Purpose: Our goal was to clarify the involvement and clinical significance of S100P in pancreatic carcinogenesis.
Experimental Design: We examined S100P expression in 45 bulk pancreatic tissues; in microdissected cells, including invasive ductal carcinoma (IDC) cells (20 sections), pancreatic intraepithelial neoplasia (PanIN) cells (12 sections), intraductal papillary mucinous neoplasm (IPMN) cells (19 sections), and normal epithelial cells (11 sections); and in pancreatic juice samples from 99 patients with pancreatic diseases (32 cancer, 35 IPMN, and 32 chronic pancreatitis samples). We used quantitative real-time reverse transcription-PCR with gene-specific priming to measure S100P in these various types of samples.
Results: In bulk tissue analyses, pancreatic cancer and IPMN expressed significantly higher levels of S100P than did nonneoplastic pancreas (P < 0.017 and P = 0.0013, respectively). Microdissection analyses revealed that IPMN expressed significantly higher levels of S100P than did IDC (P < 0.0001) and PanIN (P = 0.0031), although S100P expression did not differ between IDC and PanIN (P = 0.077). In pancreatic juice analyses, cancer and IPMN juice expressed significantly higher levels of S100P than did pancreatitis juice (both P < 0.0001). Receiver operating characteristic curve analyses revealed that measurement of S100P in pancreatic juice was useful for discriminating neoplastic disease from chronic pancreatitis (area under the curve = 0.837; 95% confidence interval, 0.749-0.903).
Conclusion:S100P may be an early developmental marker of pancreatic carcinogenesis, and measurement of S100P in pancreatic juice may be useful for early detection of pancreatic cancer or screening of early pancreatic carcinogenesis.
- pancreatic cancer
- pancreatic juice
Pancreatic cancer is the fourth most common cause of tumor-related death in the industrialized world (1, 2). It is one of the most lethal human cancers, in part because of the asymptomatic nature of the disease in the early stages and the lack of sensitive methods for diagnosis at the potentially curative stage. In particular, there are no effective biomarkers for the early diagnosis of pancreatic cancer, although CA19-9 has been used as a tumor marker of the advanced stage of this disease. Hence, tumor markers that are sensitive and specific for detection of the early stage of pancreatic cancer or carcinogenesis are needed.
Microarray analysis, which is a powerful tool for identifying genes associated with cancer, including pancreatic cancer, recently revealed that expression of S100P is increased in pancreatic cancer (3–5). S100 family proteins are small Ca2+-binding proteins of the EF-hand type and have been implicated in regulation of a variety of intracellular and extracellular processes, including cell proliferation, differentiation, and intracellular signaling (6). S100P, which is one of the least studied members of the S100 family, is expressed in breast cancer (7), prostate cancer (8), and lung cancer (9). S100P has also been reported to interact with several molecules, including CacyBP/SIP (10), receptor for advanced glycation end products (11), cytoskeletal protein ezrin (12), and S100P-binding protein Riken (S100PBPR; ref. 13). Arumugam et al. (14) recently reported that S100P expression is involved in cell growth and invasion of pancreatic cancer. These data suggest that S100P is a promising diagnostic marker and/or possible therapeutic target of pancreatic cancer. However, there are no reports describing the utility of S100P analysis in the clinical setting.
Conventional quantitative real-time reverse transcription-PCR (RT-PCR) has the potential to become a valuable analytic tool for the detection of target mRNAs from clinical samples such as body fluids and tissue biopsies. However, fragmentation of RNAs from such samples remains the most critical factor limiting this procedure. In the present study, we used one-step quantitative real-time RT-PCR with gene-specific priming, short amplicons, and normalization to reference genes to examine S100P expression in various types of clinical samples. It was recently reported that gene expression could be measured reliably from degraded RNA with this procedure (15–17). We successfully measured S100P expression in various types of clinical samples, including pancreatic bulk tissues, microdissected cells, and pancreatic juice. To clarify the involvement of S100P in pancreatic carcinogenesis, we examined S100P expression in pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN), which are thought to be precursor lesions of a subset of pancreatic cancer and compared the S100P levels with those in invasive ductal carcinoma (IDC) cells and normal epithelial cells. To assess clinical significance of S100P expression in pancreatic juice, we measured pancreatic juice samples from 99 patients with pancreatic cancer, IPMN, or chronic pancreatitis.
Materials and Methods
Cell lines, pancreatic tissues, and pancreatic juice. Fifteen pancreatic cancer cell lines—ASPC-1, BxPC-3, KP-1N, KP-2, KP-3, Panc-1, Suit-2 (Dr. H. Iguchi, National Kyushu Cancer Center, Fukuoka, Japan), MIA PaCa-2, NOR-P1 (established in our laboratory), Capan-1, Capan-2, CFPAC-1, H48N, HS766T, and SW1990 (American Type Culture Collection, Manassas, VA)—and four primary cultured pancreatic fibroblasts derived from resected pancreatic tumors were used. Cells were maintained as described previously (18). Tissue samples were obtained at the time of surgery at the Department of Surgery I, Kyushu University Hospital (Fukuoka, Japan), as described previously (19). Tissue samples were obtained from the primary tumor of each resected pancreas for a total of 11 IDC and 20 IPMN samples. Fourteen nonneoplastic tissue samples were taken from peripheral tissues away from the tumor. Tissue samples were removed as soon as possible after resection and divided into at least two bulk tissue samples. A part of each sample was embedded in optimum cutting temperature compound (Sakura, Tokyo, Japan) and snap-frozen for microdissection. The remainder was fixed in formalin, embedded in paraffin, and cut into 4-μm-thick sections for H&E staining. All tissues adjacent to the specimens were examined histologically, and the diagnosis was confirmed by experienced pathologists. Pancreatic juice samples were collected from 99 patients who had undergone endoscopic retrograde cholangiopancreatography for suspected malignancy of the pancreas at Kyushu University Hospital between January 1, 2002, and June 31, 2005. As described previously (20, 21), a 350-cm no. 6 French balloon catheter was inserted endoscopically into the pancreatic duct, and pancreatography was done. The endoscope was then withdrawn over the catheter. After i.v. injection of 1 unit/kg body weight of secretin (Eisai, Tokyo, Japan), pancreatic juice was collected for at least 15 minutes through the balloon catheter. At least three additional samples of pancreatic juice were collected from each patient at 5-minute intervals. The first sample was discarded because of contamination of the pancreatic juice by the contrast medium. For cytologic examination, the second sample was centrifuged at 400 to 700 × g for 5 minutes at 4°C, and smears were subjected to Papanicolaou staining. If epithelial cells were present, the samples were regarded as acceptable for RNA expression analyses. The third sample was centrifuged at 1,000 × g for 5 minutes at 4°C, and the cell pellet was washed once in PBS, centrifuged, and then stored at −80°C until used for RNA expression analyses. We used the same protocol for all pancreatic juice samples from patients with any of the pancreatic diseases we examined. The diagnosis of pancreatic ductal adenocarcinoma was confirmed by histologic examination of resected specimens when available, but when the case was inoperable, a clinical diagnosis was made on the basis of imaging findings. Chronic pancreatitis or IPMN was diagnosed on the basis of histologic examination of resected specimens or clinical findings at the time of the initial diagnosis and during a follow-up period of at least 12 months that included conventional diagnostic imaging. Written informed consent was obtained from all patients, and the study was approved by the surveillance committee of our institution and conducted according to the Helsinki Declaration.
Isolation of total RNA. Total RNA was extracted from bulk tissues with an RNeasy Mini kit (Qiagen, Tokyo, Japan), from cell pellets of pancreatic juice with a standard acid guanidinium thiocyanate-phenol-chloroform protocol (22) with glycogen (Funakoshi, Tokyo, Japan), and from cells isolated by laser microdissection with a PicoPure RNA Isolation kit (Arcturus Bioscience, Mountain View, CA) with DNase I (Roche Diagnostics, Mannheim, Germany) treatment according to the instructions from the manufacturer. RNA concentrations in extracts were measured with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Rockland, DE) at absorbances of 260 and 280 nm (A260/280). RNA integrity was assessed with an Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA).
Quantitative assessment of S100P by one-step real-time RT-PCR with gene-specific priming. We used one-step quantitative real-time RT-PCR with gene-specific priming to examine mRNA levels in various types of clinical samples, which contained slightly or extensively fragmented RNA. A major advantage of this technology is its ability to measure gene expression reliably from fragmented RNA through synthesis of cDNAs with gene-specific primers and use of short amplicons and normalization (15–17). We designed specific primers (S100P forward, 5′-GATGCCGTGGATAAATTGCT-3′ and reverse, 5′-AGGGCATCATTTGAGTCCTG-3′; β-actin forward, 5′-AAATCTGGCACCACACCTTC-3′ and reverse, 5′-GGGGTGTTGAAGGTCTCAAA-3′) and did BLASTN searches to ensure the specificity of each primer. To confirm that the detected signal was specific to the expected PCR product, we did RT-PCR with or without reverse transcription using each primer pair and each type of bulk tissue and then did microchip electrophoresis of the PCR products with the Agilent Bioanalyzer 2100 (Agilent). One-step quantitative real-time RT-PCR with gene-specific priming was done with a QuantiTect SYBR Green RT-PCR kit (Qiagen) with a LightCycler Quick System 350S (Roche Diagnostics). The reaction mixture was first incubated at 50°C for 15 minutes to allow for reverse transcription, where first-strand cDNA was synthesized by priming with a gene-specific primer. PCR was initiated with one cycle of 95°C for 10 minutes to activate modified Taq polymerase followed by 45 cycles of 95°C for 15 seconds, 55°C for 20 seconds, and 72°C for 10 seconds, and one cycle of 95°C for 0 seconds, 65°C for 15 seconds, and +0.1°C/s to 95°C for melting analysis to visualize nonspecific PCR products because different fragments appear as separate distinct melting peaks. Each primer set used in the present study produced a single melting peak and a single prominent band of the expected size on microchip electrophoresis. Each sample was run twice, and any sample showing >10% deviation from the RT-PCR value was tested a third time. The level of expression of the mRNA for each gene was calculated on a standard curve constructed from values for total RNA from the Capan-1 pancreatic cancer cell line. Expression of S100P was normalized to that of β-actin.
Microdissection-based quantitative analysis of S100P mRNA. Frozen tissue samples were cut into 8-μm-thick sections. One section was stained with H&E for histologic examination. IDC cells from 20 sections, PanIN cells from 12 sections, IPMN cells from 19 sections, including 16 IPMAs with mild or moderate atypia and 3 borderline IPMAs with severe atypia, and normal pancreatic ductal epithelial cells from 11 sections were isolated selectively with a laser microdissection and pressure catapulting system (P.A.L.M. Microlaser Technologies, Bernried, Germany) per the protocols of the manufacturer. After microdissection, total RNA was extracted from the selected cells and subjected to real-time RT-PCR for quantitative measurement of S100P as described previously (20, 22).
Statistical analyses. Data were analyzed by the Kruskal-Wallis test if comparisons involved three groups and by the Mann-Whitney U test if comparisons involved two groups because normal distributions were not obtained. Statistical significance was defined as P < 0.05. Because we did multiple comparisons of our real-time RT-PCR data, we conservatively used the Bonferroni correction, and the adjusted significance level was P < 0.017 for the analyses of bulk tissues and pancreatic juice and P < 0.008 for the analyses of microdissected samples. The optimal cutoff points for each marker for discriminating between pancreatic carcinoma or IPMN and chronic pancreatitis were sought by constructing receiver operating characteristic curves, which were generated by calculating the sensitivities and specificities of data for each marker at several predetermined cutoff points with the MedCalc statistical software package, version 7.6 (MedCalc, Mariakerke, Belgium; ref. 23).
Quantitative analysis of S100P mRNA levels in pancreatic cancer, IPMN, and nonneoplastic bulk pancreatic tissues. We measured S100P mRNA levels in pancreatic cancer, IPMN, and nonneoplastic bulk pancreatic tissues. To quantify S100P expression, we used β-actin as the reference gene. The level of expression of S100P normalized to that of β-actin is shown in Fig. 1 . Pancreatic cancer and IPMN bulk tissues expressed significantly higher levels of S100P than did nonneoplastic bulk pancreatic tissues (pancreatic cancer versus nonneoplastic pancreas, P < 0.017; IPMN versus nonneoplastic pancreas, P = 0.0013). S100P expression did not differ significantly between pancreatic cancer and IPMN bulk tissues. However, the median level of expression of S100P in IPMN bulk tissues was greater than that in pancreatic cancer bulk tissues (median, 2.382 versus 1.336, P = 0.27).
S100P expression in pancreatic cancer cell lines and primary cultured pancreatic fibroblasts. Results of the bulk tissue analyses suggested that S100P mRNA is expressed at higher levels in pancreatic cancer and IPMN than in nonneoplastic pancreas and that IPMN expresses similar or higher levels of S100P than pancreatic cancer. However, the bulk tissue samples used in the present study showed remarkable variation in the amount of stromal tissue. Pancreatic cancer is recognized as a desmoplastic tumor that includes abundant stromal components. In addition, we frequently observed histologically chronic pancreatitis in nonneoplastic bulk pancreatic tissues. These pancreatitis-affected lesions often included stromal components. To examine the effect of contamination of fibroblasts in bulk tissues on S100P expression profiles, we established four primary cultures of pancreatic fibroblasts from resected pancreatic tissues and measured S100P in these cells. We then compared S100P expression in these fibroblasts with that in 15 pancreatic cancer cell lines. We detected expression of S100P in all 15 pancreatic cancer cell lines. However, we found no expression of S100P in the four primary cultures of pancreatic fibroblasts examined in this study (Table 1 ). These findings suggest that analysis of bulk tissues that include a large stromal component may yield deceptively low levels of S100P and may not accurately reflect the true level of S100P expression in cancer cells, IPMN cells, and nonneoplastic ductal epithelial cells.
Quantitative analysis of S100P mRNA in IDC cells, PanIN cells, IPMN cells, and normal ductal epithelial cells. To investigate the involvement of S100P expression in carcinogenesis of pancreatic cancer, levels of S100P in PanINs (PanIN 1b and PanIN 2) and IPMN cells were compared with those in normal ductal cells and IDC cells. PanIN is thought to be a common precursor lesion of conventional pancreatic cancer (24). In particular, PanIN 1b and PanIN 2 are precursor lesions observed during the early stage of pancreatic carcinogenesis. IPMN is also a precursor lesion of another subset of pancreatic cancer (25). We used microdissection to isolate normal ductal cells, PanIN cells, IPMN cells, and IDC cells from frozen sections and measured S100P expression in these cells to avoid the effect of contamination by stromal components that we observed in bulk tissue analyses. As shown in Fig. 2 , IPMN cells expressed the highest levels of S100P between the groups (IPMN versus IDC, P < 0.0001; IPMN versus PanIN, P = 0.001; IPMN versus normal duct, P < 0.0001). IDC cells and PanIN cells expressed significantly higher levels of S100P than did normal ductal cells (IDC versus normal duct, P < 0.0001; PanIN versus normal duct, P = 0.0031). S100P expression did not differ between IDC cells and PanIN cells.
Several previous publications have suggested that expression of β-actin is altered in cancer (26). Therefore, to confirm that β-actin is a valid reference gene for normalization of expression, we normalized S100P expression to that of 18S rRNA, which is reported to be stable in pancreatic cancer (26). When S100P was normalized to 18S rRNA, IPMN cells expressed significantly higher levels of S100P than did the other three groups, and these results were similar to those when normalized to β-actin (data not shown).
Quantitative analysis of S100P mRNA levels in pancreatic juice. Before we did pancreatic juice analyses, we examined the integrity of RNA extracted from cell pellets of pancreatic juice. Fragmentation of RNAs from clinical samples, such as pancreatic juice, remains the most critical factor limiting this procedure. As expected, assessment of RNA integrity with the Agilent Bioanalyzer revealed that RNA derived from cell pellets of pancreatic juice was heavily degraded into fragments of <200 nucleotides. As a preliminary study, however, to confirm the effect of degraded RNA on one-step quantitative real-time RT-PCR with gene-specific priming, we used intact and degraded RNAs derived from cell lines and compared the results. We found that δ threshold cycle (Ct) values (difference between Ct of target gene minus Ct of reference gene) were stable, which was consistent with the results of a previously reported study (15).
We measured S100P levels in pancreatic juice samples from a total of 99 patients with different pancreatic diseases (pancreatic carcinoma, n = 32; chronic pancreatitis, n = 32; IPMN, n = 35). As shown in Fig. 3A , levels of S100P in pancreatic cancer and IPMN juice samples were significantly higher than those in chronic pancreatitis juice samples (pancreatic cancer versus chronic pancreatitis, P < 0.0001; IPMN versus chronic pancreatitis, P < 0.0001), whereas there was no significant difference in S100P expression between pancreatic cancer and IPMN juice samples (pancreatic cancer versus IPMN, P = 0.735).
The receiver operating characteristic curves for S100P expression are shown in Fig. 3B. The sensitivity to discriminate pancreatic cancer and/or IPMN from chronic pancreatitis was determined at several specificity levels. The areas under the receiver operating characteristic curve (AUC) for pancreatic cancer and chronic pancreatitis [0.829; 95% confidence interval (95% CI), 0.714-0.911] and for IPMN and chronic pancreatitis (0.824; 95% CI, 0.709-0.908) were significantly greater than those for pancreatic cancer and IPMN (AUC, 0.532; 95% CI, 0.403-0.658; AUC for pancreatic cancer and chronic pancreatitis versus AUC for pancreatic cancer and IPMN, P < 0.001; AUC for IPMN and chronic pancreatitis versus AUC for pancreatic cancer and IPMN, P = 0.001). There was no significant difference between the AUCs of pancreatic cancer and chronic pancreatitis or the AUCs of IPMN and chronic pancreatitis (P = 0.93).
These findings suggest that S100P measurement in pancreatic juice is useful for discriminating pancreatic cancer or IPMN from chronic pancreatitis but not for discriminating pancreatic cancer from IPMN. Results of our microdissection analyses also suggest that cells of premalignant lesions, such as IPMN cells and PanIN cells, express high levels of S100P similar to those of pancreatic cancer cells. Therefore, we created a neoplastic disease group, which included pancreatic cancer and IPMN, and then compared the level of S100P expression in this group with that of the chronic pancreatitis group. The neoplastic disease group showed significantly higher S100P expression than did the chronic pancreatitis group (P < 0.0001; Fig. 3C). The AUC between neoplastic disease and chronic pancreatitis was 0.837 (95% CI, 0.749-0.903). The sensitivity and specificity of S100P measurement for discriminating neoplastic disease from chronic pancreatitis were 83.6% (95% CI, 72.5-91.5) and 75.0% (95% CI, 56.6-88.5), respectively, when the cutoff point was set at 1.181 (Fig. 3C).
We used one-step quantitative real-time RT-PCR with gene-specific priming and successfully analyzed S100P levels in bulk pancreatic tissues, microdissected cells, and 99 pancreatic juice samples from patients with pancreatic cancer, IPMN, or chronic pancreatitis. In bulk tissue analyses, pancreatic cancer and IPMN expressed similar levels of S100P, which were significantly higher than the level in nonneoplastic pancreas. In microdissection analyses, IDC cells, PanIN cells, and IPMN cells expressed significantly higher levels of S100P than did normal ductal cells. In pancreatic juice analyses, pancreatic cancer and IPMN juice samples expressed significantly higher levels of S100P than did chronic pancreatitis juice samples. These data suggest that S100P expression is up-regulated during the early stage of carcinogenesis of pancreatic cancer.
Pancreatic cancer has a 5-year survival rate of <5% because the disease is typically detected at an advanced stage (27). Therefore, a method for early detection of this disease is urgently needed. Testing of pancreatic juice samples with RT-PCR to screen for changes in expression of marker transcripts is a promising diagnostic strategy for identification of persons at high risk for pancreatic cancer; however, this methodology is limited by fragmentation of RNAs from clinical samples, such as pancreatic juice. Degradation of the RNA can adversely affect the reverse transcription step. Loss of polyadenylic acid tails is the main cause of failure of the reverse transcription step with a poly-T oligomer, which is traditionally used (17). Recently, Lekanne Deprez et al. (16) also reported that gene-specific priming was most efficient for any cDNA synthesis conditions. More recently, Antonove et al. (15) reported that gene expression could be measured reliably from degraded RNA by quantitative real-time RT-PCR using short amplicons (below 200 bp) and normalization. Therefore, we used one-step quantitative real-time RT-PCR with gene-specific priming, short amplicons, and normalization in the present study and successfully measured the levels of S100P in various types of clinical samples, such as pancreatic juice.
To date, no single biomarker has been proven to have sufficient diagnostic accuracy so as to provide a stand-alone means for early detection and/or diagnosis of cancer. Thus, it is unlikely that a diagnosis and follow-up therapeutic plan would be made on the basis of a single test result. In addition, the sensitivity and specificity and AUC of pancreatic juice analyses in the present study do not seem to be high enough to distinguish pancreatic cancer or neoplastic disease from nonneoplastic disease. Because our S100P mRNA analyses with pancreatic juice yielded statistically significant results in the present study, such analysis in combination with other clinical data may aid in differential diagnosis, detection, or screening for pancreatic cancer.
In the present study, we analyzed whole-cell pellets from pancreatic juice, which contained various types of cells, including cancer cells, atypical cells, and nonneoplastic epithelial cells. Microdissection to isolate target cells in pancreatic juice may improve the diagnostic accuracy and/or sensitivity of our assay. Such studies are presently under way in our laboratory. Furthermore, Arumugam et al. (11) suggested that S100P is released from cells and acts extracellularly. Several researchers previously reported that S100P mRNA and protein expression were well correlated in pancreatic carcinogenesis (3, 5, 13, 14). Therefore, S100P protein in pancreatic juice is also a potential diagnostic marker. In the present study, all pancreatic juice samples were subjected to cytologic examination. The smear cytologic samples from patients with IPMN or pancreatic cancer typically contain more epithelial cells than do those from patients with other diseases, such as chronic pancreatitis, revealing that cellularity in IPMN and pancreatic cancer juice samples is increased. In our assay, we normalized S100P mRNA expression to that of a reference gene to control for differences in the amount or quality of total RNA isolated from different pellets. Therefore, increased cellularity of IPMN or pancreatic cancer cells in pancreatic juice was not reflected in the present results. Measurement of S100P protein in pancreatic juice may be affected by such increased cellularity of IPMN and pancreatic cancer cells. However, if an appropriate method for normalization is used, it is a possible that levels of S100P protein in pancreatic juice would be increased more significantly than those of the mRNA and might more accurately reflect cellular changes. In conclusion, results of our expression analyses suggest that S100P is an early developmental marker of pancreatic carcinogenesis and that quantification of S100P in pancreatic juice may have some advantages in detecting or screening for early pancreatic cancer and in following up individuals with high-risk factors for pancreatic cancer.
Grant support: Grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists; and the Japanese Foundation for Research and Promotion of Endoscopy Grant.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received February 7, 2006.
- Revision received May 17, 2006.
- Accepted June 5, 2006.