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Frequent deletions of tumor suppressor genes in pure pancreatic juice from patients with tumoral or nontumoral pancreatic diseases
Original Paper Received: March 29, 2001 Accepted: August 10, 2001
Pancreatology 2002;2:17–25
Frequent Deletions of Tumor Suppressor Genes in Pure Pancreatic Juice from Patients with Tumoral or Nontumoral Pancreatic Diseases Lydie Costentin a, b Philippe Pagès a, b Michèle Bouisson a Philippe Berthelémy a Louis Buscail a, b Jean Escourrou a, b, Lucien Pradayrol a Nicole Vaysse a a INSERM,
U531 Biologie et Pathologie Digestive et b Département de Gastroentérologie, CHU Rangueil, Toulouse, France
Key Words p16 W DPC4 W Pancreatic juice W Pancreatic cancer W Chronic pancreatitis
Abstract Background/Aims: K-ras codon 12 mutation is the most frequent genetic alteration in pancreatic cancer. Sensitivity and specificity of K-ras are not high enough to detect all pancreatic cancers, especially at early stage. This study investigated whether detection of p16 and/or DPC4 deletions along with K-ras mutation in DNA samples could improve the definition of patients at risk of pancreatic cancer. Methods: K-ras mutations were investigated by sequencing. p16 and DPC4 homozygous deletions were studied using comparative multiplex polymerase chain reaction of DNA in pancreatic juice sampled during endoscopic retrograde pancreatography in 57 patients with either pancreatic cancer (group I, 18 patients), chronic pancreatitis (group II, 20 patients), or nontumoral pancreatobiliary disease (group III, 19 patients). Results: The frequencies of Ki-ras mutations were 61% in group I, 10% in group II, and 10.5% in group III. The frequencies of p16 exon 2 and DPC4 deletions were, respectively, 28 and 36% in group I, 50 and 58% in group II, and 24 and 36% in group III. Conclusions: The combi-
ABC
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nation of p16 and DPC4 deletions with K-ras mutation does not improve the diagnosis of pancreatic cancer based on K-ras mutation alone. These data suggest that tumor suppressor gene inactivation can occur with a high frequency during nonmalignant pancreatic diseases. Copyright © 2002 S. Karger AG, Basel and IAP
Introduction
Pancreatic adenocarcinoma is characterized by an aggressive clinical course distinct from that observed in many other human carcinomas. Enhanced understanding of the molecular genetic events occurring during neoplastic progression in the pancreas could lead to earlier diagnosis and treatment improvement. Activating mutation in codon 12 of the K-ras protooncogene is the most common genetic abnormality described in pancreatic carcinoma patients. It is considered an initial molecular event which can be identified in pancreatic fine-needle aspirate biopsy specimens or in duodenal juice or stools of patients with pancreatic cancer (PC) [1–7]. It could be a good tool for detecting PC at an early stage. However, K-ras mutations have been found in pancreatic ductal lesions that do not harbor any malignancy
Nicole Vaysse INSERM U 531, IFR 31, Bât L3, CHU Rangueil 1, avenue J.-Poulhès F–31403 Toulouse Cedex 4 (France) Tel. +33 561 322 402, Fax +33 561 322 403, E-Mail [email protected]
Table 1. Determination of K-ras codon 12 mutation, p16 exon 2 deletion, and DPC4 exons 8–11 in pure pancreatic juice from patients with
PC (group I) Patient No.
Age years
Sex
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
72 85 60 51 73 65 64 71 46 65 64 71 45 74 72 84 60 69
female male male male female male male female male male female female male male male male male female
K-ras
p16
DPC4
RFLP
sequencing
exon 2
exon 8
exon 9
exon 10
exon 11
– + – + + – + + – – + + + + + + – –
wt GTT wt GCT GAT wt GAT GAT wt wt GAT GAT GTT CGT GAT GTT wt wt
wt wt wt wt HD wt wt wt wt wt wt wt wt wt HD HD HD HD
wt wt HD wt wt wt wt wt HD HD HD wt wt HD n.d. n.d. n.d. n.d.
wt wt HD wt wt wt wt wt HD HD HD wt wt HD n.d. n.d. n.d. n.d.
wt wt HD wt wt wt wt wt HD HD HD wt wt HD n.d. n.d. n.d. n.d.
wt wt wt wt wt wt wt wt wt wt wt wt wt wt n.d. n.d. n.d. n.d.
Follow-up period months 7† 1† lost 9† 13† 7† 3† 13† 23† 3† 1† 11† 19† 15† 12† 4† 7† 1†
HD = Homozygous deletion; n.d. = not determined for lack of material; polymerase chain reaction mediated RFLP analysis indicated K-ras wild-type (wt; –) or mutant (+) gene fragments.
[8–10]. Therefore, this molecular marker cannot be used as the only genetic marker of pancreatic ductal adenocarcinoma. Among other genetic anomalies found in PC, deletions of p16 and DPC4 and alterations of p53 are most frequently observed [11]. p16 is a tumor suppressor gene originally identified by Serrano et al. [12]. It inhibits cyclin D/CDK4 protein kinase, a key regulator of the progression of eukaryotic cells through the G1 phase of the cell cycle. Inactivation of p16 may cause abnormal cell cycling and uncontrolled cell growth [13, 14]. The p16 gene is homozygously deleted in 40% of the pancreatic carcinomas, and a similar proportion of small mutations affecting the coding sequence is observed [2, 14]. Methylation associated with silencing of p16 expression can also be found accounting for the remaining cases of PC [15]. The putative tumor suppressor gene DPC4 is involved in another tumor-suppressive pathway. It is located on chromosome 18q21 and shows homology to the Mad family of proteins which play a critical role in transforming growth factor beta superfamily signaling [16]. Homozygous deletions of DPC4 have been reported in approxi-
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Pancreatology 2002;2:17–25
mately 30% of the PCs. Sequencing of DNA derived from pancreatic carcinomas revealed that 20% of the cases with concomitant allelic loss of the DPC4 region had somatically acquired DPC4 point mutations. The biallelic inactivation of the DPC4 gene was shown in approximately 50% of the PCs [17]. p53 is altered in about 50% of the PCs. Homozygous deletions are rather common, but several mutations could be located in different exon [11]. A subset of patients with chronic pancreatitis have already been shown to carry both K-ras and p53 mutations. This study aimed at investigating whether investigation of tumor suppressor genes along with that of K-ras could improve the differential diagnosis between PC and chronic pancreatitis. Since pancreatic juice samples are readily available during endoscopic retrograde pancreatography (ERP) investigation, allowing gene studies [18], we performed a joint analysis of K-ras codon 12 mutation in combination with the detection of p16 and -DPC4 inactivation in pure pancreatic juice collected during ERP from patients with PC, chronic pancreatitis, or nontumoral pancreatobiliary diseases.
Costentin/Pagès/Bouisson/Berthelémy/ Buscail/Escourrou/Pradayrol/Vaysse
Table 2. Determination of K-ras codon 12 mutation, p16 exon 2 deletion, and DPC4 exons 8–11 in pure pancreatic juice from patients with
chronic pancreatitis (group II) Patient No.
Age years
Sex
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
50 41 55 60 37 69 51 35 44 45 48 62 40 32 66 42 55 52 61 44
male male male male male male male male male male male male male male male male male male male male
K-ras
p16
DPC4
RFLP
sequencing
exon 2
exon 8
exon 9
exon 10
exon 11
– + – + – – – – – + – – – – – + – – – –
wt wt wt wt wt wt wt wt wt GAT wt wt wt wt wt GTT wt wt wt wt
wt HD wt HD HD wt HD wt wt HD wt HD HD HD HD HD wt wt wt wt
wt HD wt HD HD HD HD wt wt HD wt HD n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
wt HD wt HD HD HD HD wt wt HD wt HD n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
wt HD wt HD HD HD HD wt wt HD wt HD n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
wt wt wt wt wt wt wt wt wt wt wt HD n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Follow-up period months 23 33 47 33 33 64 32 32 45 31 27 43 57 42 47 73 55 58 32 41
For explanation of abbreviations, see footnote to table 1.
Patients and Methods Patients Over a 6-year period (from December 1992 to December 1998), 57 patients who underwent endoscopic ductal aspiration of pancreatic cells during ERP were included in the study. ERP was performed as a diagnostic or a therapeutic purpose: a pancreatobiliary disease has been suspected in these patients because of obstructive jaundice or cholangitis or abdominal pain and/or biochemical abnormalities such as elevation of pancreatic enzyme or tumor antigen marker (carcinoembryonic antigen CA 19-9) levels and/or abnormal ultrasonography or computed tomography findings (mass vascular or lymphatic node involvement, ascitic metastases). Informed consent was obtained from all patients, and the study was approved by the ethical committee (CCPPRB Mide-Pyrénées). The patients were classified into three groups, according to clinical features, pathological criteria, and follow-up findings. Group I. Patients (n = 18; table 1) having PC; the diagnosis of PC was based upon histological or cytological findings. Group II. Patients (n = 20; table 2) having chronic pancreatitis diagnosed on the basis of one or more of the following criteria: (1) pancreatic calcifications identified by radiography, ultrasonography, computed tomography, or a combination of these; (2) pancreatic exocrine function insufficiency; (3) alterations on ERP according to the international classification of Cambridge [19], and (4) histological findings.
p16 and DPC4 in Pancreatic Juice
Group III. Patients (n = 19; table 3) suffering from nontumoral pancreatobiliary diseases: biliary acute pancreatitis (n = 11), cholangitis (n = 3), residual stones of common bile duct (n = 4), and hypercalcemic acute pancreatitis (n = 1). Additional follow-up studies included clinical examination, routine biochemical hematological profiles, and imaging studies (ultrasonography, computed tomography, and/or endoscopic ultrasonography) at 6-month intervals Pancreatic Juice Aspiration Pure pancreatic juice was collected by endoscopic cannulation using a duodenal fiberscope (JF-10; Olympus, Rungis, France). Before cholangiopancreatography; a catheter was inserted endoscopically into the main pancreatic duct for pure pancreatic juice aspiration. A total of 0.5–3 ml of pure pancreatic juice was aspirated and immediately stored at –20 ° C until DNA extraction. In order to verify the pancreatic origin of the sample, contrast medium was injected into the main pancreatic duct immediately after pancreatic juice aspiration, and the position of the catheter was assessed under fluoroscopy and radiography. Cell Lines The human pancreatic carcinoma derived cell lines Capan-1, AsPc-1, and BxPc3 were analyzed. Cells were cultured in RPMI 1640 medium with 10% fetal bovine serum, 50 U/ml penicillin, and 50 Ìg/ml streptomycin (Life-Technologies, Gaithersburg, Md.,
Pancreatology 2002;2:17–25
19
Table 3. Determination of K-ras codon 12 mutation, p16 exon 2 deletion, and DPC4 exons 8–11 in pure pancreatic juice from patients with
nontumoral pancreatobiliary diseases (group III) Patient No.
Age years
Sex
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
31 51 60 48 70 61 57 72 84 46 59 76 24 76 67 74 54 73 54
female male female male male female female male male male male female female male female female female male male
K-ras
p16
DPC4
RFLP
sequencing
exon 2
exon 8
exon 9
exon 10
exon 11
– + – – – – + – – – – – – + – – – + –
wt wt wt wt wt wt wt wt wt wt wt wt wt GAT wt wt wt GTT wt
wt wt wt wt wt wt wt n.d. wt n.d. wt wt wt HD wt HD HD HD wt
wt wt HD wt wt wt wt wt HD HD wt wt wt HD n.d. n.d. n.d. n.d. n.d.
wt wt HD wt wt wt wt wt HD HD wt wt HD HD n.d. n.d. n.d. n.d. n.d.
wt wt HD wt wt wt wt wt HD HD wt wt wt HD n.d. n.d. n.d. n.d. n.d.
wt wt wt wt wt wt wt wt wt wt wt wt wt HD n.d. n.d. n.d. n.d. n.d.
Follow-up period months 86 33 57 61 65 54 87 43 49 49 47 21 23 32 54 43 36 45 54
For explanation of abbreviations, see footnote to table 1.
USA). It has been previously reported that Capan-1 has a homozygous p16 deletion and a point mutation in codon 343 of DPC4; that AsPc-1 has a 2-bp p16 deletion frame shift and a point mutation in condon 100 of DPC4, and that BxPc3 has homozygous deletion of DPC4 [20, 21; Pancreas Cancer Web: www.path.jhu.edu/pancreas]. DNA Extraction Intraductal aspirates of pure pancreatic juice were centrifugated at 1,500 g for 20 min. DNA was extracted from the pellet by lysing cells in distilled water at 100 ° C during 10 min. Genomic DNA of cell lines was isolated by proteinase K digestion and sequential phenolchloroform extraction. K-ras Mutation Analysis K-ras codon 12 mutations were identified [22] using a two-step enriched-nested polymerase chain reaction (PCR) amplification, followed by restriction fragment length polymorphism (RFLP), using the restriction endonuclease Bst N1 from Bacillus stearothermophilus N (Biolabs). PCR products were run out on a 15% polyacrylamide gel. Ras mutations were sequenced using an automated sequencer (ABI 373A; Applied Biosystems, Foster City, Calif., USA) and a prism dye terminator cycle sequencing kit. Homozygous Deletions Analysis Because all samples contained contaminating normal cells, a comparative multiplex PCR technique was used to assay for homozygous deletions of the p16 or DPC4 genes. Exon 2 of p16 was coam-
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Pancreatology 2002;2:17–25
plified with the exon 1 of the K-ras gene, whereas exons 8 and 10 of DPC4 were coamplified with the exon 1 of K-ras, and exons 9 and 11 of DPC4 were coamplified with the GAPDH gene. Standards were made by mixing normal DNA and DNA from cells homozygously deleted for p16 or DPC4 to simulate a p16 or DPC4 loss of 0, 25, 50, and 100%. A homozygous deletion was defined as a 75% reduction of the p16 or DPC4 bands in the presence of a clear control band. Each sample showing a homozygous deletion was independently retested twice with both pairs of primers. For p16 deletions, the PCR reaction mixture in a final volume of 50 Ìl consisted of PCR buffer (50 mmol/l KCl, 10 mmol/l Tris-HCl, 0.01% gelatin, and 0.1% Triton X-100), 1.5 mmol/l MgCl2, 250 Ìmol/l dNTPs, 5% dimethylsulfoxide, 1.25 U Taq polymerase (Ampli Taq Gold; Perkin-Elmer, Branchburg Park, N.J., USA), and 0.5 Ìmol/l of each specific primer (table 4). Five Ìl of human crude lysates or 250 ng of genomic DNA of cell lines was added. Cycling was performed on an automatic cycler (Biometra, Göttingen, Germany). Initial denaturation at 95 ° C for 10 min was followed by 35 cycles of PCR: 45 s at 95 ° C, 45 s at 55 ° C, and 1 min at 72 ° C, followed by a final elongation at 72 ° C for 5 min. Negative controls without added DNA were carried out to discard the possibility of accidental contamination by previously amplified fragments. PCR products were analyzed on 8% nondenaturing polyacrylamide gel electrophoresis. The gels were stained with ethidium bromide and exposed to ultraviolet light. To identify homozygous deletions of DPC4, first amplifications were performed in 50-Ìl volumes containing PCR buffer as de-
Costentin/Pagès/Bouisson/Berthelémy/ Buscail/Escourrou/Pradayrol/Vaysse
Table 4. Primers used for PCR
Gene
Product Primers size, bp
K-ras
242
5)-GTGTATTAACCTTATGTGTGAC-3) (forward) 5)-TATCTGTATCAAAGAATGGTCC-3) (reverse)
K-ras (exon 1) 156
5)-ACTGAATATAAACTTGTGGTAGTTGGACCT-3) (forward) 5)-CAAAGAATGGTCCTGCACCAGT-3) (reverse)
GAPDH
380
5)-TCCATGACAACTTTGGCATCGTGG-3) (forward) 5)-GTTGCTGTTGAAGTCACAGGAGAC-3) (reverse)
p16 (exon 2)
459
5)-CTCTACACAAGCTTCCTTTCC-3) (forward) 5)-GGGCTGAACTTTCTGTGCTGG-3) (reverse)
Exon 8
355
5)-TGTTTTGGGTGCATTACATTTC-3) (forward) 5)-CAATTTTTTAAAGTAACTATCTGAC-3) (reverse)
Exon 9
254
5)-TTCCTAAGGTTGCACATAGGC-3) (forward) 5)-CTTCCACCCAGATTTCAATTC-3) (reverse)
Exon 10
293
5)-AGGCATTGGTTTTTAATGTATG-3) (forward) 5)-CTGCTCAAAGAAACTAATCAAC-3) (reverse)
Exon 11
296
5)-CTGATGTCTTCCAAACTCTTTTCTG-3) (forward) 5)-TGTATTTTGTAGTCCACCATC-3) (reverse)
DPC4
scribed above, 3 mmol/l MgCl2 250 Ìmol/l dNTPs, 1.25 U of Taq polymerase, and 0.52 Ìmol/l of K-ras exon 1 primer and 0.52 Ìmol/l of DPC4 exon 8 and 10 primers. Second amplifications were performed in 50-Ìl volumes containing PCR buffer, 2.25 mmol/l MgCl2, 10% dimethylsulfoxide, 250 Ìmol/l dNTPs, 1.25 U of Taq polymerase, and 0.24 Ìmol/l of GAPDH primers and 0.52 Ìmol/l of DPC4 exon 9 and 11 primers. After an initial denaturation at 95 ° C for 10 min, the PCR reactions were carried out in a Biometra cycler at 95 ° C for 45 s, at 54 ° C for 45 s, and at 72 ° C for 1 min, for 45 cycles followed by a final elongation at 72 ° C for 5 min. Negative controls and PCR product analysis were performed as described above. Statistical Analysis Groups were compared by the use of the ¯2 method or the Yates test. p ! 0.05 was considered significant.
Results
K-ras Mutation Analysis K-ras mutations were found in 11 of 18 (61%, CI 38– 84%) patients with PC (table 1). On the other hand, K-ras gene mutations were found in 2 of 20 patients (10%; CI 0–23%) with chronic pancreatitis (table 2) and in 2 of 19 patients (10.5%, CI 0–25%) with nontumoral pancreatobiliary disease (table 3). A significantly higher rate of codon 12 mutations was observed in patients with PC (p !
p16 and DPC4 in Pancreatic Juice
0.001). Sensitivity and specificity of K-ras mutation detection for PC diagnosis were 61 and 89.7%, respectively. Homozygous Deletions Analysis In conditions suitable for coamplification of p16 and K-ras genes, a clear band with the expected size for p16 exon 2 amplicon was only visible for DNA extracted from AsPc-1 cells, but not for DNA extracted from Capan-1. K-ras exon 1 fragment (156 bp) was present in AsPc-1 and Capan-1 (fig. 1A). For the BxPc3 cell line, multiplex PCR failed to generate a specific DPC4 amplification product, whereas the specific K-ras and GAPDH products were readily detectable, as shown in figure 1B. Conversely, DPC4 and the two reference genes were successfully amplified from Capan-1 and AsPc-1 DNA, as expected. By mixing a known amount of DNA from a cell line that has a homozygous deletion with a known amount of normal DNA as indicated in Patients and Methods, we showed that p16 or DPC4 homozygous deletion was detected when the proportion of contaminating DNA was lower than 25%. Results are shown for DPC4 in figure 2. Results for p16 are in agreement with previous published results [23].
Pancreatology 2002;2:17–25
21
Fig. 1. p16 and DPC4 deletion analysis by comparative multiplex PCR. A Exon 2 of the p16 gene was coamplified with the exon 1 of
the K-ras gene. In conditions suitable for coamplification of p16 and K-ras genes, a clear band with the expected size for p16 exon 2 amplicon (459 bp) was visible for DNA extracted from AsPc-1 cells (lanes 2 and 3), whilst no band was seen for DNA extracted from Capan-1 (lanes 4 and 5). K-ras exon 1 fragment (156 bp) was always present. Lane 1 was loaded with PGEM size markers. B Exons 8 and 10 of the DPC4 gene (355 and 293 bp, respectively) were coamplified with the exon 1 of the K-ras gene (156 bp), while exons 9 and 11 of the DPC4 gene (296 and 254 bp, respectively) were coamplified with the
GAPDH gene (380 bp). Negative controls without added DNA were carried out to discard the possibility of accidental contamination by previously amplified fragments (lanes 3 and 5). The quality of DNA was assured by successful amplification of GAPDH, resulting in a slightly larger PCR product than expected for the DPC4-specific amplification. For the BxPc3 cell line, multiplex PCR failed to generate a specific DPC4 amplification product (lanes 2 and 7), whereas the specific K-ras and GAPDH products were readily detectable. Conversely, DPC4 and the two reference genes were successfully amplified from AsPc1 DNA, as expected (lanes 1 and 6). Lane 4 was loaded with DNA ladder (100 bp).
Fig. 2. Dilution studies of normal DPC4
gene with deleted exons 8 and 10. DPC4 gene at different concentrations. DPC4 exons 8 and 10 were coamplified with K-ras gene. DNA from BxPc3 harbored DPC4-deleted exons 8 and 10. Normal DPC4 gene was from normal pancreas. Lane T: negative control without added DNA; lanes 1–6: DNA from BxPc3 cells, 100% (lane 1), 875% (lane 2), 75% (lane 3), 50% (lane 4), 25% (lane 5), and 0% (lane 6), was mixed with normal DNA, 0% (lane 1), 125% (lane 2), 25% (lane 3), 50% (lane 4), 75% (lane 5), and (100% lane 6); lane M: DNA molecular weight marker.
The PCR-based deletion analysis assay revealed deletions of p16 exon 2 in 5 of 18 patients (28%, CI 0–49%) with PC. Only 3 patients had K-ras mutation associated with p16 deletion. Likewise, p16 deletion was found in 10 of 20 patients (50%, CI 28–72%) with chronic pancreatitis and in 4 of 17 patients (24%, CI 0–44%) in group III. In
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Pancreatology 2002;2:17–25
these last two groups all patients with K-ras codon 12 mutation had p16 exon 2 deletion too, whereas the inverse was not true. There was a significant difference between group II and group III for p16 exon 2 deletion (p ! 0.05). Sensitivity and specificity of p16 exon 2 test for the PC diagnosis were 27.7 and 65.8%, respectively.
Costentin/Pagès/Bouisson/Berthelémy/ Buscail/Escourrou/Pradayrol/Vaysse
Table 5. Alterations of p16, DPC4 and K-ras genes in pancreatic juice from patients with PC, chronic pancreatitis, and nontumoral pancreatobiliary diseases
PC (group I)
Chronic pancreatitis (group II)
Pancreatobiliary diseases (group III)
K-ras codon 12 mutations (wt = GGT) % (95% CI)
n = 18; mutation = 11 61 (38–84)
n = 20; mutation = 2 10 (0–23)
n = 19; mutation = 2 10.5 (0–25)
p16 exon 2 % (95% CI)
n = 18; deletion = 5 28 (0–49)
n = 20; deletion = 10 50 (28–72)
n = 17; deletion = 4 24 (0–44)
DPC4 % (95% CI)
n = 14; deletion = 5 36 (0–79)
n = 13; deletion = 8 58 (21–96)
n = 14; deletion = 5 36 (0–79)
The PCR-based deletion analysis assay revealed deletions of DPC4 exons 8–10 in 5 of 14 patients (36%, CI 0–79%) with PC. No exon 11 deletion was found in this group. Moreover DPC4 exon 8–10 deletions were found in 7 of 12 patients (58%, CI 21–96%) with chronic pancreatitis. Finally, DPC4 exons 8–10 were found to be deleted in 4 of 14 patients in group III. One patient in group II and another patient in group III showed a DPC4 exon 11 deletion associated with exon 8–10 deletions. Furthermore, 1 patient in group III had an isolated deletion of DPC4 exon 9. There was no significant difference between the different groups for DPC4 deletions. Sensitivity and specificity for PC diagnosis were 36 and 54%, respectively. A number of experiments was lacking. This is due to the low number of cells present in some samples and the number of amplifications including retesting when a deletion was found. To exclude the possibility of differential amplification with varying concentrations of starting DNA template, we studied serial dilutions with normal DNA, ranging from 10 to 50 ng of template DNA. The ratio of p16 or DPC4 to reference gene (K-ras or GAPDH) was equivalent in ten normal DNA samples and in samples in which the starting DNA template concentration was varied in the range cited above (data not shown). Detection of the reference gene was used to indicate that the DNA material was sufficient.
(range 38–67) months in group III; all patients in these groups were free from pancreatic or biliary tract malignancy at the end of the study.
Discussion
Follow-Up Follow-up was performed until June 2000. One patient dropped out of the study. In group I, the median follow-up duration was 9 (range 2–16) months; all patients were dead at the end of the study. The median follow-up periods were 44 (range 30–56) months in group II and 50
Up to now, K-ras oncogene codon 12 mutation is the most frequently used genetic diagnostic tool investigated to screen high-risk groups for PC. Recently, an association between coffee drinking and K-ras mutation has been reported in PC patients [24], supporting the idea that constituants of coffee might be involved in the genesis of exocrine PC. In our study, sensitivity and specificity of K-ras mutations for PC were 61 and 89.7%, respectively, confirming previous results [25, 26]. This diagnostic performance indicated that the K-ras mutation still remained the best marker of PC in pancreatic juice. However, several studies found K-ras codon 12 mutations in nonneoplasic pancreatic diseases or in normal pancreata [8, 9, 27, 28], indicating these mutations lack specificity for PC diagnosis. A major concept emerging from the molecular analysis of human cancers is that cell progression to malignancy requires several hints: oncogene mutation and tumor suppressor gene inactivation. Alterations in at least four pathways seem to be needed [29]. It has been shown that a vast majority (about 80%) of pancreatic tumors have a fingerprint, including the activation of Kras and the inactivation of a number of tumor suppressor genes [17, 30]. Although p53 is an attractive target for the detection of pancreatic cancer, p53 mutation analysis is laborious. Therefore, the combination of K-ras with the study of genes belonging to different suppressive pathways such as p16 and DPC4 might be helpful. Indeed, the
p16 and DPC4 in Pancreatic Juice
Pancreatology 2002;2:17–25
23
deletion rate we observed for p16 exon 1 and DPC4 exons 8–10 in pancreatic juice from patients with PC was in the range of that observed in microdissected human tumors [31]. Previous studies [8] indicate that the frequency of K-ras mutations in chronic pancreatitis is about 10%. As expected, 2 patients out of 20 in group II harbored K-ras mutations. Moreover a high incidence of p16 and DPC4 inactivations was also observed in DNA amplified from pancreatic juice of patients with chronic pancreatitis in group II. This is consistent with the inactivation of p53 observed in a number of patients with chronic pancreatitis without morphological evidence of PC [32]. Tumor suppressor gene alterations in chronic pancreatitis could not be simply considered a genetic change preceding neoplasia. The high frequency of p16 and DPC4 alterations in chronic pancreatitis does not reflect the rather low relative risk of PC (4% over 20 years) observed in chronic pancreatitis patients [33]. More probably, tumor suppressor gene alterations in chronic pancreatitis might be in relation with the occurrence of chronic inflammatory processes developed during the course of the disease. Chronic pancreatitis lesions are surrounded by inflammatory tissues, producing cytokines that exert their proliferative activity on epithelial cells, inducing ductal hyperproliferation [8, 34, 35]. Alteration of tumor suppressor genes in a nonmalignant disease has recently been demonstrated in ulcerative colitis. Allelic deletions of APC, DCC p53, and p16 genes have been detected not only in carcinoma patients, but also in the epithelium of chronic colitis patients and in the normal epithelium [36]. Also, microsatellite instability has been detected in patients with chronic ulcerative colitis [36] or pancreatitis [37], indicating that the DNA mismatch repair system may be saturated under some circumstances. It is tempting to suggest that in some conditions chronic inflammation may ini-
tiate genetic alterations such as microsatellite instability and tumor suppressor gene inactivation. The genetic profiles of chronic pancreatitis indicate a lower incidence of Ki-ras mutations and a higher incidence of p16 and DPC4 alterations than observed in PC. This molecular heterogeneity may be helpful to classify distinct clinical categories and treatment responses. In group III some patients harbored deletions and/or K-ras mutation. Most of them suffered from acute pancreatitis, cholangitis, or bile duct stone. This is in agreement with p16 alterations in normal tissue [36]. This study reports a demonstration of K-ras mutations and p16 and DPC4 deletions in nonmalignant diseases. Our series is limited regarding the number of patients with PC and of those with chronic pancreatitis. Data are missing for DPC4. However, the frequent alterations of p16 and DPC4 in chronic pancreatitis and alterations found in nontumoral acute pancreatobiliary diseases or in patients without pancreatic disease involve strong restrictions in their use for PC diagnosis. As a consequence, the combination of tumor suppressor genes with K-ras did not improve sensitivity and specificity characteristics of K-ras analysis alone for the diagnosis of PC. K-ras codon 12 mutation analysis in pure pancreatic juice still represents the best available molecular genetic marker for PC diagnosis. The molecular classification of pancreatic diseases on the basis of high-scale gene expression is expected to be necessary to discriminate chronic pancreatitis and PC.
Acknowledgment This work was supported by a ‘Programme Hospitalier de Recherche Clinique’ grant (Nb UA392).
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6 Smit VT, Boot AJ, Smith AM, Fleuren GJ, Cornelisse CJ, Bos JL: K-Ras codon 12 mutations occur very frequently in pancreatic adenocarcinomas. Nucleic Acids Res 1988;16: 7773–7782. 7 Tada M, Omata M, Kawai S, Shaisho H, Ohto M, Saikki RK: Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma. Cancer Res 1993;53:2472–2474. 8 Löhr M, Maisonneuve P, Lowenfels AB: K-ras mutations and benign pancreatic disease. Int J Pancreatol 2000;27:93–103.
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9 Luttges J, Reinecke-Lutge A, Mollmann B, Menke MA, Clemens A, Klimpfinger M, Sipos B, Kloppel G: Duct changes and K-ras mutations in the disease-free pancreas: Analysis of type-age relation and spatial distribution. Virchows Arch 1999;435:461–468. 10 Tada M, Ohashi M, Shiratori Y, Okudaira T, Komatsu Y, Kawabe T: Analysis of K-ras gene mutation in hyperplastic duct cells of the pancreas without pancreatic disease. Gastroenterology 1996;110:227–231. 11 Kern SE: Advances from genetic clues in pancreatic cancer. Curr Opin Oncol 1998;10:74– 80. 12 Serrano M, Hannon JH, Beach D: A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 1993;366:704–707. 13 Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshmam K, Tavtigian SV, Stockert E, Day RS 3rd, Johnson BE, Skolnick MH: A cell cycle regulator potentially involved in genesis of many tumor types. Science 1994;264:436– 440. 14 Naumann M, Savitskaia N, Eilert C, Schramm A, Kalthoff H, Schmiegel W: Frequent codeletion of p16/MTS1 and p15/MTS2 and genetic alterations in p16/MTS1 in pancreatic tumors. Gastroenterology 1996;110:1215–1224. 15 Schutte M, Hruban RH, Geradts J, Maynard R, Hilgers W, Rabindran SK, Moskaluk CA, Hahn SA, Schwarte-Waldhoff I, Schmiegel W, Baylin SB, Kern SE, Herman JG: Abrogation of the Rb/p16 tumor suppressive pathway in virtually all pancreatic carcinomas. Cancer Res 1997;57:3126–3130. 16 Hahn SA, Schutte M, Shamsul Hoque ATM, Moskaluk CA, Da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo CJ, Hruban RH, Kern SE: DPC4 a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996;271:350–353. 17 Rozenblum E, Schutte M, Goggins M, Hahn S, Panzer S, Zahurak M, Goodman SN, Sohn TA, Hruban RH, Yeo CJ, Kern SE: Tumor-suppressive pathways in pancreatic carcinoma. Cancer Res 1997;57:1731–1734.
p16 and DPC4 in Pancreatic Juice
18 Berthelémy P, Bouisson M, Escourrou J, Vaysse N, Rumeau J, Pradayrol L: Identification of K-ras mutations in pancreatic juice in the early diagnosis of pancreatic cancer. Ann Intern Med 1995;123:188–191. 19 Axon AT, Classen M, Cotton PB: Pancreatography in chronic pancreatitis: International definitions. Gut 1984;25:1107–1112. 20 Caldas C, Hahn SA, Hruban RH, Redston MS, Yeo CJ, Kern SE: Detection of K-ras mutation in stool of patients with pancreatic adenocarcinoma and pancreatic ductal hyperplasia. Cancer Res 1994;54:3568–3573. 21 Schutte M, Hruban RH, Hedrick L, Cho KR, Nadasdy GM, Weinstein CL, Bova GS, Isaacs WB, Cairns P, Nawroz H, Sidransky D, Casero RA Jr, Meltzer PS, Hahn SA, Kern SE: DPC4 gene in various tumor types. Cancer Res 1996; 56:2527–2530. 22 Kahn SM, Jiang W, Culbertson TA, Weinstein IB, Williams GM, Tomita N, Ronai Z: Rapid and sensitive nonradioactive detection of mutant K-ras genes via ‘enriched’ PCR amplification. Oncogene 1991;6:1079–1083. 23 Neumeister P, Hoefler G, Beham-Schmid C, Schmidt H, Apfelbeck U, Schaider H, Linkesch W, Sill H: Deletion analysis of the p16 tumor suppressor gene in gastrointestinal mucosaassociated lymphoid tissue lymphomas. Gastroenterology 1997;1123:1871–1875. 24 Porta M, Malats N, Alguacil J, Ruiz L, Jaroid M, Carrato A, Rifa J, Guarner L: Coffee, pancreatic cancer and K-ras mutations: Updating the research agenda. J Epidemiol Community Health 2000;54:656–659. 25 Watanabe H, Sawabu N, Ohta H, Saatomura Y, Yamakawa O, Motto Y: Identification of Kras oncogene mutations in pure pancreatic juice of patients with ductal pancreatic cancer. Jpn J Cancer Res 1993;84:961–965. 26 Tada M, Omaha M, Ohto M: Clinical applications of ras gene mutation for diagnosis of pancreatic carcinoma. Gastroenterology 1991;100: 233–238. 27 Kimura W, Zhao B, Futakawa N, Makuchi M: Significance of K-ras codon 12 point mutation in pancreatic juice in the diagnosis of carcinoma of the pancreas. Hepatology 1999;4:532– 534.
28 Yanagisawa A, Ohtake K, Ohashi K, Hori M, Kitagawa T, Sugano H, Kato Y: Frequent c-Kiras oncogene activation in mucous cell hyperplasia of pancreas suffering from chronic inflammation. Cancer Res 1993;53:953–956. 29 Weitzmann JB, Yaniv M: Rebuilding the road to cancer. Nature 1999;400:401–402. 30 Tascilar M, Caspers E, Sturm PDJ, Goggins M, Hruban RH, Offerhaus GJA: Role of tumor markers and mutations in cells and pancreatic juice in the diagnosis of pancreatic cancer. Ann Oncol 1999;10(suppl):107–110. 31 Bartsch D, Shevlin DW, Tung WS, Kisker O, Wells SA Jr, Goodfellow PJ: Frequent mutations of CDKN2 in primary pancreatic adenocarcinomas. Cancer 1995;14:189–195. 32 Gansauge S, Schmid RM, Müller J, Adler G, Mattfeldtt T, Beger HG: Genetic alterations in chronic pancreatitis: Evidence for early occurrence of p53 but not K-ras mutations. Br J Surg 1998;85:337–340. 33 Lowenfels AB, Maisonneuve P, Cavallini G, Ammann RW, Lankisch PG, Andersen JR, Dimagno EP, Andren-Sandberg A, Domellof L, International Pancreatitis Study Group: Pancreatitis and the risk of pancreatic cancer. N Engl J Med 1993;328:1433–1437. 34 Cubila AL, Fitzgerald PJ: Morphological lesions associated with human primary invasive nonendocrine pancreas cancer. Cancer Res 1976;36:2690–2698. 35 Kozuka S, Sassa R, Taki T, Masamoto K, Nagasawa S, Saga S, Hasegawa K, Takeuchi M: Relation of pancreatic duct hyperplasia to carcinoma. Cancer 1979;43:1418–1428. 36 Park WS, Pham T, Wang C, Pack S, Mueller E, Mueller J, Vortmeyer A, Zhuang Z, Fogt F: Loss of heterozygosity and microsatellite instability in non-neoplastic mucosa from patients with chronic ulcerative colitis. Int J Mol Med 1998;2:221–224. 37 Brentnall TA, Chen R, Lee JG, Kimmey MB, Bronner MP, Haggit RC, Kowdley KV, Hecker LM, Byrd DR: Microsatellite instability and Kras mutations associated with pancreatic adenocarcinoma and pancreatitis. Cancer Res 1995;55:4264–4267.
Pancreatology 2002;2:17–25
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Pancreatology 2002;2:17–25
Frequent Deletions of Tumor Suppressor Genes in Pure Pancreatic Juice from Patients with Tumoral or Nontumoral Pancreatic Diseases Lydie Costentin a, b Philippe Pagès a, b Michèle Bouisson a Philippe Berthelémy a Louis Buscail a, b Jean Escourrou a, b, Lucien Pradayrol a Nicole Vaysse a a INSERM,
U531 Biologie et Pathologie Digestive et b Département de Gastroentérologie, CHU Rangueil, Toulouse, France
Key Words p16 W DPC4 W Pancreatic juice W Pancreatic cancer W Chronic pancreatitis
Abstract Background/Aims: K-ras codon 12 mutation is the most frequent genetic alteration in pancreatic cancer. Sensitivity and specificity of K-ras are not high enough to detect all pancreatic cancers, especially at early stage. This study investigated whether detection of p16 and/or DPC4 deletions along with K-ras mutation in DNA samples could improve the definition of patients at risk of pancreatic cancer. Methods: K-ras mutations were investigated by sequencing. p16 and DPC4 homozygous deletions were studied using comparative multiplex polymerase chain reaction of DNA in pancreatic juice sampled during endoscopic retrograde pancreatography in 57 patients with either pancreatic cancer (group I, 18 patients), chronic pancreatitis (group II, 20 patients), or nontumoral pancreatobiliary disease (group III, 19 patients). Results: The frequencies of Ki-ras mutations were 61% in group I, 10% in group II, and 10.5% in group III. The frequencies of p16 exon 2 and DPC4 deletions were, respectively, 28 and 36% in group I, 50 and 58% in group II, and 24 and 36% in group III. Conclusions: The combi-
ABC
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Accessible online at: www.karger.com/journals/pan
nation of p16 and DPC4 deletions with K-ras mutation does not improve the diagnosis of pancreatic cancer based on K-ras mutation alone. These data suggest that tumor suppressor gene inactivation can occur with a high frequency during nonmalignant pancreatic diseases. Copyright © 2002 S. Karger AG, Basel and IAP
Introduction
Pancreatic adenocarcinoma is characterized by an aggressive clinical course distinct from that observed in many other human carcinomas. Enhanced understanding of the molecular genetic events occurring during neoplastic progression in the pancreas could lead to earlier diagnosis and treatment improvement. Activating mutation in codon 12 of the K-ras protooncogene is the most common genetic abnormality described in pancreatic carcinoma patients. It is considered an initial molecular event which can be identified in pancreatic fine-needle aspirate biopsy specimens or in duodenal juice or stools of patients with pancreatic cancer (PC) [1–7]. It could be a good tool for detecting PC at an early stage. However, K-ras mutations have been found in pancreatic ductal lesions that do not harbor any malignancy
Nicole Vaysse INSERM U 531, IFR 31, Bât L3, CHU Rangueil 1, avenue J.-Poulhès F–31403 Toulouse Cedex 4 (France) Tel. +33 561 322 402, Fax +33 561 322 403, E-Mail [email protected]
Table 1. Determination of K-ras codon 12 mutation, p16 exon 2 deletion, and DPC4 exons 8–11 in pure pancreatic juice from patients with
PC (group I) Patient No.
Age years
Sex
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
72 85 60 51 73 65 64 71 46 65 64 71 45 74 72 84 60 69
female male male male female male male female male male female female male male male male male female
K-ras
p16
DPC4
RFLP
sequencing
exon 2
exon 8
exon 9
exon 10
exon 11
– + – + + – + + – – + + + + + + – –
wt GTT wt GCT GAT wt GAT GAT wt wt GAT GAT GTT CGT GAT GTT wt wt
wt wt wt wt HD wt wt wt wt wt wt wt wt wt HD HD HD HD
wt wt HD wt wt wt wt wt HD HD HD wt wt HD n.d. n.d. n.d. n.d.
wt wt HD wt wt wt wt wt HD HD HD wt wt HD n.d. n.d. n.d. n.d.
wt wt HD wt wt wt wt wt HD HD HD wt wt HD n.d. n.d. n.d. n.d.
wt wt wt wt wt wt wt wt wt wt wt wt wt wt n.d. n.d. n.d. n.d.
Follow-up period months 7† 1† lost 9† 13† 7† 3† 13† 23† 3† 1† 11† 19† 15† 12† 4† 7† 1†
HD = Homozygous deletion; n.d. = not determined for lack of material; polymerase chain reaction mediated RFLP analysis indicated K-ras wild-type (wt; –) or mutant (+) gene fragments.
[8–10]. Therefore, this molecular marker cannot be used as the only genetic marker of pancreatic ductal adenocarcinoma. Among other genetic anomalies found in PC, deletions of p16 and DPC4 and alterations of p53 are most frequently observed [11]. p16 is a tumor suppressor gene originally identified by Serrano et al. [12]. It inhibits cyclin D/CDK4 protein kinase, a key regulator of the progression of eukaryotic cells through the G1 phase of the cell cycle. Inactivation of p16 may cause abnormal cell cycling and uncontrolled cell growth [13, 14]. The p16 gene is homozygously deleted in 40% of the pancreatic carcinomas, and a similar proportion of small mutations affecting the coding sequence is observed [2, 14]. Methylation associated with silencing of p16 expression can also be found accounting for the remaining cases of PC [15]. The putative tumor suppressor gene DPC4 is involved in another tumor-suppressive pathway. It is located on chromosome 18q21 and shows homology to the Mad family of proteins which play a critical role in transforming growth factor beta superfamily signaling [16]. Homozygous deletions of DPC4 have been reported in approxi-
18
Pancreatology 2002;2:17–25
mately 30% of the PCs. Sequencing of DNA derived from pancreatic carcinomas revealed that 20% of the cases with concomitant allelic loss of the DPC4 region had somatically acquired DPC4 point mutations. The biallelic inactivation of the DPC4 gene was shown in approximately 50% of the PCs [17]. p53 is altered in about 50% of the PCs. Homozygous deletions are rather common, but several mutations could be located in different exon [11]. A subset of patients with chronic pancreatitis have already been shown to carry both K-ras and p53 mutations. This study aimed at investigating whether investigation of tumor suppressor genes along with that of K-ras could improve the differential diagnosis between PC and chronic pancreatitis. Since pancreatic juice samples are readily available during endoscopic retrograde pancreatography (ERP) investigation, allowing gene studies [18], we performed a joint analysis of K-ras codon 12 mutation in combination with the detection of p16 and -DPC4 inactivation in pure pancreatic juice collected during ERP from patients with PC, chronic pancreatitis, or nontumoral pancreatobiliary diseases.
Costentin/Pagès/Bouisson/Berthelémy/ Buscail/Escourrou/Pradayrol/Vaysse
Table 2. Determination of K-ras codon 12 mutation, p16 exon 2 deletion, and DPC4 exons 8–11 in pure pancreatic juice from patients with
chronic pancreatitis (group II) Patient No.
Age years
Sex
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
50 41 55 60 37 69 51 35 44 45 48 62 40 32 66 42 55 52 61 44
male male male male male male male male male male male male male male male male male male male male
K-ras
p16
DPC4
RFLP
sequencing
exon 2
exon 8
exon 9
exon 10
exon 11
– + – + – – – – – + – – – – – + – – – –
wt wt wt wt wt wt wt wt wt GAT wt wt wt wt wt GTT wt wt wt wt
wt HD wt HD HD wt HD wt wt HD wt HD HD HD HD HD wt wt wt wt
wt HD wt HD HD HD HD wt wt HD wt HD n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
wt HD wt HD HD HD HD wt wt HD wt HD n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
wt HD wt HD HD HD HD wt wt HD wt HD n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
wt wt wt wt wt wt wt wt wt wt wt HD n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Follow-up period months 23 33 47 33 33 64 32 32 45 31 27 43 57 42 47 73 55 58 32 41
For explanation of abbreviations, see footnote to table 1.
Patients and Methods Patients Over a 6-year period (from December 1992 to December 1998), 57 patients who underwent endoscopic ductal aspiration of pancreatic cells during ERP were included in the study. ERP was performed as a diagnostic or a therapeutic purpose: a pancreatobiliary disease has been suspected in these patients because of obstructive jaundice or cholangitis or abdominal pain and/or biochemical abnormalities such as elevation of pancreatic enzyme or tumor antigen marker (carcinoembryonic antigen CA 19-9) levels and/or abnormal ultrasonography or computed tomography findings (mass vascular or lymphatic node involvement, ascitic metastases). Informed consent was obtained from all patients, and the study was approved by the ethical committee (CCPPRB Mide-Pyrénées). The patients were classified into three groups, according to clinical features, pathological criteria, and follow-up findings. Group I. Patients (n = 18; table 1) having PC; the diagnosis of PC was based upon histological or cytological findings. Group II. Patients (n = 20; table 2) having chronic pancreatitis diagnosed on the basis of one or more of the following criteria: (1) pancreatic calcifications identified by radiography, ultrasonography, computed tomography, or a combination of these; (2) pancreatic exocrine function insufficiency; (3) alterations on ERP according to the international classification of Cambridge [19], and (4) histological findings.
p16 and DPC4 in Pancreatic Juice
Group III. Patients (n = 19; table 3) suffering from nontumoral pancreatobiliary diseases: biliary acute pancreatitis (n = 11), cholangitis (n = 3), residual stones of common bile duct (n = 4), and hypercalcemic acute pancreatitis (n = 1). Additional follow-up studies included clinical examination, routine biochemical hematological profiles, and imaging studies (ultrasonography, computed tomography, and/or endoscopic ultrasonography) at 6-month intervals Pancreatic Juice Aspiration Pure pancreatic juice was collected by endoscopic cannulation using a duodenal fiberscope (JF-10; Olympus, Rungis, France). Before cholangiopancreatography; a catheter was inserted endoscopically into the main pancreatic duct for pure pancreatic juice aspiration. A total of 0.5–3 ml of pure pancreatic juice was aspirated and immediately stored at –20 ° C until DNA extraction. In order to verify the pancreatic origin of the sample, contrast medium was injected into the main pancreatic duct immediately after pancreatic juice aspiration, and the position of the catheter was assessed under fluoroscopy and radiography. Cell Lines The human pancreatic carcinoma derived cell lines Capan-1, AsPc-1, and BxPc3 were analyzed. Cells were cultured in RPMI 1640 medium with 10% fetal bovine serum, 50 U/ml penicillin, and 50 Ìg/ml streptomycin (Life-Technologies, Gaithersburg, Md.,
Pancreatology 2002;2:17–25
19
Table 3. Determination of K-ras codon 12 mutation, p16 exon 2 deletion, and DPC4 exons 8–11 in pure pancreatic juice from patients with
nontumoral pancreatobiliary diseases (group III) Patient No.
Age years
Sex
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
31 51 60 48 70 61 57 72 84 46 59 76 24 76 67 74 54 73 54
female male female male male female female male male male male female female male female female female male male
K-ras
p16
DPC4
RFLP
sequencing
exon 2
exon 8
exon 9
exon 10
exon 11
– + – – – – + – – – – – – + – – – + –
wt wt wt wt wt wt wt wt wt wt wt wt wt GAT wt wt wt GTT wt
wt wt wt wt wt wt wt n.d. wt n.d. wt wt wt HD wt HD HD HD wt
wt wt HD wt wt wt wt wt HD HD wt wt wt HD n.d. n.d. n.d. n.d. n.d.
wt wt HD wt wt wt wt wt HD HD wt wt HD HD n.d. n.d. n.d. n.d. n.d.
wt wt HD wt wt wt wt wt HD HD wt wt wt HD n.d. n.d. n.d. n.d. n.d.
wt wt wt wt wt wt wt wt wt wt wt wt wt HD n.d. n.d. n.d. n.d. n.d.
Follow-up period months 86 33 57 61 65 54 87 43 49 49 47 21 23 32 54 43 36 45 54
For explanation of abbreviations, see footnote to table 1.
USA). It has been previously reported that Capan-1 has a homozygous p16 deletion and a point mutation in codon 343 of DPC4; that AsPc-1 has a 2-bp p16 deletion frame shift and a point mutation in condon 100 of DPC4, and that BxPc3 has homozygous deletion of DPC4 [20, 21; Pancreas Cancer Web: www.path.jhu.edu/pancreas]. DNA Extraction Intraductal aspirates of pure pancreatic juice were centrifugated at 1,500 g for 20 min. DNA was extracted from the pellet by lysing cells in distilled water at 100 ° C during 10 min. Genomic DNA of cell lines was isolated by proteinase K digestion and sequential phenolchloroform extraction. K-ras Mutation Analysis K-ras codon 12 mutations were identified [22] using a two-step enriched-nested polymerase chain reaction (PCR) amplification, followed by restriction fragment length polymorphism (RFLP), using the restriction endonuclease Bst N1 from Bacillus stearothermophilus N (Biolabs). PCR products were run out on a 15% polyacrylamide gel. Ras mutations were sequenced using an automated sequencer (ABI 373A; Applied Biosystems, Foster City, Calif., USA) and a prism dye terminator cycle sequencing kit. Homozygous Deletions Analysis Because all samples contained contaminating normal cells, a comparative multiplex PCR technique was used to assay for homozygous deletions of the p16 or DPC4 genes. Exon 2 of p16 was coam-
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Pancreatology 2002;2:17–25
plified with the exon 1 of the K-ras gene, whereas exons 8 and 10 of DPC4 were coamplified with the exon 1 of K-ras, and exons 9 and 11 of DPC4 were coamplified with the GAPDH gene. Standards were made by mixing normal DNA and DNA from cells homozygously deleted for p16 or DPC4 to simulate a p16 or DPC4 loss of 0, 25, 50, and 100%. A homozygous deletion was defined as a 75% reduction of the p16 or DPC4 bands in the presence of a clear control band. Each sample showing a homozygous deletion was independently retested twice with both pairs of primers. For p16 deletions, the PCR reaction mixture in a final volume of 50 Ìl consisted of PCR buffer (50 mmol/l KCl, 10 mmol/l Tris-HCl, 0.01% gelatin, and 0.1% Triton X-100), 1.5 mmol/l MgCl2, 250 Ìmol/l dNTPs, 5% dimethylsulfoxide, 1.25 U Taq polymerase (Ampli Taq Gold; Perkin-Elmer, Branchburg Park, N.J., USA), and 0.5 Ìmol/l of each specific primer (table 4). Five Ìl of human crude lysates or 250 ng of genomic DNA of cell lines was added. Cycling was performed on an automatic cycler (Biometra, Göttingen, Germany). Initial denaturation at 95 ° C for 10 min was followed by 35 cycles of PCR: 45 s at 95 ° C, 45 s at 55 ° C, and 1 min at 72 ° C, followed by a final elongation at 72 ° C for 5 min. Negative controls without added DNA were carried out to discard the possibility of accidental contamination by previously amplified fragments. PCR products were analyzed on 8% nondenaturing polyacrylamide gel electrophoresis. The gels were stained with ethidium bromide and exposed to ultraviolet light. To identify homozygous deletions of DPC4, first amplifications were performed in 50-Ìl volumes containing PCR buffer as de-
Costentin/Pagès/Bouisson/Berthelémy/ Buscail/Escourrou/Pradayrol/Vaysse
Table 4. Primers used for PCR
Gene
Product Primers size, bp
K-ras
242
5)-GTGTATTAACCTTATGTGTGAC-3) (forward) 5)-TATCTGTATCAAAGAATGGTCC-3) (reverse)
K-ras (exon 1) 156
5)-ACTGAATATAAACTTGTGGTAGTTGGACCT-3) (forward) 5)-CAAAGAATGGTCCTGCACCAGT-3) (reverse)
GAPDH
380
5)-TCCATGACAACTTTGGCATCGTGG-3) (forward) 5)-GTTGCTGTTGAAGTCACAGGAGAC-3) (reverse)
p16 (exon 2)
459
5)-CTCTACACAAGCTTCCTTTCC-3) (forward) 5)-GGGCTGAACTTTCTGTGCTGG-3) (reverse)
Exon 8
355
5)-TGTTTTGGGTGCATTACATTTC-3) (forward) 5)-CAATTTTTTAAAGTAACTATCTGAC-3) (reverse)
Exon 9
254
5)-TTCCTAAGGTTGCACATAGGC-3) (forward) 5)-CTTCCACCCAGATTTCAATTC-3) (reverse)
Exon 10
293
5)-AGGCATTGGTTTTTAATGTATG-3) (forward) 5)-CTGCTCAAAGAAACTAATCAAC-3) (reverse)
Exon 11
296
5)-CTGATGTCTTCCAAACTCTTTTCTG-3) (forward) 5)-TGTATTTTGTAGTCCACCATC-3) (reverse)
DPC4
scribed above, 3 mmol/l MgCl2 250 Ìmol/l dNTPs, 1.25 U of Taq polymerase, and 0.52 Ìmol/l of K-ras exon 1 primer and 0.52 Ìmol/l of DPC4 exon 8 and 10 primers. Second amplifications were performed in 50-Ìl volumes containing PCR buffer, 2.25 mmol/l MgCl2, 10% dimethylsulfoxide, 250 Ìmol/l dNTPs, 1.25 U of Taq polymerase, and 0.24 Ìmol/l of GAPDH primers and 0.52 Ìmol/l of DPC4 exon 9 and 11 primers. After an initial denaturation at 95 ° C for 10 min, the PCR reactions were carried out in a Biometra cycler at 95 ° C for 45 s, at 54 ° C for 45 s, and at 72 ° C for 1 min, for 45 cycles followed by a final elongation at 72 ° C for 5 min. Negative controls and PCR product analysis were performed as described above. Statistical Analysis Groups were compared by the use of the ¯2 method or the Yates test. p ! 0.05 was considered significant.
Results
K-ras Mutation Analysis K-ras mutations were found in 11 of 18 (61%, CI 38– 84%) patients with PC (table 1). On the other hand, K-ras gene mutations were found in 2 of 20 patients (10%; CI 0–23%) with chronic pancreatitis (table 2) and in 2 of 19 patients (10.5%, CI 0–25%) with nontumoral pancreatobiliary disease (table 3). A significantly higher rate of codon 12 mutations was observed in patients with PC (p !
p16 and DPC4 in Pancreatic Juice
0.001). Sensitivity and specificity of K-ras mutation detection for PC diagnosis were 61 and 89.7%, respectively. Homozygous Deletions Analysis In conditions suitable for coamplification of p16 and K-ras genes, a clear band with the expected size for p16 exon 2 amplicon was only visible for DNA extracted from AsPc-1 cells, but not for DNA extracted from Capan-1. K-ras exon 1 fragment (156 bp) was present in AsPc-1 and Capan-1 (fig. 1A). For the BxPc3 cell line, multiplex PCR failed to generate a specific DPC4 amplification product, whereas the specific K-ras and GAPDH products were readily detectable, as shown in figure 1B. Conversely, DPC4 and the two reference genes were successfully amplified from Capan-1 and AsPc-1 DNA, as expected. By mixing a known amount of DNA from a cell line that has a homozygous deletion with a known amount of normal DNA as indicated in Patients and Methods, we showed that p16 or DPC4 homozygous deletion was detected when the proportion of contaminating DNA was lower than 25%. Results are shown for DPC4 in figure 2. Results for p16 are in agreement with previous published results [23].
Pancreatology 2002;2:17–25
21
Fig. 1. p16 and DPC4 deletion analysis by comparative multiplex PCR. A Exon 2 of the p16 gene was coamplified with the exon 1 of
the K-ras gene. In conditions suitable for coamplification of p16 and K-ras genes, a clear band with the expected size for p16 exon 2 amplicon (459 bp) was visible for DNA extracted from AsPc-1 cells (lanes 2 and 3), whilst no band was seen for DNA extracted from Capan-1 (lanes 4 and 5). K-ras exon 1 fragment (156 bp) was always present. Lane 1 was loaded with PGEM size markers. B Exons 8 and 10 of the DPC4 gene (355 and 293 bp, respectively) were coamplified with the exon 1 of the K-ras gene (156 bp), while exons 9 and 11 of the DPC4 gene (296 and 254 bp, respectively) were coamplified with the
GAPDH gene (380 bp). Negative controls without added DNA were carried out to discard the possibility of accidental contamination by previously amplified fragments (lanes 3 and 5). The quality of DNA was assured by successful amplification of GAPDH, resulting in a slightly larger PCR product than expected for the DPC4-specific amplification. For the BxPc3 cell line, multiplex PCR failed to generate a specific DPC4 amplification product (lanes 2 and 7), whereas the specific K-ras and GAPDH products were readily detectable. Conversely, DPC4 and the two reference genes were successfully amplified from AsPc1 DNA, as expected (lanes 1 and 6). Lane 4 was loaded with DNA ladder (100 bp).
Fig. 2. Dilution studies of normal DPC4
gene with deleted exons 8 and 10. DPC4 gene at different concentrations. DPC4 exons 8 and 10 were coamplified with K-ras gene. DNA from BxPc3 harbored DPC4-deleted exons 8 and 10. Normal DPC4 gene was from normal pancreas. Lane T: negative control without added DNA; lanes 1–6: DNA from BxPc3 cells, 100% (lane 1), 875% (lane 2), 75% (lane 3), 50% (lane 4), 25% (lane 5), and 0% (lane 6), was mixed with normal DNA, 0% (lane 1), 125% (lane 2), 25% (lane 3), 50% (lane 4), 75% (lane 5), and (100% lane 6); lane M: DNA molecular weight marker.
The PCR-based deletion analysis assay revealed deletions of p16 exon 2 in 5 of 18 patients (28%, CI 0–49%) with PC. Only 3 patients had K-ras mutation associated with p16 deletion. Likewise, p16 deletion was found in 10 of 20 patients (50%, CI 28–72%) with chronic pancreatitis and in 4 of 17 patients (24%, CI 0–44%) in group III. In
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Pancreatology 2002;2:17–25
these last two groups all patients with K-ras codon 12 mutation had p16 exon 2 deletion too, whereas the inverse was not true. There was a significant difference between group II and group III for p16 exon 2 deletion (p ! 0.05). Sensitivity and specificity of p16 exon 2 test for the PC diagnosis were 27.7 and 65.8%, respectively.
Costentin/Pagès/Bouisson/Berthelémy/ Buscail/Escourrou/Pradayrol/Vaysse
Table 5. Alterations of p16, DPC4 and K-ras genes in pancreatic juice from patients with PC, chronic pancreatitis, and nontumoral pancreatobiliary diseases
PC (group I)
Chronic pancreatitis (group II)
Pancreatobiliary diseases (group III)
K-ras codon 12 mutations (wt = GGT) % (95% CI)
n = 18; mutation = 11 61 (38–84)
n = 20; mutation = 2 10 (0–23)
n = 19; mutation = 2 10.5 (0–25)
p16 exon 2 % (95% CI)
n = 18; deletion = 5 28 (0–49)
n = 20; deletion = 10 50 (28–72)
n = 17; deletion = 4 24 (0–44)
DPC4 % (95% CI)
n = 14; deletion = 5 36 (0–79)
n = 13; deletion = 8 58 (21–96)
n = 14; deletion = 5 36 (0–79)
The PCR-based deletion analysis assay revealed deletions of DPC4 exons 8–10 in 5 of 14 patients (36%, CI 0–79%) with PC. No exon 11 deletion was found in this group. Moreover DPC4 exon 8–10 deletions were found in 7 of 12 patients (58%, CI 21–96%) with chronic pancreatitis. Finally, DPC4 exons 8–10 were found to be deleted in 4 of 14 patients in group III. One patient in group II and another patient in group III showed a DPC4 exon 11 deletion associated with exon 8–10 deletions. Furthermore, 1 patient in group III had an isolated deletion of DPC4 exon 9. There was no significant difference between the different groups for DPC4 deletions. Sensitivity and specificity for PC diagnosis were 36 and 54%, respectively. A number of experiments was lacking. This is due to the low number of cells present in some samples and the number of amplifications including retesting when a deletion was found. To exclude the possibility of differential amplification with varying concentrations of starting DNA template, we studied serial dilutions with normal DNA, ranging from 10 to 50 ng of template DNA. The ratio of p16 or DPC4 to reference gene (K-ras or GAPDH) was equivalent in ten normal DNA samples and in samples in which the starting DNA template concentration was varied in the range cited above (data not shown). Detection of the reference gene was used to indicate that the DNA material was sufficient.
(range 38–67) months in group III; all patients in these groups were free from pancreatic or biliary tract malignancy at the end of the study.
Discussion
Follow-Up Follow-up was performed until June 2000. One patient dropped out of the study. In group I, the median follow-up duration was 9 (range 2–16) months; all patients were dead at the end of the study. The median follow-up periods were 44 (range 30–56) months in group II and 50
Up to now, K-ras oncogene codon 12 mutation is the most frequently used genetic diagnostic tool investigated to screen high-risk groups for PC. Recently, an association between coffee drinking and K-ras mutation has been reported in PC patients [24], supporting the idea that constituants of coffee might be involved in the genesis of exocrine PC. In our study, sensitivity and specificity of K-ras mutations for PC were 61 and 89.7%, respectively, confirming previous results [25, 26]. This diagnostic performance indicated that the K-ras mutation still remained the best marker of PC in pancreatic juice. However, several studies found K-ras codon 12 mutations in nonneoplasic pancreatic diseases or in normal pancreata [8, 9, 27, 28], indicating these mutations lack specificity for PC diagnosis. A major concept emerging from the molecular analysis of human cancers is that cell progression to malignancy requires several hints: oncogene mutation and tumor suppressor gene inactivation. Alterations in at least four pathways seem to be needed [29]. It has been shown that a vast majority (about 80%) of pancreatic tumors have a fingerprint, including the activation of Kras and the inactivation of a number of tumor suppressor genes [17, 30]. Although p53 is an attractive target for the detection of pancreatic cancer, p53 mutation analysis is laborious. Therefore, the combination of K-ras with the study of genes belonging to different suppressive pathways such as p16 and DPC4 might be helpful. Indeed, the
p16 and DPC4 in Pancreatic Juice
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deletion rate we observed for p16 exon 1 and DPC4 exons 8–10 in pancreatic juice from patients with PC was in the range of that observed in microdissected human tumors [31]. Previous studies [8] indicate that the frequency of K-ras mutations in chronic pancreatitis is about 10%. As expected, 2 patients out of 20 in group II harbored K-ras mutations. Moreover a high incidence of p16 and DPC4 inactivations was also observed in DNA amplified from pancreatic juice of patients with chronic pancreatitis in group II. This is consistent with the inactivation of p53 observed in a number of patients with chronic pancreatitis without morphological evidence of PC [32]. Tumor suppressor gene alterations in chronic pancreatitis could not be simply considered a genetic change preceding neoplasia. The high frequency of p16 and DPC4 alterations in chronic pancreatitis does not reflect the rather low relative risk of PC (4% over 20 years) observed in chronic pancreatitis patients [33]. More probably, tumor suppressor gene alterations in chronic pancreatitis might be in relation with the occurrence of chronic inflammatory processes developed during the course of the disease. Chronic pancreatitis lesions are surrounded by inflammatory tissues, producing cytokines that exert their proliferative activity on epithelial cells, inducing ductal hyperproliferation [8, 34, 35]. Alteration of tumor suppressor genes in a nonmalignant disease has recently been demonstrated in ulcerative colitis. Allelic deletions of APC, DCC p53, and p16 genes have been detected not only in carcinoma patients, but also in the epithelium of chronic colitis patients and in the normal epithelium [36]. Also, microsatellite instability has been detected in patients with chronic ulcerative colitis [36] or pancreatitis [37], indicating that the DNA mismatch repair system may be saturated under some circumstances. It is tempting to suggest that in some conditions chronic inflammation may ini-
tiate genetic alterations such as microsatellite instability and tumor suppressor gene inactivation. The genetic profiles of chronic pancreatitis indicate a lower incidence of Ki-ras mutations and a higher incidence of p16 and DPC4 alterations than observed in PC. This molecular heterogeneity may be helpful to classify distinct clinical categories and treatment responses. In group III some patients harbored deletions and/or K-ras mutation. Most of them suffered from acute pancreatitis, cholangitis, or bile duct stone. This is in agreement with p16 alterations in normal tissue [36]. This study reports a demonstration of K-ras mutations and p16 and DPC4 deletions in nonmalignant diseases. Our series is limited regarding the number of patients with PC and of those with chronic pancreatitis. Data are missing for DPC4. However, the frequent alterations of p16 and DPC4 in chronic pancreatitis and alterations found in nontumoral acute pancreatobiliary diseases or in patients without pancreatic disease involve strong restrictions in their use for PC diagnosis. As a consequence, the combination of tumor suppressor genes with K-ras did not improve sensitivity and specificity characteristics of K-ras analysis alone for the diagnosis of PC. K-ras codon 12 mutation analysis in pure pancreatic juice still represents the best available molecular genetic marker for PC diagnosis. The molecular classification of pancreatic diseases on the basis of high-scale gene expression is expected to be necessary to discriminate chronic pancreatitis and PC.
Acknowledgment This work was supported by a ‘Programme Hospitalier de Recherche Clinique’ grant (Nb UA392).
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