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Keywords:

  • MUC1;
  • MUC5AC;
  • pancreatic cancer;
  • pancreatic juice

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Pancreatic juice is a promising type of diagnostic sample for pancreatic cancer, and members of the mucin (MUC) family are diagnostic candidates. To evaluate the utility of MUC family members as diagnostic markers, we measured MUC mRNA expression in pancreatic tissues and pancreatic juice obtained from patients with different pancreatic diseases as well as in pancreatic cancer cell lines by real-time PCR. Furthermore, to support the possibility of early diagnosis by quantification of MUC1 and MUC5AC, immunohistochemistry and microdissection-based quantitative analysis of mRNA were carried out. There was no significant correlation between MUC1 and MUC5AC expression in cell lines. When β-actin was used as a reference gene, median MUC1 and MUC5AC mRNA expression levels were remarkably greater in tumoral tissues than in non-tumoral tissues, but median MUC4 and MUC6 mRNA expression levels were not. Receiver operating characteristic curve analysis showed that quantitative analysis of MUC1 and MUC5AC mRNA in pancreatic juice is better diagnostic modality than that of MUC4 and MUC6 mRNA. Immunohistochemistry showed that MUC1 and MUC5AC were highly expressed in invasive ductal carcinomas (IDC) and moderately expressed in high-grade pancreatic intraepithelial neoplasia (PanIN); no staining was observed in normal ducts. Analysis of cells isolated by microdissection showed stepwise upregulation of MUC1 and MUC5AC in the development of high-grade PanIN to IDC. Our results suggest that MUC1 and MUC5AC are upregulated stepwise in pancreatic carcinogenesis and that quantitative assessment of MUC1 and MUC5AC mRNA in pancreatic juice has high potential for preoperative diagnosis of pancreatic cancer. © 2005 Wiley-Liss, Inc.

Pancreatic cancer is the fifth leading cause of cancer death and has the lowest survival rate of any solid cancer.1, 2 Curative resection is the only way to improve prognosis; other conventional therapeutic approaches, including chemotherapy and radiotherapy, show limited efficacy.3, 4 Despite improvements in diagnostic imaging, diagnosis before surgery remains difficult due to the inaccessibility of the organ and the highly malignant nature of the disease. The vast majority of patients with pancreatic cancer suffer a poor clinical course. There are 2 difficulties in preoperative diagnosis: distinguishing pancreatic carcinoma from other disorders such as pancreatitis and detecting it early enough to perform curative resection.

Endoscopic retrograde cholangiopancreatography (ERCP) combined with cytology of pancreatic juice is currently used as a minimally invasive diagnostic tool to distinguish pancreatic cancer from other disorders.5, 6 The sensitivity of cytology is not satisfactory, however, varying from 30–80%.7, 8 It has also been reported that detection of mutations in K-ras, p53 and other oncogenes in pancreatic juice is useful for preoperative diagnosis.9, 10, 11, 12 K-ras mutation occurs at a specific codon (codon 12 or 13), whereas mutations in other oncogenes, such as p53, occur as numerous hot spots. Detection of these mutations may be too complex and time-consuming to introduce into clinical practice. Detection of K-ras mutations has been used in a large clinical study.13 The sensitivity of detection of ras mutation in pancreatic juice was 38.1%,13 however, which is inconsistent with previous reports describing a sensitivity of 79–90%.9, 10, 11, 12 These conflicting data indicate the difficulty of introducing assays for the detection of DNA mutations into clinical practice. Novel biomarkers or diagnostic strategies are needed urgently to distinguish pancreatic cancer from other disorders preoperatively and, if possible, to detect early pancreatic cancer.

Mucins (MUC) are high molecular weight glycoproteins. Fourteen mucin genes have been described currently, and 8 are now well characterized, including MUC 1–4, MUC5B, MUC5AC, MUC6 and MUC7.14, 15, 16 Alterations in the expression of MUC have been reported in both pre-neoplastic and neoplastic lesions.17, 18, 19 In addition, recent studies have reported that MUC are expressed differently between non-neoplastic and neoplastic pancreatic lesions. Overexpression of MUC1 and MUC6 and de novo expression of MUC4 and MUC5AC in pancreatic cancer have been observed at RNA and protein levels.18, 20, 21, 22, 23 DNA microarray technology has been applied to pancreatic cancer and has shown that MUC4 and MUC5AC are upregulated in pancreatic cancer compared to levels in normal pancreatic tissues.24, 25 Thus, MUC are promising diagnostic markers for pancreatic cancer.

In our study, we measured the expression of MUC family mRNA in pancreatic cell lines, tissues and juice quantitatively by real-time PCR. MUC1 and MUC5AC mRNA levels were significantly increased in tumoral tissues of pancreas and in pancreatic juice from patients with pancreatic cancer. The preoperative diagnostic abilities of quantitative analysis of MUC1, MUC4, MUC5AC and MUC6 mRNA in pancreatic juice were compared by evaluation of receiver operating characteristic (ROC) curves.26 In addition, to investigate whether quantification of MUC1 and MUC5AC in pancreatic juice is useful for detection of early pancreatic cancer, immunohistochemistry (IHC) studies and microdissection-based quantitative analysis of mRNA were carried out on samples of normal ducts, high-grade pancreatic intraepithelial neoplasia (PanIN) and invasive ductal carcinomas (IDC).

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell lines, pancreatic tissues and pancreatic juice

The following 15 pancreatic cancer cell lines were used: 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 (Japanese Cancer Resource Bank, Tokyo, Japan), NOR-P1 (established at our laboratory), Capan-1, Capan-2, CFPAC-1, H48N, HS766T and SW1990 (American Type Culture Collection, Manassas, VA). A human pancreatic duct epithelial cell line (HPDE) immortalized by transduction with the E6/E7 genes of human papilloma virus 16 was kindly provided by Dr. M.-S. Tsao (University of Toronto, Canada). Cells were maintained as described previously.27, 28 Tissue samples were obtained at the time of surgery at Kyushu University Hospital, Fukuoka, Japan. Thirty-two tumoral tissue samples were obtained from cancerous lesions of resected pancreases with primary pancreatic ductal carcinoma, and 42 non-tumoral tissue samples were taken from the peripheral soft tissue away from the tumors. The tissue samples were removed as soon as possible after resection and divided into more than 3 large tissue samples. The first samples were stored at −80°C for analysis of whole tissue samples. The second samples were embedded in OCT compound (Sakura, Tokyo, Japan) and snap-frozen for analysis by microdissection. The third samples were fixed in formalin, embedded in paraffin, and cut into 4-μm thick sections for IHC or hematoxylin and eosin (H&E) staining. All tissues adjacent to the specimens were examined histologically, and the diagnosis was confirmed. Pancreatic juice samples were collected from 63 patients who underwent ERCP for suspected malignancy of the pancreas at Kyushu University Hospital from January 1, 2002, to October 31, 2004, as described previously.29, 30 We used pellets of cellular material from pancreatic juice for preparation of RNA.

The diagnosis of pancreatic ductal adenocarcinoma was confirmed by histologic examination of resected specimens where they were available. In inoperable cases, clinical diagnosis was made on the basis of imaging findings. The diagnosis of pancreatitis or cholelithiasis was made on the basis of either histologic examination of resected specimens or clinical observation with conventional diagnostic imaging for at least 6 months. Written informed consent was obtained from all patients, and the study was conducted according to the Helsinki Declaration.

Quantitative assessment of MUC levels by real-time PCR

We designed a real-time PCR protocol for the quantitative analysis of MUC family members (MUC1, MUC4, MUC5AC and MUC6) and housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin). We designed specific primers (MUC1 forward primer, 5′-agacgtcagcgtgagtgatg-3′; reverse primer, 5′-gacagccaaggcaatgagat-3′; MUC4 forward primer, 5′-gggaagaaaggcccaactac-3′; reverse primer, 5′-ctatgctgacgggttggaat-3′; MUC5AC forward primer, 5′-ccttcgacggacagagctac-3′; reverse primer, 5′-tctcggtgacaacacgaaag-3′; MUC6 forward primer, 5′-ccaatccccaagctcattta-3′; reverse primer, 5′-tggtgcctgtactggtgtgt-3′; GAPDH forward primer, 5′-caatgaccccttcattgacc-3′; reverse primer, 5′-gatctcgctcctggaagatg-3′; and β-actin forward primer, 5′-aaatctggcaccacaccttc-3′; reverse primer, 5′-ggggtgttgaaggtctcaaa-3′), carried out BLAST searches to ensure the gene specificity of these primers. Quantitative RT-PCR was carried out with a Quantitect SYBR Green RT-PCR kit (QIAGEN, Tokyo, Japan) and a LightCycler Quick System 350S (Roche Diagnostics, Mannheim, Germany), as described previously.31 In brief, the total volume of the reaction mixture was 20 μl, and it contained 10 μl of 2× SYBR Green Buffer, 0.2 μl of RT Mix, 1 μl of each primer (10 μM) and 1 μl of total RNA (0.01 μg/μl). The reaction mixture was first incubated at 50°C for 15 min to allow for reverse transcription. PCR was then initiated at 95°C for 10 min to activate modified Taq polymerase, followed by a 45-cycle amplification (95°C for 15 sec, 55°C for 20 sec and 72°C for 10 sec) and 1 cycle (95°C for 0 sec, 65°C for 15 sec and 0.1°C/sec to 95°C) for melting analysis. Each sample was run twice. In addition, all samples showing over 10% deviation in their values were tested in a third run. Deviation in concentration was calculated after use of the calibration curve. The mRNA expression of each gene was calculated on a standard curve constructed with the use of total RNA from the Capan-1 pancreatic cancer cell line. The threshold for observation of product ranged from 20–33 cycles for MUC1 and MUC4, 20–35 cycles for MUC5AC, 20–31 cycles for MUC6 and 15–30 cycles for β-actin and GAPDH primers. For relative quantification, the expression of each gene was normalized against that of the indicated housekeeping gene and expressed as the ratio of relative expression of each target gene mRNA to that of each housekeeping gene mRNA.

IHC studies

Tissue sections of formalin-fixed, paraffin-embedded specimens were deparaffinized in xylene and rehydrated in graded alcohols. Sections to be analyzed for MUC1 and MUC5AC staining were microwaved in 1 mM EDTA, pH 8.0, for 10 min, endogenous peroxidase was blocked with 3% hydrogen peroxide in PBS, nonspecific binding was blocked by a 20 min incubation in protein blocking solution consisting of PBS plus 1.5% normal goat serum (DAKO, Glostrup, Denmark), and then the sections were incubated with the appropriate dilution of mouse monoclonal MUC1 or MUC5AC antibody (Novocastra Laboratories, Ltd., Newcastle upon Tyne, UK) overnight at 4°C. The sections were then incubated in the appropriate dilution of biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA) for 30 min with a Vectastain Elite ABC kit (Vector Laboratories). Positive reactions were visualized by incubation of the slides with stable 3,3′-diaminobenzidine tetrahydrochloride (Dojindo laboratories, Kumamoto, Japan). The sections were rinsed with distilled water and counterstained with hematoxylin for 10 sec.

Evaluation of antibody reactivity

The degree of monoclonal anti-MUC1 or anti-MUC5AC reactivity in each tissue section was scored by the percentage of stained normal or neoplastic epithelial cells in the section (0 points for no staining, 1 point for <20% of cells staining, 2 points for 20–75% of cells staining and 3 points for >75% of cells staining), and the intensity of immunoreactivity was graded on a scale from 0–3. The total score was obtained as the product of intensity and extent of staining. Negative sections had a score of 0, weakly positive sections had a score of 1–3, moderately positive sections had a score of 4–6 and strongly positive sections had a score >6. Experienced pathologists carried out this evaluation.

Microdissection-based quantitative analysis of mRNA

Frozen tissue samples were cut into 8-μm thick sections. One section was stained with H&E for histologic examination. IDC cells, PanIN cells and normal pancreatic ductal epithelial cells were isolated selectively using laser microdissection and a pressure catapulting system (LMPC; Palm Microlaser Technologies AG, Bernried Germany) in accordance with the manufacturer's protocols. After microdissection, total RNA was extracted from the selected cells as described previously32 and was subjected to real-time PCR for quantitative measurement of MUC1 and MUC5AC.

Statistical analysis

Data were analyzed by Mann-Whitney U-test and Spearman rank correlation test because normal distribution was not obtained. Statistical significance was defined as <0.05, but because we carried out multiple comparisons on our real-time PCR data of 4 genes in whole tissue and pancreatic juice, Bonferroni's correction was used; thus, the adjusted significance level was <0.0125 in the analyses of pancreatic whole tissue and juice. The optimal cut-off points for each marker for discriminating between pancreatic carcinoma and other benign diseases were sought by constructing ROC curves, which were generated by calculating the sensitivities and specificities of each marker at several predetermined cut-off points.33

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Comparison of expression of MUC mRNA in pancreatic cancer cell lines and in HPDE

We measured MUC1, MUC4, MUC5AC and MUC6 mRNA levels in 15 pancreatic cancer cell lines and in HPDE cells by real-time PCR. Expression profiles of MUC mRNA in pancreatic cancer cell lines and HPDE are shown in Table I. In HPDE, only MUC1 mRNA was detectable. In all pancreatic cancer cell lines except Suit-2, the expression of MUC1 mRNA was greater than that in HPDE. The expression of MUC4 mRNA was detectable and was measured quantitatively in 11 pancreatic cancer cell lines. The expression of MUC5AC mRNA was detectable and was measured quantitatively in 13 pancreatic cancer cell lines. The expression of MUC6 mRNA was detectable and was measured quantitatively in all pancreatic cancer cell lines except NOR-P1. There was no significant correlation between MUC1 and MUC5AC mRNA levels in these pancreatic cancer cell lines (Fig. 1; p = 0.4749). We found no significant correlation between any other genes examined. (data not shown). These data suggest that the genes examined in our study may be independent markers for diagnosis of pancreatic cancer.

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Figure 1. No correlation between MUC1 and MUC5AC mRNA levels in 16 cell lines. The relative expression of MUC1 and MUC5AC in 15 pancreatic cell lines and HPDE was normalized to β-actin. There was no significant correlation between the expression of MUC1 and MUC5AC.

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Table I. Expression Profiles Of MUC mRNAs in Pancreatic Cancer Cell Lines and HPDE1
 MUC1MUC4MUC5ACMUC6
  • 1

    Expression of each gene was normalized to that of beta-actin.–

  • 2

    Values are expressed relative to 1.000 for expression in Capan-1 cells. HPDE, human pancreatic ductal epithelial cell line; ND, not detected.

HPDE0.862NDNDND
Panc-15.62ND0.0900.517
MIA PaCa-24.250.3940.0070.181
ASPC-133.780.3950.0030.930
Suit-20.180.0830.0120.269
SW199013.10ND0.0370.658
KP-1N1.380.5500.0165.360
BxPC33.801.4360.0221.217
Capan-22.420.7431.0240.991
CFPAC48.000.6230.7110.140
KP-3130.380.939ND3.149
NOR-P19.16NDNDND
H48N8.1020.1250.2132.785
HS766T1.00ND0.7710.301
KP-28.250.5020.0261.276
Capan-11.001.0001.0001.000

Notably, relative MUC mRNA expression levels were generally considerably lower in cultured cells than in tissues or juice samples (Table I, Fig. 2b, Fig. 3a). This may be due to differences in β-actin mRNA expression between cultured cells and cells derived from tissue or juice. Several studies have suggested that β-actin expression is affected by cell cycle and experimental conditions.34, 35, 36 Cultured cells are highly proliferative compared to primary tumors. Therefore, when normalized to β-actin, relative expression of target mRNA may decrease in cultured cells compared to that in tissues.

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Figure 2. Relative expression of MUC in tumoral and non-tumoral whole tissues. (a) There was a significant difference in the relative expression of MUC1 after Bonferroni's correction but not of MUC4, MUC5AC or MUC6 between tumoral and non-tumoral tissues when GAPDH was used as the reference gene. (b) When β-actin was used as the reference gene, the median relative expression of MUC1 and MUC5AC was 7.2- and 2.8-fold greater in tumoral tissues, respectively, than in non-tumoral tissues, whereas those of MUC4 and MUC6 in tumoral tissues was similar to that in non-tumoral tissues. *p < 0.05; **p < 0.0125.

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Figure 3. Relative expression of MUC in pancreatic juice normalized to β-actin and ROC curve analysis. (a) The relative expression of MUC1, MUC4, MUC5AC and MUC6 in pancreatic juice was normalized to β-actin expression. There were significant differences in the expression of MUC1 and MUC5AC between carcinoma juice and non-neoplastic samples after Bonferroni's correction (**p < 0.0125). (b) The sensitivity of each marker was determined at several specificity levels. ROC curve analysis showed that the discriminative abilities of MUC1 and MUC5AC were greater than that of MUC4 or MUC6.

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Quantitative analysis of MUC mRNA in tumoral and non-tumoral pancreatic tissues

We measured MUC1, MUC4, MUC5AC and MUC6 mRNA levels in tumoral and non-tumoral pancreatic whole tissues. To quantify target gene expression, 2 major housekeeping genes (GAPDH and β-actin) were used for relative quantification. The results of relative quantification normalized to the indicated housekeeping genes are shown in Figure 2a (GAPDH) and Figure 2b (β-actin). When normalized to GAPDH, a significant difference between tumoral and non-tumoral tissues was found only in MUC1 mRNA levels (Fig. 2a; p = 0.0017). When normalized to β-actin, median expression levels of MUC1 and MUC5AC mRNA were 7.2- and 2.8-fold greater in tumoral tissues, respectively, than in non-tumoral tissues, although the difference in MUC5AC mRNA expression levels was not significant after Bonferroni's correction (level of significance adjusted to p < 0.0125) (Fig. 2b; p = 0.0033 for MUC1, p = 0.0223 for MUC5AC). Schek et al.37 and Crnogorac-Jurcevic et al.38 also reported that the expression of GAPDH increases in pancreatic cancer. These data suggest that β-actin may be a better reference gene than GAPDH for diagnosis by quantification of MUC mRNA in pancreatic tissues. Therefore, in subsequent analyses of pancreatic juice, β-actin was used as the reference gene.

There were no significant differences in MUC4 and MUC6 mRNA levels between tumoral and non-tumoral tissues (p = 0.5385 for MUC4, p = 0.5858 for MUC6, even when normalized to β-actin). This was inconsistent with previously published findings.18, 20 Swartz et al.39 and other investigators18, 20, 40 reported detection of MUC4 or MUC6 expression in PanIN, reactive epithelium, squamous metaplasia or mononuclear cells. In our present study, we investigated H&E staining of adjacent tissue of non-tumoral tissues and found reactive epithelial change or appearance of mild pancreatitis with mononuclear cells in some samples, possibly due to sampling from site distal to the tumor. It is also possible that these large samples included PanIN lesions. In addition, the tumoral samples used in our present study showed remarkable variation in the amount of stromal tissue present in the tumors. As described previously,20 this variation affects relative mRNA expression levels in tumoral samples. The use of whole tissues or bulky tumors for analysis may contribute to conflicting results between our present study and previous studies.

Quantitative analysis of MUC1, MUC4, MUC5AC and MUC6 mRNA expression in pancreatic juice

We measured MUC1, MUC4, MUC5AC and MUC6 in 63 pancreatic juice samples, including samples from 24 pancreatic carcinomas and 39 pancreases with non-neoplastic disease (pancreatitis, n = 33; cholelithiasis, n = 6). The amount of total RNA extracted from pancreatic juice samples was too small to measure quantitatively because few cells are present in pancreatic juice. Therefore, an adequate reference gene was necessary for relative quantification of target gene expression. Based on our present analysis of tissue samples, the β-actin gene was used as the reference gene for relative quantification. Relative expression levels of MUC1 and MUC5AC were significantly greater in carcinoma samples than in pancreatitis-affected or cholelithiasis-affected samples after Bonferroni's correction (Fig, 3a; p < 0.0001 for MUC1, p < 0.0001 for MUC5AC). There were, however, no significant differences in relative MUC4 and MUC6 expression between carcinoma juice and pancreatitis-affected or cholelithiasis-affected samples after Bonferroni's correction (p = 0.0185 for MUC4, p = 0.0304 for MUC6).

The ROC curves for MUC 1, MUC4, MUC5AC and MUC6 mRNA expression are presented in Figure 3b. The sensitivity of each marker was determined at several specificity levels. The area under the curve (AUC) was 0.864 for MUC1 (95% confidence interval [CI] = 0.748–0.939) and 0.762 for MUC5AC (95% CI = 0.632–0.864). For MUC4, the AUC was 0.686 (95% CI = 0.550–0.801), and for MUC6, the AUC was 0.646 (95% CI = 0.510–0.767). A significant difference between AUC for MUC1 and MU6 was observed (difference between areas, 0.217; 95% CI = 0.048–0.386; p = 0.012). These data showed that the discriminative abilities of MUC1 and MUC5AC were greater than that of MUC4 or MUC6.

In our study, cytologic Class IV or V41 was considered positive for a diagnosis of malignancy. The cytologic sensitivity for diagnosis of pancreatic cancer was only 22.0% (95% CI = 14.7–29.3), which was similar to that cited in previous reports,29 although the specificity was 100%. ROC curve analyses showed the sensitivity and specificity of MUC1 analysis to be 41.7% (95% CI = 22.1–63.3) and 97.4% (95% CI = 86.5–99.6), respectively, when the cut-off point was set at 264.18. MUC5AC analysis showed similar sensitivity and specificity when the cut-off point was set at 82.33. These data suggest that analysis of MUC1 and MUC5AC may offer some advantage over cytology alone.

IHC studies and microdissection-based quantitative analysis of mRNA shows differential expression of MUC1 and MUC5 AC in IDC, high-grade PanIN and normal ducts

To clarify whether quantification of MUC1 and MUC5AC in pancreatic juice may lead to early detection of pancreatic cancer, the expression of protein and mRNA levels of these genes in high-grade PanIN was compared to those in normal ducts or IDC. Representative IHC data obtained with the anti-MUC1 and anti-MUC5AC antibodies are shown in Figure 4. Strong MUC1 and MUC5AC staining was identified in 52.2% and 48.3% of IDC, respectively, whereas in >70% of normal ducts, no expression of either gene was identified (Tables II, III). Importantly, most high-grade PanIN showed weak or modest expression of both genes.

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Figure 4. IHC studies. Immunoreactivity for MUC1 (a–c) and MUC5AC (d–f). Representative images of IDC (a,d), high-grade PanIN (b,e) and normal ducts (c,f) (original magnification = 400×). Differential expression of MUC1 and MUC5AC protein levels was found among IDC, high-grade PanIN and normal ducts.

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Table II. MUC1 Immunoreactivity in Pancreatic Tissue
 Normal (n = 27)PanINs (n = 12)IDC (n = 23)
Negative20 (74.1%)1 (8.3%)0 (0%)
Weak5 (18.5%)9 (75%)1 (4.3%)
Moderate2 (7.4%)2 (16.7%)10 (43.5%)
Strong0 (0%)0 (0%)12 (52.2%)
Table III. MUC5AC Immunoreactivity in Pancreatic Tissue
 Normal (n = 29)PanIN (n = 11)IDC (n = 29)
Negative24 (82.8%)0 (0%)0 (0%)
Weak4 (13.8%)4 (36.4%)5 (17.2%)
Moderate1 (3.4%)7 (63.6%)10 (34.5%)
Strong0 (0%)0 (0%)14 (48.3%)

By microdissection, normal duct cells, PanIN cells and IDC cells were isolated from frozen sections and were subjected to quantification of MUC1 or MUC5AC mRNA. As with the protein levels observed in the IHC study, MUC1 and MUC5AC mRNA were expressed differentially in normal duct cells, PanIN cells and IDC cells (Fig. 5a,b). In particular, there were significant differences in MUC5AC mRNA levels among normal duct cells, PanIN cells and IDC cells (p = 0.0248 between PanIN cells and IDC cells; p = 0.006 between PanIN cells and normal duct cells).

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Figure 5. Microdissection-based quantitative analysis of MUC5AC (a) and MUC1 (b) mRNA. By microdissection, IDC cells, high-grade PanIN cells and normal ductal cells were isolated from frozen sections, and total RNA extracted from these cells was subjected to quantitative analysis of MUC1 and MUC5AC mRNA by real-time PCR. Differential expression of MUC1 and MUC5AC mRNA levels was found among IDC, high-grade PanIN and normal ducts.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This is the first report of quantitative analysis of MUC1 and MUC5AC mRNA levels in pancreatic juice for preoperative diagnosis of pancreatic cancer. In our present study, ROC curve analyses of MUC1 and MUC5AC mRNA showed the usefulness of quantifying these 2 markers for diagnosis of pancreatic cancer and showed some advantage over cytologic examination alone. In addition, in support of the possibility that quantification of MUC1 and MUC5AC may lead to the detection of early pancreatic cancer, IHC and microdissection-based quantitative analysis of mRNA showed that MUC1 and MUC5AC are differentially expressed in normal ducts, high-grade PanIN and IDC.

A cut-off point with high values can be selected if high specificity is needed, although sensitivity decreases. If pancreatic cancer is diagnosed, the patient may require quite invasive surgery compared to other disease. Therefore, the diagnosis of pancreatic cancer should be of high specificity. In this respect, quantitative analysis of MUC genes may be superior to qualitative analysis of DNA markers.

In addition, in comparison to DNA mutation assays, quantitative analysis of mRNA with real-time PCR is much more suitable to clinical practice due to its simplicity and rapidity. We can also quantify multiple target genes at a time with real-time PCR, suggesting that this analysis is also suitable for the evaluation of aberrant mRNA expression during pancreatic carcinogenesis, possibly leading to improved diagnostic accuracy. In comparison to analysis of cell pellets, microdissection to isolate target cells in pancreatic juice will make it much easier to detect differential expression between non-malignant and malignant cells as in our present study, leading to decreased overlap between cancer and control samples. Microdissection studies are currently in progress in our laboratory.

Early diagnosis is necessary to improve the prognosis of patients with pancreatic cancer. Maitra et al.42 and Kim et al.18 carried out IHC and reported that overexpression of MUC1 and de novo expression of MUC5AC were observed at all stages of PanIN and invasive ductal adenocarcinoma, which was confirmed by our present IHC and microdissection-based quantitative mRNA analysis. Notably, however, our present data showed differential expression of MUC1 and MUC5AC between normal ducts, high-grade PanIN and IDC, suggesting that alteration of MUC1 and MUC5AC expression may be an early event in pancreatic carcinogenesis and that expression of these genes may increase stepwise with disease progression. Therefore, accurate quantitative, not qualitative, analysis of MUC1 and MUC5AC mRNA levels in pancreatic juice may provide for the discrimination of early pancreatic cancer from premalignant lesions.

In our study, most of the samples were from patients with advanced cancer (Stages III or IV). Only one tumoral tissue sample and one pancreatic juice sample were obtained from 2 separate patients with Stage II pancreatic cancer. Therefore, we could not show the clear evidence of early diagnosis in the present study. From a clinical standpoint, however, we need not only early detection of pancreatic cancer but also differential diagnosis from other diseases. Even advanced pancreatic cancer is occasionally difficult to distinguish from chronic pancreatitis. The results of analysis of pancreatic juice in our study suggest the usefulness of quantitative analyses of MUC1 and MUC5AC for discrimination of advanced pancreatic cancer from non-neoplastic lesions including chronic pancreatitis.

In conclusion, our data suggest that quantitative analysis of MUC1 and MUC5AC mRNA levels in pancreatic juice is a promising approach for preoperative diagnosis of pancreatic cancer and possibly for diagnosis of early pancreatic cancer.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We are grateful to M. Ohta (Department of Clinical Pathology, Kyushu University) for skillful cytologic examination and analysis.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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