Pyrophosphatase 1 expression is associated with future recurrence and overall survival in Chinese patients with intrahepatic cholangiocarcinoma

The inorganic pyrophosphatase gene (PPA1) encodes inorganic pyrophosphatase, an enzyme that catalyzes the hydrolysis of inorganic pyrophosphate to orthophosphate, and has been revealed to be dysregulated in several types of human cancer. However, the role of PPA1 in intrahepatic cholangiocarcinoma (ICC) has not yet been determined. The present study detected PPA1 expression and investigated its clinical significance in ICC. Tissue microarray blocks containing 93 ICC specimens were constructed. The protein expression of PPA1 in these specimens was detected by immunohistochemistry. PPA1 was overexpressed in 49.5% of the ICC specimens and was significantly associated with large tumor size, positive margins, T stage, lymph nodal metastases, poorly differentiated tumors and advanced disease stage. Furthermore, PPA1 expression was an indicator of future recurrence and poor survival in patients with ICC. Increased expression of PPA1 is a common event in human ICC and is significantly associated with a poor outcome in patients with ICC, suggesting a potential role for PPA1 in the development and progression of ICC.


Introduction
Cholangiocarcinoma includes 3 categories based on the anatomical location of the origin within the biliary system: Intrahepatic, hilar and distal (1).These malignancies are slow-growing but aggressive and are associated with a very poor prognosis with a median survival of 6-12 months from the point of diagnosis (2).Intrahepatic cholangiocarcinoma (ICC) is the second most common primary hepatic malignancy following hepatocellular carcinoma worldwide, which also has a higher incidence in East Asia (3,4).In recent decades, the incidence and mortality rates of ICC have been progressively increasing (5,6).Curative resection appears to be the most effective treatment for ICC (7,8).Unfortunately, the resectability rate remains low due to the late presentation of the disease and the invasion of tumor cells into the blood and lymphatic vessels (9).Furthermore, the etiology of ICC remains unknown, as no identifiable risk factors are identified in 90% of patients with ICC.Predisposing factors include primary hepatolithiasis, sclerosing cholangitis, choledochal cysts, primary biliary cirrhosis and infection with Clonorchis sinensis or Opisthorchis viverrini, as well as inflammatory bowel disease and chronic pancreatitis (10,11).Furthermore, genetic factors regulating the progression and prognosis of ICC remain to be investigated.To date, certain key molecules may aid in diagnosing and predicting the prognosis and future recurrence of ICC (12)(13)(14).
The Inorganic pyrophosphatase gene (PPA1) encodes the enzyme, inorganic pyrophosphatase (PPase), which catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to orthophosphate.PPi is a high-energy phosphate compound involved in multiple cell metabolism process, including polysaccharide, nucleic acid and protein synthesis (15).The present study revealed that PPase was associated with the molting and development of roundworm Ascaris (16), and was necessary for the growth of E. coli (17).Additionally, the expression of PPA1 in aging rat liver was higher than that in young rats (18,19).Notably, PPA1 was overexpressed in various types of human tumor, including lung adenocarcinoma, breast cancer, hepatocellular carcinoma and primary colorectal cancer (20)(21)(22)(23)(24).However, it remains unclear whether or not PPA1 serves an oncogenic role in ICC, and the mechanisms responsible for this also require further investigation.
The present study aimed to detect the expression of PPA1 in human resected ICC and to analyze the correlation between PPA1 expression and the malignant behaviors of ICC, in order to predict the outcomes of patients with ICC.

Materials and methods
Patient specimens and tissue microarray construction.A total of 93 patients with ICC and 25 with benign diseases at the Eastern Hepatobiliary Hospital (Shanghai, China; 75 with ICC and 25 with non-cancerous bile duct tissues) and Changhai Hospital (Shanghai, China; 18 with ICC), between January 2005 and December 2008, were enrolled in the present study.The mean age of these patients was 55 years, ranging from 31 to 79 years.The patient medical records were reviewed to obtain data and detailed information is listed in Table I.Of the enrolled patients, 39 (41.9%) were male and 54 (58.1%) were female, and 51 (54.8%) were diagnosed with stage III/IV disease, while 42 (45.2) exhibited stage I/II disease.All the enrolled patients were available for follow-up and none of the patients had received preoperative treatment, either radiotherapy or chemotherapy.
Tissue microarray blocks of benign diseases and tumor tissue specimens were constructed using a manual arrayer (Beecher Instruments, Sun Prairie, WI, USA).Each block had at least one 1.5-mm core of nonneoplastic mucosal tissue and two 1.5-mm cores of primary tumor tissue (25).All the tissue specimens in the present study were obtained with written informed consent and the use of the tissues and clinical information was approved by the Eastern Hepatobiliary Hospital and Changhai Hospital Institutional Review Boards.
Immunohistochemistry and evaluation of immunostaining.4-µm thick sections of the paraffin-embedded tissue microarrays (TMAs) were deparaffinized in xylene, and then rehydrated in graded concentrations of ethyl alcohol (100, 95, 75%), then water.TMA sections were microwave-treated in 0.01 mol/l citrate buffer (pH 6.0) at 99˚C for 4 min.TMAs were placed in 3% H 2 O 2 for 10 min to inhibit endogenous peroxide activity, washed 3 times with phosphate-buffered saline (PBS) for 3 min and blocked in goat serum (SP KIT-B1 Fuzhou Maixin Biotechnology Development Co., Ltd., Fuzhou, China) at room temperature for 10 min.Anti-PPA1 primary antibody (cat.no.H62; dilution, 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), was incubated with the sections for at 4˚C for 24 h.TMAs were then washed 3 times with PBS buffer for 10 min.A streptavidin-peroxidase kit (cat.no.KIT-9720; Maixin Biotech Co., Ltd., Fuzhou, China) was used to visualize antibody binding, according to the manufacturer's instructions.The sections were counterstained with hematoxylin for 3 min at room temperature and washed in water for 3 min.These TMAs were rehydrated in a descending series of alcohol (85, 95, and 100%; 2 min each) and Xylene for 1 min.A semiquantitative scoring system was used to evaluate the expression of PPA1 under an Olympus CX31 microscope (Olympus, Center Valley, PA, USA), as previously described (26).A light microscope was used at a magnification is of x40 or x200.Staining intensity was divided into: Negative, 0; weak, 1; moderate, 2; and intense, 3. The percentage of positively stained cells was scored as 0-1 (0-100%).Theoretically, a weighted score ranging between 0 (0% of cells stained) and 3 (100% of the cells stained at 3+ intensity) was generated for each case.A score >0 was considered as positive.
Statistical analysis.Categorical data were analyzed using the χ 2 test.Survival rates were calculated using the Kaplan-Meier method.The Cox proportional hazards model for multivariate survival analysis was used to assess predictors associated with tumor recurrence and survival.P<0.05 was considered to indicate a statistically significant difference.Statistical analyses and graphics were performed using the SPSS 19.0 statistical package (IBM Corp., Armonk, NY, USA) (27).

Expression of PPA1 in patients with ICC.
Representative images of PPA1 immunostaining in human ICC and paired adjacent non-neoplastic tissues are presented in Fig. 1.No staining or weak staining of PPA1 (21.7%; 5/23) was observed in the cytoplasm of cholangiocytes of normal bile ducts and benign diseases (Fig. 1A).In tumor cells, however, strong PPA1-positive staining was preferentially localized to the cytoplasm.Positive expression of PPA1 was observed in 46/93 (49.5%) of the primary ICC tissues (Fig. 1B-D).
Furthermore, it was also revealed that overexpression of PPA1 was correlated with advanced tumor (T), node (N), and Tumor-Node-Metastasis (TNM) stages according to the 8th edition AJCC staging manual (28).Positive PPA1 expression was observed more frequently in large ICC tumors (invasion level T3-T4) (66.1%) than in small ICC tumors (invasion level T1-T2; 16.1%; P<0.001), and more frequently in cases with regional  lymph node metastasis (64.7%) than in N0-stage tumors (31.0%;P=0.001).With regards to the TNM stage, overexpression of PPA1 was significantly associated with advanced disease stages: 31% at stage I/II and 64.7% at stage III/IV (P=0.001).

PPA1 expression is associated with decreased overall survival (OS) in patients with ICC.
The median cumulative overall survival in patients with resected ICC was 33 months.Kaplan-Meier analyses revealed that the ICC patients with positive PPA1 expression had a poor prognosis compared with those with negative PPA1 expression (P<0.001;Fig. 2A).Furthermore, subgroup analysis of PPA1 expression according to TNM stage was performed.The outcome of patients with PPA1-positive expression was worse in those with stage I/II disease than in those without PPA1 expression with stage I/II disease (51.6 vs. 30.5 months; P=0.009; Fig. 2B).In patients with stage III/IV disease, patients with PPA1-positive expression tended to have a worse outcome than those without PPA1 expression, but this difference was no statistically significant (30.2 vs. 20.8months; P= 0.138; Fig. 2C).Additionally, univariate analysis revealed that all other factors were significantly associated with patient overall survival (Table II), including gender (P<0.001),tumor size (P=0.008),positive tumor margins (P<0.001),T stage (P<0.001),regional lymph node metastasis (P<0.001),TNM stage (P<0.001) and differentiation (P<0.001).Subsequent multivariate analysis using the Cox proportional hazards model demonstrated that T stage was an independent prognostic indicator of OS in patients with ICC (Table II).
PPA1 expression is associated with recurrence in patients with ICC.Among the 93 patients with ICC, 90 experienced recurrence following resection.Patients with PPA1-positive tumors exhibited higher chanceto recurrence compared with those with PPA1-negative tumors (P<0.001;Fig. 3A).Furthermore, in patients with stage I/II disease, Kaplan-Meier survival analyses according to TNM stage revealed that those with PPA1-negative tumors exhibited a significantly longer disease-free survival (DFS) than those with PPA1-positive tumors (50.2 vs. 26.7 months; P= 0.008; Fig. 3B).However, this difference was not statistically significant in patients with stage III/IV disease (PPA1-negative vs. PPA1-positive: 27.5 vs. 19.6 months; P=0.184; Fig. 3C).In addition, the univariate Table II.Univariate and multivariate analysis of variables associated with overall survival in patients with intrahepatic cholangiocarcinoma.analysis revealed that age (P=0.011),sex (P=0.005),tumor size (P=0.003),positive margin (P<0.001),T stage (P<0.001),lymph node metastasis (P<0.001),disease stage (P<0.001) and differentiation (P<0.001) were significant predictors of tumor recurrence (Table III).Multivariate analysis using the Cox proportional hazards model demonstrated that PPA1 expression, T stage and differentiation were independent predictors of tumor recurrence (Table III).

Discussion
The expression profile of the PPA1 gene has been investigated in lung adenocarcinoma, breast cancer, hepatocellular carcinoma, primary colorectal cancer and gastric cancer, and the PPA1 protein has been revealed to be overexpressed in these types of cancer (20)(21)(22)(23)(24)29).Given that ICC has a high mortality rate worldwide and that early tumor invasion, wide metastases and recurrence result in a poor prognosis for patients with this disease (2), the present study aimed to investigate the potential role of PPA1 in ICC.It was revealed that PPA1 was highly overexpressed in ICC specimens compared with expression in adjacent non-neoplastic tissues and benign disease tissues, and overexpression of PPA1 was significantly correlated with malignant behaviors of ICC including tumor size, tumor margins, T stage, lymph node metastasis, differentiation and TNM stage.Additionally, patients with PPA1-overexpression exhibited poorer OS rates and higher rates of recurrence than patients with low PPA1 expression.Therefore, to the best of our knowledge, these data serve as the first evidence that PPA1 may serve an important role in ICC development and progression.A number of previous studies have examined clinicopathological prognostic factors for ICC following surgical resection, including sex, age at diagnosis, tumor size, tumor margins, depth of tumor invasion, differentiation, lymph node metastasis and TNM stage (30)(31)(32)(33).Furthermore, it was well-known that lymph Table III.Univariate and multivariate analysis of variables associated with recurrence in patients with intrahepatic cholangiocarcinoma.------------------------------------------------ It is known that poorly-differentiated tumors proliferate and metastasize more frequently than well-differentiated tumors.In the present study, PPA1 was expressed more frequently in poorly-differentiated tumors than in well/moderately-differentiated tumors.In addition, positive PPA1 expression was observed more frequently at late stages than at early stages of the disease, suggesting that PPA1 may serve an important role in promoting the growth and development of ICC.However, there are several limitations to the present study.To begin with, the number of ICC samples was small.Additionally, the underlying molecular mechanisms of PPA1 in ICC progression and prognosis were not thoroughly investigated.However, it may be concluded that PPA1 may serve an important role in ICC by regulating energy metabolism and signaling pathways (34,35).

P-value -
In summary, the present study demonstrated that PPA1 was overexpressed in human ICC and that the level of PPA1 expression may be associated with adverse clinical outcomes in patients with ICC.Furthermore, overexpression of PPA1 was a promising prognostic and predictive factor in Chinese patients with ICC.These observations support PPA1 as an indicator of a worse outcome for patients with ICC and as a potential biomarker in predicting the outcome of patients.

Figure 2 .
Figure 2. Kaplan-Meier analysis of the OS in patients with ICC according to the expression of PPA1.(A) OS were significantly shorter in patients with positive PPA1 expression (median survival, 23.3 months) than in those with negative PPA1 (median survival, 45.1 months; P<0.001).(B) OS were significantly shorter in patients exhibiting stage I/II disease with positive PPA1 expression (median survival, 30.5 months) than in those with negativePPA1 expression (median survival, 51.6 months; P= 0.009).(C) OS were shorter in patients exhibiting stage III/IV disease with positive PPA1 expression (median survival, 20.8 months) than in thosewith negative PPA1 expression, but with no statistical significance (median survival, 30.2 months; P= 0.138).OS, overall survival; ICC, intrahepatic cholangiocarcinoma; PPA1, inorganic pyrophosphatase; TNM, Tumor-Node-Metastasis.

Figure 3 .
Figure 3. Kaplan-Meier of DFS in patients with ICC according to the expression of PPA1.(A) DFS were significantly shorter in patients with positive PPA1 (median survival, 22.2 months) than in those with negative PPA1 expression (median survival, 42.9 months; P<0.001).(B) DFS were significantly shorter in patients exhibiting stage I/II disease with positive PPA1 expression (median survival, 26.7 months) than in those with negative PPA1 expression (median survival, 50.2 months; P= 0.008).(C) DFS were shorter in patients exhibiting III/IV stage disease with positive PPA1 expression (median survival, 19.6 months) than in those with negative PPA1 expression, but with no statistical significance (median survival, 27.5 months; P= 0.184).DFS, disease-free survival; ICC, intrahepatic cholangiocarcinoma; PPA1, inorganic pyrophosphatase; TNM, Tumor-Node-Metastasis.

Table I .
Correlation between PPA1 expression and clinicopathological parameters of intrahepatic cholangiocarcinoma.
Notably, only in the relatively early disease stages, patients with positive PPA1 expression experienced significantly shorter OS and higher recurrence rates than those with negative PPA1 expression, indicating that early detection may be an effective way to prolong the outcome of patients with ICC.
(31,33)fidence interval; T, tumor; TNM, Tumor-Node-Metastasis; PPA1, inorganic pyrophosphatase.nodemetastases,TNM stage and tumor margins are important indicators for predicting tumor recurrence and OS(31,33).In the present study, it was revealed that age, sex, tumor size, tumor margins, T stage, lymph node metastasis, differentiation, TNM stage and PPA1 expression levels were predictors of survival in a group of Chinese patients with ICC.Additionally, T stage was revealed to be an independent predictor of OS.Additionally, tumor recurrence, T stage, PPA1 and differentiation may predict future recurrence independently.