, gender, tumor grade, or tumor size (Table 3).

A basic multivariate Cox regression analysis model

was designed by the forward logistic regression (LR)

procedure after including the traditional prognostic and

predictive factors such as age, gender, tumor size, tumor

grade, and UICC tumor stage. Only PAI-2 mRNA value

(HR = 3.08; P = 0.002,) and UICC tumor stage classifi-

cation (HR = 0.31; P = 0.001,) were shown to indepen-

dently predict survival. Age, tumor grade, uPA, uPAR,

and PAI-1 mRNA values and IHC score for PAI-2 did not

contribute to the model (Table 3). However, PAI-2 IHC

score did demonstrate independent prediction

(HR = 2.72; P = 0.025) when the model was tested in the

absence of PAI-2 mRNA values. Again, uPA, uPAR, and

PAI-1 did not contribute to the model in the absence of

PAI-2 mRNA values.

Table 1.The ratio of mRNA expression for urokinase-type plasminogenactivator (uPA), uPA receptor (uPAR), plasminogen activatorinhibitor type 1 (PAI-1) and PAI-2 messenger ribonucleic acid(mRNA) between malignant and benign and their adjacent

uninvolved pancreatic tissues

mRNA ratiomedian (min–max)in PC tumours

(n = 46)

mRNA ratiomedian (min–max)in benign tumours

(n = 12)

P value(PC vs.benign)

uPA 7.6 (0.1–592.5)** 1.4 (0.4–7.4) 0.004uPAR 9.6 (0.2–275.4)** 1.7 (0.1–15.7) 0.025PAI-1 3.3 (0.1–124.3)** 1.4 (0.2–57.2) NSPAI-2 1.9 (0.1–510.8)** 1.8 (0.1–22.0)* NS

PC: pancreatic cancer; NS: not significant.* P values at 0.05 level and ** P values at 0.01 level between

pancreatic cancer and its adjacent uninvolved tissue or between


Correlation Between PAI-2 mRNA Expressionand Corresponding Protein Levels from IHC

There was a strong positive correlation between PAI-2

mRNA expression and protein expression as determined

by IHC (r = 0.401, P = 0.006) using Spearman�scorrelation analysis. The patient with the highest PAI-2

mRNA expression value (510.8-fold increase) also had

the highest PAI-2 protein expression value (IHC 4+), as

determined by a pathologist blinded to the mRNA data.

Similarly, all patients with IHC 3+ protein expression

values of PAI-2 had greater than a 50-fold increase in

PAI-2 mRNA.

DISCUSSION

Overexpression of the uPA system in PC tissue29 has

been confirmed by mRNA measurement and correlated

with outcome. Tumors with high expression of uPA,

uPAR, and PAI-1 were more likely presenting with ad-

vanced tumor stage and shorter overall survival. Con-

versely, high PAI-2 expression was more likely to be

present in smaller tumors and independently predicted

longer overall patient survival. In contrast, mRNA

expression levels of uPA, uPAR, or PAI-1 within tumor

tissues did not independently predict survival. It is not

clear whether the high expression of PAI-2 indicates a

better prognosis due to the protective effect of the pro-

tein or because PAI-2 overexpression occurs in less

aggressive tumors. The results highlight the importance

of the uPA/uPAR mechanism in the migratory behavior

of PC and are consistent with those of Taeuchi and

colleagues29 who used IHC to demonstrate overex-

pression of PAI-2 protein. However, Q-RT-PCR is a

more precise tool for choosing at-risk patients. The

concordance between the IHC score for PAI-2 expres-

sion and Q-RT-PCR would allow for IHC, which is a

cheaper and simpler method, to be used as a measure

of prognosis in PC.

PAI-2 was most concentrated in the tumor cells and

was absent in background staining, highlighting the

specificity of this antibody for PAI-2 and confirming that

the protein is produced by the tumor cells and not in the

adjacent stroma. PAI-2 is localized in cancer cells both

in tumors where PAI-2 is a predictor of a good prognosis

(e.g. breast38 and NSLC39) as well as tumors where

PAI-2 predicts poor prognosis (e.g. colon40 and endo-

metrial cancer41). Interestingly, IHC for PAI-2 was neg-

ative both in benign pancreatic ducts and in the

separate group of pancreatic carcinomas with a very

poor prognosis.

Figure 2. Overall survival of patientswith pancreatic adenocarcinoma withrespect to: A urokinase-typeplasminogen activator (uPA), B. uPAreceptor (uPAR), C. plasminogenactivator inhibitor type 1 (PAI-1), andD. PAI-2 messenger ribonucleic acid(mRNA) expression, [low expression(solid line) and high expression(dashed line)] (log-rank test).


Various other primary cancers overexpress uPA,

uPAR, and PAI-1, and this has been correlated with

survival. It is of particular note that in breast cancer,42

the accumulation of uPA-PAI-1 complexes inside the

tumor cells was predictive of poor outcome. This was

observed when the tumor was small. In this study, the

tumor tissue levels of uPA, uPAR or PAI-1 were asso-

ciated with poor survival but failed to reach independent

statistical significance. Thus, it is not certain whether the

expression of uPA, uPAR, and PAI-1 are elevated as a

consequence of the tumor�s invasive status or whether

such expression is the cause of invasion. We were un-

able to confirm the findings of Cantero et al.27 that the

elevation of both uPA and uPAR together improved the

prediction of outcome. We observed strong correlation

between tumor tissue levels of uPA, uPAR, and PAI-1

with UICC tumor stage. It is interesting that although the

JPS is more predictive of survival, the UICC system

provided a stronger statistical model and has therefore

been used in this paper (details not shown in this pa-

per). This is probably because there were only 2 and 5

patients with stages I and II, respectively, due to the

stringent criteria of the JPS system.

Strong uPA and uPAR staining was seen in tumor

cells and weaker staining in the intervening stroma. This

accentuation of staining implies that uPA and uPAR are

primarily produced in tumor cells but not the intervening

stroma, while the background staining implies that these

proteins may have their primary effect in the adjacent

stroma. Of significance was the positive staining of

some benign epithelial elements adjacent to the tumor in

the presence of negative staining of these same benign

elements away from the tumor. This may indicate that

these benign cells take up uPA and uPAR produced by

tumor cells. Alternatively, these cells may be exposed to

certain growth factors or inflammatory mediators in the

tumor microenvironment, inducing the production of uPA

and uPAR. Factors other than malignant transformation

are known to stimulate promotion of the local uPA/

uPAR/PAI-1 system in the pancreas. Transforming

growth factor (TGF)-beta-1 stimulates the release of

PAI-1 in the pancreas, while other agents release PAI-1

in the kidney and spleen.43 The physiological regulation

of PAI-1 expression is likely to involve a complex inter-

action of transcription factors and DNA sequences.

Overexpression of these proteins also occurs in pan-

creatic inflammatory conditions such as chronic pan-

creatitis where it is presumed that the stimulus is from

local factors.44

Demonstrating the relationship of the uPA system to

PC progression opens new areas of exploration to

Table 2.Correlation between urokinase-type plasminogen activator (uPA) system, tumor stage, tumor grade, age, and gender

uPA uPAR PAI-1 PAI-2 P2-IHC Stage Grade T-size Age

uPAR 0.827**PAI-1 0.641** 0.738**PAI-2 )0.206 )0.124 )0.118PAI-2 IHC )0.037 0.014 )0.037 0.401**Stage 0.439** 0.444** 0.248 )0.028 )0.199Grade 0.226 0.251 0.226 0.070 0.110 0.311T-size 0.118 0.211 0.295* )0.387** )0.193 0.175 0.181Age )0.073 )0.049 )0.033 0.005 0.243 0.01 )0.068 )0.146Gender )0.051 0.109 0.130 0.070 0.063 0.213 0.256 0.019 0.248

uPAR: urokinase-type plasminogen activator receptor; PAI-1: plasminogen activator inhibitor type 1; PAI-2: plasminogen acti-vator inhibitor type 2; IHC: immunohistochemistry; T: tumor

*Correlation is significant at the 0.01 level (two tailed).**Correlation is significant at the 0.001 level (two tailed).

Figure 3. Overall survival of patients with pancreatic adeno-carcinoma with respect to immunohistochemical (IHC) score forplasminogen activator inhibitor type 2 (PAI-2) [negative (solidline) and positive (dashed line) score] (log-rank test).


determine how this information may be used for the pa-

tient�s benefit. It would be interesting to explore whether

influencing the uPA system can reduce the metastasizing

capacity of PC. There may be importance in providing

exogenous PAI-2 to patients as a targeting agent either

independently or conjugated with other cytotoxic agents.

For example, it has been shown in a mouse PC model

that PAI-2 can be used as a carrier of alpha therapy with

a dose-related effect.45

The measurement of components of the uPA system,

particularly PAI-2, has important implications for indi-

vidual patients. The excellent correlation between Q-RT-

PCR data and IHC for PAI-2 using paraffin-embedded

tissue, a commercially available antibody, and the sim-

ple methods outlined in this paper indicates that the

assessment of PAI-2 status of pancreatic carcinomas

can be reliably performed in any surgical pathology

laboratory with IHC facilities. PAI-2 testing is therefore

potentially available to any surgeon performing pancre-

atic surgery. Further understanding of the uPA system in

the PC setting may lead to novel therapies for this

intractable disease. In the mean time, staining for PAI-2

has a powerful prognostic value that should be used to

help doctors decide upon multimodal treatment and fol-

low-up policy.

ACKNOWLEDGEMENTS

We thank Dr. Jaswinder S. Samra for kindly providing

pancreatic samples, and Prof. Barry Allen for his

insightful discussions concerning PAI-2. This study was

supported by the Cancer Surgery Research Foundation

(CanSur) and G.J. Tattersall�s Pty Ltd.

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Table 3.Cox univariate and multivariate analysis for survival

Cutoff values Number (group)

Univariate analysis survivalbenefit

Multivariate analysis survivalbenefit

P HR 95% CI P HR 95% CI

Age <55, 55–70, >70 7, 17, 22 0.12 1.47 0.90–2.40Gender M:F 28:18 0.54 1.24 0.63–2.42Tumor stage (UICC) I, II, III, IV 17, 17, 1, 11 0.019 0.416 0.19–0.86 0.001 2.014 1.32–3.05Tumor stage (JPS) I, II, III, IV 2, 5, 11, 28 0.056 0.51Tumor grade Well, moderate, Poor 4, 31, 11 0.760 1.09 0.61–1.96Tumor size <3,>3 cm 21, 25 0.310 1.41 0.73–2.74uPA mRNA <,>6-fold 19, 27 0.087 1.84 0.92–3.69uPAR mRNA <,>6-fold 21, 25 0.033 0.48 0.24–0.94PAI-1 mRNA <,>2-fold 19, 27 0.080 1.88 0.93–3.85PAI-2 mRNA <,>2-fold 25, 21 0.007 2.58 1.29–5.12 0.001 0.24 0.11–0.52PAI-2 IHC 1 & 2+, 3 & 4+ 34, 11 0.053 2.29 0.99–5.31 0.025a 2.72a 1.13–6.53a

HR: hazard ratio; 95% CI: 95% confidence interval; UICC: Internationale Contre le Cancer (International Union Against Cancer);JPS: Japan Pancreas Society; uPA: urokinase-type plasminogen activator; mRNA messenger ribonucleic acid; uPAR: uPAreceptor; PAI-1: plasminogen activator inhibitor type 1; PAI-2: plasminogen activator inhibitor type 2; IHC: immunohistochemistry.

aIncluded in equation when PAI-2 mRNA excluded.


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High Expression of Plasminogen Activator Inhibitor-2 (PAI-2) is a Predictor of Improved Survival in Patients with Pancreatic Adenocarcinoma