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¶
From the Institute of Pathology*
and the
Department of Surgery,
the University Clinic
of Regensburg, Regensburg, Germany; the Institute of
Pathology,§
Klinikum Kassel, Kassel, Germany;
the Department of Surgery,
University Clinic
Witten-Herdecke, Wuppertal, Germany; and the Department of Molecular
Genetics and Molecular Diagnosis,¶
the City of
Hope National Medical Center and Beckman Research Institute, Duarte,
California
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although much is known about the invasive tumors, little is known about the genetic alterations in progenitor lesions. K-ras mutations have been found in preneoplastic intraductal lesions (PILs) and appear to be early events in pancreatic carcinogenesis.9-11 To date, analysis of the p53 gene in PILs has only been performed by immunohistochemistry and often without a precise morphological definition of preneoplastic lesions according to the World Health Organization International Histological Classification of Tumors.12
The available data are controversial and suggest mutational inactivation of p53 at either an early13 or late stage of pancreatic carcinogenesis.14-16 In two recent studies that addressed more precisely the different histological forms of preneoplastic lesions in pancreatic carcinoma, p16INK4 has been found inactivated in both low-grade and high-grade ductal lesions.17,18 Alterations of DPC4 in PILs have not yet been described.
For analysis of loss of heterozygosity (LOH), a homogeneous population of tumor cells or epithelial cells from PILs is required. This can only be achieved by precise microdissection, usually of ~50 to 200 cells, which allows few specific (nested) polymerase chain reaction (PCR) amplifications. Because LOH studies need to be done with multiple markers, preamplification of DNA by whole genome amplification could be very helpful. For this purpose, we established a protocol which allows multiple DNA analyses of single cells or small cell groups by conducting whole genome amplification followed by locus-specific PCR of multiple specific sites.19
Microsatellite analysis was performed with multiple markers mapping p16INK4, DPC4, and p53. With this technique, we obtained information about allelic deletion of up to three tumor suppressor genes within a single lesion. In addition, p53 immunohistochemistry was correlated with the molecular data.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twenty-nine archival cases of pancreatic adenocarcinoma from the
University Clinic of Regensburg were selected for the presence of PILs
associated with invasive carcinoma. One case of chronic pancreatitis
and one case of nesidioblastosis with PILs were included as controls of
nonneoplastic disease. According to the World Health Organization
Classification12
and a recently published
study,17
PILs were classified on the basis of their growth
pattern and on the degree of nuclear atypia and were divided into five
groups (Figure 1
, Table 1
). Normal appearing epithelia with
cuboidal to low columnar cells with round-to-oval nuclei without signs
of atypia were graded PIL grade 0. Flat focal lesions with uniformly
tall columnar mucin-filled cells without signs of nuclear atypia were
graded PIL grade 1. Focal lesions graded PIL grade 2 consisted of
mucin-filled cells with basally located nuclei without signs of atypia
and a papillary growth pattern, the papillary folds typically showing a
fibrovascular stalk. If these lesions exhibited mild nuclear atypia
like larger sized round or oval nuclei and a more pronounced chromatin
structure with clumped or dense hyperchromatic chromatin pattern, the
lesion was graded PIL grade 3. When the epithelial lining showed
irregular budding and bridging and signs of severe nuclear atypia like
prominent nucleoli, hyperchromasia, loss of polarity, irregular size
and contours, the lesion was graded PIL grade 4. In all lesions
described, signs of invasion into the surrounding tissue were absent.
To avoid examination of infiltrating cancers extending into ducts
thereby mimicking a PIL, the first and the last sections were stained
with H 38 E to ensure proper alignment of serial sections. If in
the last section signs of invasive carcinoma were discovered, the
sections were excluded from the study. In addition, only tissue blocks
were processed for which a distance of at least 10 mm from the invasive
carcinoma was described in the pathology report. For each patient, at
least one example of nonneoplastic tissue was microdissected as a
normal control.
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Five-micron serial sections of formalin-fixed, paraffin-embedded tissue were deparaffinized by incubating the slides in xylene for 2 x 15 minutes and rehydrating in 99.9% ethanol for 2 x 10 minutes, in 96% ethanol for 2 x 10 minutes, and in 70% ethanol for 2 x 10 minutes.
Microdissection
Methylene blue-stained sections were microdissected (Figure 2)
using a joystick hydraulic
micromanipulator (Leitz, Wetzlar, Germany). Between 50 and 200 cells
were collected with sterile needles (microlance3R; Becton Dickinson,
Franklin Lakes, NJ) and transferred into 10 µl of TL-buffer (1
x Taq PCR buffer, from Life Technologies, Eggenstein,
Germany, including 4 mg/ml of Proteinase K and 0.5% Tween 20 from
Merck, Darmstadt, Germany). In tumors, the microdissected samples were
enriched for a neoplastic cellularity of at least 60% to avoid
false-negative results in LOH analysis because of contamination by
normal stromal cells present in tumors. Cell lysis was performed by
incubation for 16 hours at 50°C and a 10-minute inactivation step at
94°C.
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As a first step, whole genome amplification was performed by using
an improved primer extension preamplification (I-PEP)-PCR as described
recently19
using a MJR PTC200 thermocycler (Biozym,
Oldenburg, Germany). Briefly, I-PEP PCR was set up by adding 50 µl
I-PEP mix (final concentration: 0.05 mg/ml gelatin, 16 µmol/L
(N)15 random primer, 0.1 mmol/L dNTP, 3.6 U
Taq Expand High Fidelity polymerase, 2.5 mmol/L
MgCl2, in 1x PCR buffer No. 3 from Boehringer
Mannheim, Mannheim, Germany) to 10 µl of lysed cells. PCR was run for
50 cycles. Step 1: 92°C for 90 seconds; step 2: 92°C for 40
seconds; step 3: 37°C for 2 minutes; step 4: ramp 0.1°C per 1
second to 55°C; step 5: 55°C for 4 minutes; step 6: 68°C for 30
seconds; step 7: go to step 2, 49 times; step 8: 68°C for 15 minutes;
step 9: 4°C. The presence and relative quantity of PCR product was
ascertained by resolution on a 2% agarose gel. Specific single-round
PCR (0.2 mmol/L dNTP, 0.3 µmol/L primers, 0.5 U Taq Expand
High Fidelity polymerase) was done using 3-µl aliquots of the
preamplified DNA in a final volume of 20 µl in a MJ Research
Thermocycler (PTC100, MJ Research, Watertown, MA) for 50 cycles: 94°C
for 1 minute, 50 to 60°C for 1 minute, 72°C for 1 minute, followed
by a final extension at 72°C for 8 minutes as described
previously.20,21
Primers used are given in Table 2
. Amplified microsatellites (3 µl)
were analyzed by 6.7% polyacrylamide/50% urea gel electrophoresis (1
hour, 1500V, 50°C) in a SequiGen sequencing gel chamber (BioRad,
Hercules, CA) and by silver nitrate staining as described
previously.22
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LOH was diagnosed when a significantly lower ratio in the signal intensity (<50%) was observed in one of the two alleles in the PIL/tumor sample compared to the matched normal sample after amplification with an informative microsatellite marker. All results of LOH or homozygous deletion were confirmed by at least one repetition of PCR on the same DNA sample or on DNA obtained from serial sections.
Immunohistochemistry
Five-micron sections of formalin-fixed, paraffin-embedded tissue blocks were stained with p53-antibody (clone Bp53–12; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) according to the manufacturer’s instruction. Appropriate positive tissue controls were included for p53 staining in every experiment. p53 immunoreactivity was evaluated according to Baas et al23 with tumors showing high labeling index (>30%), low labeling index (>1%, <30%), and no positivity (<1%).
Statistical Analysis
The Fisher’s exact test24 was used to calculate P values using Stat Xact software (Cytel Corporation, Cambridge, MA). A P value of <0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Table 2
gives the informative rate and the rate of amplification
that could be achieved by the microsatellite markers. These markers
have been found suitable for chromosomal mapping for detection of LOH
and homozygous deletions of p16INK425
and DPC4.26
In our hands, the
microsatellite markers D9S1751 and D9S1748
(p16INK4) and CU18007 and D18S63
(DPC4) gave reliable results. For multiplex PCR,
D9S1751 was found to be most suitable. p53 markers D17Sp53
and TP53alk showed a 97% rate of amplification and were informative in
77% and 72% of cases, respectively.
Microsatellite Analysis of Pancreatic Carcinoma
At least two and up to seven tumor loci were microdissected per
case (Table 3)
. LOH of
p16INK4 was found in nine of 22 cases
(41%), and eight of 22 tumors (36%) showed homozygous deletion in
multiplex PCR (Figure 3)
. LOH of
DPC4 was present in 15 out of 25 cases (60%), and
homozygous deletion was detected in four out of 25 cases (16%).
p53 showed LOH in 22 out of 27 cases (81%).
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. Overall, the loss of all three tumor suppressor genes was found in 7 out of 29 cases (24%), and two of three tumor suppressor genes were lost in 11 out of 29 cases (38%). Only one of 29 carcinomas (4%) showed no alteration by microsatellite analysis at the three loci investigated but this case did show strong staining for the p53 protein in immunohistochemistry, which is in favor of the presence of a mutated p53 gene in this case. Furthermore, in 24 out of 29 tumors (83%) microdissection revealed the presence of genetic heterogeneity in at least one tumor suppressor gene indicating the presence of different tumor cell subclones. No correlation could be observed between marker loss and either malignancy grade or tumor stage.
Microsatellite Alterations in PIL
From a total of 331 microdissected PILs, the DNA of 277 PILs was
suitable for microsatellite analysis (Figures 4 and 5)
.
In total, 163 of the 277 PILs (59%) showed LOH. Two PILs grade 4
(0.7%) exhibited a homozygous deletion at the
p16INK4 locus. In two of 22 PILs (9%)
having histologically normal epithelium (grade 0), LOH of
p53 was detected in two different patients; neither stained
positively for p53 protein.
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. Deletions of two
of the three tumor suppressor genes was first observed in
histologically altered epithelium (grade 1), and triple lesions were
first observed in PILs having moderate (grade 3) to severe (grade 4)
nuclear atypia. Single lesions were found in 15 of 81 PILs (19%)
without nuclear atypia compared to 75 of 196 PILs (38%) showing
nuclear atypia. Multiple deletions (double or triple) were found in
five of 81 PILs (6%) without nuclear atypia and in 17 of 196 PILs
(9%) showing nuclear atypia (Figure 6)
. Of the total 22 PILs with
multiple lesions, 17 (77%) were found in PILs showing nuclear atypia.
The trend of multiple lesions associated with a PIL with nuclear atypia
was found to be not statistically significant (P
= 0.538).
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. In the two cases with chronic pancreatitis and nesidioblastosis, a total of 21 PILs were microdissected. Seven of these PILs were grade 3. LOH was not detected in a single lesion.
p53 Immunohistochemistry in Tumors and in PILs
Twenty-one of 29 carcinomas were available for p53 immunostaining. Sixteen of the 21 carcinomas (76%) showed moderate to strong expression of p53 protein, 13 (81%) of these had LOH at the p53 locus. In five carcinomas that stained negative for p53, LOH of p53 was detected in three cases (60%). Three of the 16 tumors (19%) with p53 protein stabilization showed no LOH of p53.
A total of 151 PILs were stained for p53 protein stabilization (Table 4)
. 33 PILs (22%) revealed weak to
strong p53 protein staining (Figure 7)
.
In comparison, 42 of 151 PILs (28%) showed LOH of p53.
Sixteen (38%) of these PILs accompanied positive p53
immunohistochemistry. Of the PILs without nuclear atypia, only two
(both grade 2) of 52 PILs (4%) showed a weak staining for p53 protein.
All other PILs with p53 protein stabilization had low to severe signs
of nuclear atypia. Five of 52 PILs (10%) without nuclear atypia and
absence of p53 protein expression showed p53 LOH. Twenty-one
of the 99 PILs (21%) showing nuclear atypia and negative staining for
p53 protein revealed LOH of p53. On the other hand, 15 of 99
PILs (15%) having nuclear atypia showed p53 protein expression in the
absence of LOH. Two of 35 PILs grade 3 (6%) showed both positive
staining for p53 and LOH, whereas 14 of 64 PILs grade 4 (22%)
exhibited positive staining for p53 in addition to LOH. Of the 33 PILs
that stained positive for p53, 16 (48%) revealed LOH at the
p53 locus.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We demonstrated multiple genetic alterations within a single tumor in one to three tumor suppressor genes, revealing widespread genetic heterogeneity in pancreatic carcinoma. Considerable intratumoral genetic heterogeneity in primary pancreatic cancers has been described before by using cytogenetic techniques.6,31 In these studies, up to 76% of tumors were found to harbor up to 39 related and 54 unrelated clones. However, intratumoral genetic heterogeneity was not found in a study where xenografted pancreatic carcinomas were microdissected.32 The reason for this discrepancy remains obscure, but xenografting of primary tumors may introduce some bias in the results obtained. For example, a selection for subclones within a cancer cell population may be the reason for a variation of mutational profile or expression patterns of various genes in primary tumors versus xenografts.33-37
In our study, the presence of LOH in one tumor focus and the simultaneous presence of both alleles in a different tumor focus was shown not to be because of sample contamination by normal cells. A point of concern during amplification of single or few cells is the occurrence of allele drop out, especially if tissue sections are microdissected because chromosomes or parts of chromosomes could get lost during specimen cutting.38-40 The I-PEP PCR technique used in our study has been extensively evaluated to demonstrate the reliability, reproducibility, and limitations of this method for mutation analysis of single or several cells obtained in routine tumor pathology.19 In this study we have demonstrated accurate biallelic amplification of various microsatellite markers by I-PEP even in 30 cells microdissected from formalin-fixed, paraffin-embedded normal tissues. In light of the data presented we believe that our LOH data reflect true mutational events, as we always amplified 50 or more cells thereby minimizing chances for differential allelic amplification or loss.
Our detected frequency of deletions at the p16INK4 and DPC4 locus was lower than those reported by others previously using different techniques such as xenografting of tumors with subsequent tumor microdissection5,30,32 and higher for the p16INK4 locus than the frequency of deletions obtained by conventional microdissecting of primary tumors.41,42 A major advantage of our microdissection technique is that the frequency of deletions may resemble more closely the in vivo situation as compared to tumor xenografts, where (eg, p53) mutations are over-represented (up to 100%).33 Single foci of LOH or simultaneous LOH and homozygous deletion within one tumor area would have been overlooked in conventional studies using less precise microdissection techniques or only one tumor focus for mutation analysis, as it has been shown previously for prostate cancer.43 Consequently, differences in the methodological approaches used may account, in part, for the findings obtained.
This is the first study to systematically investigate LOH in three tumor suppressor genes in a large number of preneoplastic lesions in pancreatic carcinoma. For tumor suppressor genes, the only data that are available so far have reported p16INK4 mutations together with K-ras mutations in a small number of cases.17 An immunohistochemical analysis of p16 protein expression in a wide variety of preneoplastic lesions described loss of p16 protein expression in both low-grade and high-grade duct lesions.18 For p53, only immunohistochemical analyses have been performed so far.13,16,44,45 For DPC4, no data have been reported yet.
Molecular analysis of precursor lesions are of significant interest, because of the need 1) to understand of the genetic alterations that occur during the multistep carcinogenic process; 2) to develop a pancreatic cancer-specific expression profile; and 3) to create a screening method that could be developed to detect preneoplastic lesions as early as possible to identify patients with a high risk of developing cancer.
The analysis of K-ras mutations as a screening method for patients at risk of developing pancreatic cancer has been questioned, because K-ras mutations are often detected in PILs from healthy individuals not at risk for developing cancer.46-48 Clear evidence has accumulated that PILs are true neoplastic precursor lesions in pancreatic carcinoma.17,18,49 Our molecular data strongly support these findings, demonstrating genetic alterations of p16INK4, DPC4, and p53 genes occurring early in carcinogenesis of pancreatic carcinoma. Our findings show that more than one genetic alteration of the carcinoma can be found in adjacent PILs and that a rough correlation between the grade of histological atypia and the number of accumulated mutations exists.
Most interestingly, we found LOH of p53 in two independent PILs that had histological normal epithelium, whereas LOH of p16INK4 and DPC4 could not be detected within truly normal epithelium in our series. Very recently, Gansauge et al50 found p53 mutations by sequence analysis in normal appearing epithelium in eight of 80 cases of chronic pancreatitis, highlighting the role of p53 mutations in pancreatic carcinogenesis.
Although deletion of all three tumor suppressor genes could be detected at the earliest stages of histological change, there was a statistically significant tendency for p53 and DPC4 mutations to accumulate in PILs with nuclear atypia, whereas no statistically significant difference could be found for p16INK4. This suggests an earlier accumulation of p16INK4 mutations in pancreatic tumorigenesis compared to DPC4 and p53 mutations. This observation is of importance in regard to molecular screening of patients being at risk of developing pancreatic adenocarcinoma. The significance of our observations in regard to the development of pancreatic carcinoma has yet to be established, and further studies of larger series of tissues including chronic pancreatitis may be informative.
Because we found in PILs only a low number of double and triple lesions compared to a high number of single lesions, the sensitivity of our methods for detecting double lesions is poor. But the detection of a PIL harboring multiple lesions seems to be associated with high-grade atypia, although this trend is not statistically significant. When double lesions were analyzed for combinations of tumor suppressor genes, we found the most frequent combination to be p53 LOH and DPC4 LOH, followed by the combination of p53 with p16INK4 and then by p16INK4 and DPC4. These findings, along with previous data, suggest an important role for mutations of p53 during carcinogenesis in pancreatic carcinoma, as is true in many other malignancies.51 Rozenblum et al5 reported a high concordance of DPC4 and p16INK4 inactivation in pancreatic cancer, suggesting that the inactivation of p16INK4 increases the selective advantage of subsequent mutation of DPC4. It is unclear why we did not find a concordance of p16INK4 and DPC4 mutations in PILs, although a clonal outgrowth of tumor cells having p16INK4 and DPC4 mutations might occur at later stages of tumorigenesis among lesions with p53 mutations. In addition, the number of PILs with inactivation of the p16INK4 gene is probably underestimated in this study, because methylation analysis was not performed.
A question that is not addressed in our study is the mutational status
of the allele that is not affected by LOH and the mutational status of
the epithelium that had no detectable LOH. As has been shown for
preneoplastic lesions in Barrett’s esophagus,52
LOH of
one allele may not be necessarily accompanied by a mutation of the
other allele to produce loss of functional protein. To determine more
precisely the time point of complete mutational inactivation of tumor
suppressor genes during tumorigenesis of the pancreas, sequence
analysis and methylation analysis need to be performed in precursor
lesions. In most tumors, p53 protein stabilization indicates loss of
function of the protein on the basis of mutational inactivation of one
allele in conjunction with the loss of the wild-type allele. Because we
found only 22% of the PILs investigated had positive p53
immunohistochemistry compared to 28% of PILs with LOH of
p53, a possible explanation might be the alteration of one
allele and the presence of a functionally intact second allele, which
may not lead to p53 protein stabilization. In contrast to our tumor
samples, positive p53 immunohistochemistry in PILs was less often
accompanied with LOH at the p53 locus. One explanation for
this might be the observation that PILs with positive p53
immunohistochemistry often showed a mixed staining pattern (Figure 7)
,
indicating the presence of cells with at least one functional allele in
close approximation with epithelial cells that have lost function of
both alleles. Such a mixed cell population may have masked the
detection of LOH in some cases.
Our data may have impact on the screening for true precancerous lesions in chronic pancreatitis, which is considered to be a common condition predisposing to pancreatic carcinoma.53 The predictive value of K-ras mutations is still highly controversial, and immunohistochemical detection of p53 protein stabilization seems not to be useful because p53 protein stabilization in chronic pancreatitis appears to be because of the accumulation of wild-type p53 protein.54
In conclusion, by using precise microdissection techniques and a novel PCR protocol, we show 1) that genetic heterogeneity is a common feature of carcinomas of the exocrine pancreas; 2) LOH of the p16INK4, DPC4, and p53 genes can be found in ductal lesions with low-grade dysplasia and even rarely in normal epithelium surrounding invasive carcinoma, suggesting that the latter represents a true preneoplastic lesion that can be screened for by simple microsatellite analysis; and 3) immunohistochemical expression of p53 protein accompanied LOH at the p53 locus in 81% of invasive carcinoma but only in up to 22% in PILs.
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Acknowledgements |
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Footnotes |
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Accepted for publication March 10, 2000.
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