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Int. J. Cancer: 70, 661–667 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
Ki-ras MUTATIONS IN EXOCRINE PANCREATIC CANCER: ASSOCIATION
WITH CLINICO-PATHOLOGICAL CHARACTERISTICS AND WITH TOBACCO
AND ALCOHOL CONSUMPTION
Núria MALATS1, Miquel PORTA,1* Josep M.a COROMINAS1, Josep L. PIÑOL1, Juli RIFÀ2 and Francisco X. REAL1 for the PANK-ras I
Project Investigators
1Institut Municipal d’Investigació Mèdica, Hospital del Mar, Universitat Autònoma de Barcelona, Barcelona, Spain
2Hospital Son Dureta, Palma de Mallorca, Spain
The aims of this study were (i) to assess the prevalence and
spectrum of codon 12 Ki-ras mutations in patients diagnosed
with exocrine pancreatic cancer (EPC) in 2 general hospitals
between 1980 and 1990, (ii) to analyze the association of this
genetic alteration with clinical and pathological characteristics, and (iii) to determine the association of Ki-ras mutations
with tobacco and alcohol consumption. DNA was amplified
from paraffin-embedded tissue samples and mutations in
codon 12 of Ki-ras were detected using the artificial RFLP
technique. Cox proportional-hazards regression and unconditional logistic regression were applied. Codon 12 Ki-ras
mutations were detected in 30 of 51 cases for which molecular results were available. The amino-acid substitutions were
Asp (8), Val (6), and Arg (3). A double mutation, including
always a Val, was detected in 5 cases. None of the 4 nonductal pancreatic neoplasms were mutated. The mutation
prevalence was 79% in metastases and 54% in primary tumors. The risk of a mutated tumor was 3 times higher in
alcohol drinkers than in non-drinkers, and a linear trend was
apparent. When age, gender, hospital, and tobacco and
alcohol consumption were taken into account, a high risk for
mutations was detected in patients who only smoked and in
patients who only drank, but less so in patients who both
smoked and drank. These results raise novel hypotheses
regarding the role of tobacco and alcohol in EPC. Int. J.
Cancer, 70:661–667, 1997.
r 1997 Wiley-Liss, Inc.
Advances in the genetic analysis of human cancer provide a
basis for its molecular classification, the development of novel diagnostic and therapeutic strategies, and the identification of putative
etiological clues. Exocrine pancreas cancer (EPC) is the tumor for
which fewer therapeutic advances have been made in the last 50
years, and its mortality remains similar to its incidence (Warshaw
and Fernandez del Castillo, 1992; Urrutia and DiMagno, 1996).
After Almoguera et al. (1988) reported a high prevalence of
mutations in codon 12 of Ki-ras in EPC, about 50 reports have
described Ki-ras mutations in this tumor, with prevalence ranging
from 47% (Tabata et al., 1993) to 100% (Tada et al., 1990; Caldas
et al., 1994). The basis for such variation is unclear, and individual
studies are not strictly comparable due to significant differences in
strategies used to select patients, number of cases studied, type of
sample analyzed, and mutation-detection techniques. Only 7 of the
studies analyzed more than 40 patients (Grünewald et al., 1989;
Capellà et al., 1991; Hruban et al., 1993; Motojima et al., 1993;
Finkelstein et al., 1994; Scarpa et al., 1994; Pellegata et al., 1994).
Furthermore, most published series provide scanty information on
the selection criteria used, and only one described the population
study base and analyzed the differences between cases with and
without molecular results (Hruban et al., 1993). While selection
biases are generally important in molecular studies, they may be of
even greater relevance in EPC, since the proportion of patients
undergoing histological confirmation is often low and varies
widely. Thus, EPC may be one of the tumor types with a higher rate
of diagnostic misclassification (Porta et al., 1994). Moreover, few
studies have assessed the associations between Ki-ras mutations
and the clinical and pathological characteristics of cases (Grünewald
et al., 1989; Nagata et al., 1990; Hruban et al., 1993; Motojima et
al., 1993; Scarpa et al., 1994; Pellegata et al., 1994; Finkelstein et
al., 1994).
It is plausible that lifestyle and environmental factors may cause
EPC through the activation of Ki-ras. The molecular abnormalities
of tumors may reflect exposure to carcinogens involved in their
development, as described for acute myeloid leukemia (Taylor et
al., 1992). Similarly, Ki-ras-mutated EPC and wild-type EPC may
result from different etiologic agents. The actual causes of EPC
remain largely unknown (Boyle et al., 1989; Haddock and Carter,
1990; Warshaw and Fernández del Castillo, 1992). Tobacco has
been consistently found to be a risk factor for EPC (Boyle et al.,
1989; Haddock and Carter, 1990; Bueno de Mesquita et al., 1991;
Howe et al., 1991; Fujji et al., 1990), but only 2 studies have
analyzed the relationship between smoking and Ki-ras mutations in
EPC, and their results are contradictory (Nagata et al., 1990;
Hruban et al., 1993). Although alcohol consumption has been
reported to be a risk factor for EPC, its role remains controversial
(Boyle et al., 1989; Haddock and Carter, 1990; Bueno de Mesquita
et al., 1992). The possible association between alcohol drinking
and Ki-ras mutations in EPC has not been studied.
The aims of this study were (i) to determine the prevalence and
the spectrum of codon-12 Ki-ras mutations in all patients diagnosed with EPC in 2 general hospitals between 1980 and 1990, (ii)
Abbreviations: EPC, exocrine pancreatic cancer; PCR, polymerase chain
reaction; RFLP, restriction-fragment-length polymorphism; OR, odds ratio;
CI, confidence interval.
Results of this study were presented to the II Congresso Ibero-Americano
de Epidemiologı́a (Salvador de Bahia, Brazil, April 24–28, 1995), the 27th.
Meeting of the European Pancreatic Club (Barcelona, June 28–30, 1995)
(Digestion 1995; 56(4):302), the 6th. Meeting of the Spanish Association
for Cancer Research (Barcelona, September 24–27, 1995) (Oncology
Reports 1995; 2 [Suppl]: 929), and the 14th. International Scientific
Meeting of the International Epidemiological Association (Nagoya, Japan,
August 27–30, 1996).
Contract grant sponsor: Fondo de Investigación Sanitaria (FIS), contract
grant numbers 91/0595, 92/0007; Contract grant sponsor: Fundación Salud
2.000 (Grant Serono); Contract grant sponsor: Institut Municipal
d’Investigació MeTdica (IMIM), contract grant number 875138/9; Contract
grant sponsor: Comissió Interdepartamental de Recerca i Innovació tecnològica (CIRIT), contract grant numbers GRQ93-9301, SGR 434.
PANK-ras I Project Investigators: M. Porta and F.X. Real (Principal
Investigators), N. Malats, J.L. Piñol, J.M. Corominas, J. Rifà, M. Andreu,
M. Conangla, T. Thomson, M. Gallén, R. Solà and F. Tous.
*Correspondence to: Institut Municipal d’Investigació Mèdica, Hospital
del Mar, Universitat Autònoma de Barcelona, Carrer del Dr. Aiguader #80,
E-08003 Barcelona, Spain. Fax: 34-3-2213237.
Received 28 June 1996; revised 25 November 1996
MALATS ET AL.
662
to analyze the association between this genetic alteration and
clinical and pathological characteristics of the cases, and (iii) to
assess the association of Ki-ras mutations with tobacco and alcohol
consumption.
MATERIAL AND METHODS
Patients and information
The methodology of the PANK-ras I study has been described
elsewhere (Porta et al., 1994; Malats et al., 1995). Briefly, cases
diagnosed with EPC (n 5 149) between 1980 and 1990 were
identified through the cancer registries at Hospital del Mar
(Barcelona) and Hospital Son Dureta (Palma de Mallorca), Spain.
Sociodemographic, lifestyle, clinical and pathological information was obtained from clinical and pathological records through a
structured data form. Age was calculated from date of birth to date
of diagnosis. Prior medical history of diabetes mellitus, acute and
chronic pancreatitis, gallstones, duodenal ulcer, and psychiatric
disorders was recorded as present, if mention was made in clinical
records, and as absent otherwise. Tumor site was coded according
to the International Classification of Diseases for Oncology (ICD-O).
Disease extent was classified as local when the tumor was localized
within the pancreas, regional when it was locally advanced or
showed regional lymph-node involvement, and metastatic when
distant metastases were demonstrated. Tumors were graded as
well-, moderately, and poorly differentiated following ICD-O
criteria. Treatment intention was categorized as curative, palliative,
and symptomatic. Survival was determined as the period between
date of most-likely diagnosis and death/last control. Smoking
history was recorded as a dichotomous variable (ever/never).
Alcohol consumption was registered in 4 categories: no consumption, light (,30 g per day), moderate (30–80 g per day), and heavy
(.80 g per day).
Tissue specimens
An exhaustive search for histological material from all patients
included in the study was carried out. Samples of primary and of
metastatic lesions were obtained. Paraffin-embedded tissue was
available from 56 EPC patients (64.4% of the 87 cases with
histological confirmation). Tissue blocks from the remaining 31
patients could not be retrieved. From each specimen, 20 sections of
5 µ thickness were cut, and placed on glass slides. The first, 10th
and 20th sections were stained with hematoxylin/eosin and used for
histological evaluation. All cases were reviewed by a pathologist
(J.M.C.) who was unaware of the original diagnosis and of the
results of the molecular analysis. The pathologist defined tumor (T)
and non-tumor (N) areas by microscopic examination. The material
corresponding to the T and N areas was scraped, always starting
from the N area. The percentage of tumor cells present in the T area
was assessed by 2 independent investigators (J.M.C. and F.X.R.).
In general, the area analyzed was 1 cm2.
Detection of c-Ki-ras mutations
Care was taken to avoid contamination during all steps of
amplification and analysis. Tissue scrapings were de-paraffinized in
xylene and washed with 95% ethanol; tissue pellets were desiccated and heated at 95°C for 10 min. DNA was purified using a
commercial kit (LINUS, Cultek, Madrid, Spain) and amplified in 2
steps by nested PCR. The primers and conditions for DNA
amplification used in the study have been described (Berrozpe et
al., 1994; Malats et al., 1995). This technique was able to detect
one homozygous mutated cell in the presence of 102 normal cells.
DNA was successfully amplified from 51 of 56 cases from which
tissue blocks were available. The primers used in the second,
nested, PCR introduced an artificial restriction-endonuclease site
for BstNI. The 103-bp products of this amplification reaction were
digested overnight. Wild-type sequences were cleaved, resulting in
2 fragments of 82 and 21 bp, whereas codon-12 mutated sequences
were not. Products were analyzed by acrylamide-gel electrophoresis and ethidium-bromide staining. Analyses were restricted to
codon 12 because mutations in other codons are very rare in
pancreatic cancer (reviewed in Caldas and Kern, 1995).
To characterize the nucleotide substitution in codon 12, all
mutated samples were further analyzed using a similar RFLP-based
approach, as described elsewhere (Berrozpe et al., 1994; Malats et
al., 1995). GGT = AGT (Gly = Ser), GGT = GCT (Gly = Ala),
and GGT = TGT (Gly = Cys) mutations were not studied because
of their low prevalence in EPC (reviewed in Caldas and Kern,
1995).
DNA from oral mucosal scrapings was used as normal control,
and DNA from pancreas-cancer cell lines or tumors (Berrozpe et
al., 1994) was used as control for the Asp, Val and Arg mutations.
Interpretation of electrophoresis of digestion products’ was
performed by consensus of 2 investigators (NM and AS). When the
results were discordant, the analysis was repeated and the results
were evaluated again. The reliability of the digestion results was
further assessed by an independent investigator (F.X.R.). Agreement was high ($95%) for all enzyme digestions.
Statistical methods
Comparison of 2 qualitative variables was performed using
Pearson’s x2 test or, alternatively, with Fisher’s exact test when
$20% of cells had expected counts of less than 5. Odds ratios (OR)
were used to estimate the magnitude of associations between
variables; when the observed number of cases was zero in one cell
of the contingency table, Woolf-Haldane’s correction was applied.
The logit estimator of the OR was calculated with precision-based
confidence intervals (CI). Adjusted OR and CI were estimated by
unconditional logistic regression. Student’s t-test or MannWhitney’s U-test were used to analyze the relationship between a
categorical variable with 2 levels, and a normally or non-normally
distributed quantitative variable, respectively. Kaplan-Meier plots
and the log-rank test were used to evaluate the association between
mutations and survival. Cox proportional-hazards regression was
applied to analyze survival, taking into account possible confounder variables. All p values are 2-sided. Statistical analysis was
performed using Macintosh statistical packages STATVIEW 4.01
(Abacus Concepts, Berkeley, CA) and SPSS 4.0 (SPSS, Chicago,
IL), and IBM PC packages EGRET (Statistics and Epidemiologic
Research, Seattle, WA) and SAS (SAS Institute, Cary, NC).
RESULTS
Prevalence and spectrum of Ki-ras mutations
The characteristics of the 51 patients from whom results on
Ki-ras mutations were available were similar to those of patients
from whom molecular results were not available. As might be
expected, Ki-ras mutations could be determined in a larger
proportion of cases treated with curative intention ( p , 0.001).
Codon-12 Ki-ras mutations were detected in 30 of 51 EPC cases
(59%; 95% CI, 44.9–70.0). The proportion of tumor cells in the
tissue area analyzed was similar for mutated and wild-type tumors
(data not shown). The spectrum of mutations was as follows: 8
cases (27%) had an Asp substitution, 6 cases (20%) had a Val
substitution, 3 cases (10%) had an Arg substitution, and in 8 cases
the type of mutation could not be identified. A double mutation was
detected in 5 cases (17%), of which 3 corresponded to primary
tumors and 2 to metastases (Table I). A Val substitution was found
in all 5 cases.
Pathological and clinical characteristics
Regarding the pathological characteristics of tumors (Table II),
frequency of mutations was similar among cancers of the head,
body, and tail of the pancreas. Whereas 29 of 46 ductal-type
adenocarcinomas harboured Ki-ras mutations (63%), none of 4
non-ductal pancreatic neoplasms (1 acinar-cell carcinoma and 3
anaplastic carcinomas) were mutated ( p 5 0.026). Among ductaltype adenocarcinomas, the prevalence of mutations was 79% in
metastatic-tissue samples and 54% in primary-tumor samples (OR,
3.21, p 5 0.082) (Table II). No association was observed between
Ki-ras MUTATIONS IN EXOCRINE PANCREATIC CANCER
differentiation degree and Ki-ras mutations ( p 5 0.255). In 4
cases, samples from primary tumor and from a metastatic lesion
were analyzed. No consistent pattern was apparent (lower half of
Table I).
Although not statistically significant, a higher frequency of
mutations was observed in cases from Hospital del Mar ( p 5 0.123).
Patients less than 56 years old were 3 times more likely to have a
mutated tumor than older patients (OR, 3.00; p 5 0.128) (Table
III). Mutations were not significantly associated with gender, past
medical history, family history of cancer, and interval from first
symptom to diagnosis. The mutation prevalence was similar across
tumor-stage and treatment-intention strata. Survival was also
similar in cases with mutated and wild-type tumors ( p 5 0.116)
(Figure 1). After adjustment for possible confounders such as
gender, hospital, stage, and first-symptom diagnosis interval through
Cox’s proportional-hazards regression analysis, Ki-ras mutations
did not show a prognostic value in these patients.
In order to explore the degree of certainty with which EPC cases
were diagnosed, a diagnostic-certainty classification was developed (Porta et al., 1994). It was based mainly on the following 2
criteria: (i) presence of a discrete tumor mass in the pancreas vs.
infiltration to adjacent sites and/or distant metastases, and (ii)
availability of pathological sample from pancreas vs. from other
sites. Cases were classified in 2 groups: a group with a higher
probability of having EPC, and a group with a lower probability.
TABLE I – DESCRIPTION OF CASES WITH A BI-MUTATIONAL PATTERN
(SECTION A) AND PATIENTS FROM WHOM PRIMARY AND METASTATIC
SAMPLES WERE ANALYZED (SECTION B)1
Section
A
A, B
B
Patient
Primary-tumor tissue
45602 sample 1
sample 2
256201 sample 1
sample 2
261068
NA
287127
NA
Mutation frequency was similar in the 2 diagnostic-certainty
groups (Table III).
Tobacco and alcohol consumption
In the crude analysis, the risk of having a mutated EPC was 1.5
times higher for smokers than for non-smokers ( p 5 0.511). The
risk was also 3 times higher for alcohol drinkers (.30 g per day)
than for non-drinkers (#30 g per day) ( p 5 0.079) (Table III). A
similar risk was observed when ever-drinkers were compared with
never-drinkers (OR, 2.67; p 5 0.132). When alcohol consumption
was classified in 4 categories, a direct relationship was also
observed ( p 5 0.071). This pattern was evidenced across strata of
all other study variables except for tobacco: moderate and heavy
alcohol consumption was positively associated to Ki-ras mutations
only among non-smokers. In comparison with patients who did not
smoke and did not drink, cases who only smoked had an OR of 5.00
( p 5 0.170), those who only drank had an OR of 16.00 ( p 5 0.024),
and patients who both smoked and drank had an OR of 3.67
( p 5 0.102) (Table IV). This pattern remained substantially unaltered when light drinkers were not included in the reference group.
When age, gender and hospital were adjusted for, the association
between alcohol and Ki-ras mutations was again detected in
patients who only smoked (OR, 12.92; p 5 0.079) and in patients
who only drank (OR, 22.08; p 5 0.025), but less so in patients who
both smoked and drank (OR, 8.38; p 5 0.106). The tobaccoalcohol interaction term in the logistic regression model was
statistically significant ( p 5 0.044); the calibration (HosmerLemeshow test, p 5 0.319) and discrimination tests (ROC 5 77%)
of this multiplicative model indicated that it fitted the data well.
DISCUSSION
Metastatic tissue
[Val]
[Val, Arg]
[Val]
[Asp]
663
NA
NA
sample 1 [Val, Asp]
sample 1 [Val]
sample 2 [Arg]
sample 4
275846 sample 1 [Val, Arg]
sample 2
sample 3
47420 sample 1
sample 2 [unknown]
75964 sample 1 [unknown] sample 2
282223 sample 1
sample 2
1Brackets indicate that the sample was mutated and, if known, the
specific amino-acid substitution. A blank space indicates that no
mutation was detected. NA, not available.
Although the prevalence of Ki-ras mutations in EPC has been
the object of several reports, this study provides valuable additional
information for the following reasons: (i) it precisely defined the
study base by including all patients diagnosed of EPC in a specified
period of time in 2 general hospitals that are not referral centers for
this tumor; (ii) it compared demographical and clinicopathological
characteristics of patients with and without molecular results; and
(iii) it assessed the prevalence of Ki-ras mutations among diagnosticcertainty categories, an important issue given the difficulties in conclusively establishing the diagnosis of EPC (Porta et al., 1994, 1996).
Of 149 potentially eligible patients, 87 (58.4%) were histologically confirmed. The figure is not particularly low for EPC
(Carriaga and Henson, 1995; Porta et al., 1996). Ki-ras mutations
could be determined for 51 patients, representing 58.6% of the 87
cases with histological confirmation. The proportion of cases from
which tumor tissue is available largely depends on the origin and
TABLE II – PATHOLOGICAL CHARACTERISTICS AND Ki-ras MUTATIONS
Total n (%)
Total
Sub-site
head
body
tail
unspecified
Histology
non-ductal type
ductal type
Differentiation degree2
well-differentiated
moderately differentiated
poorly differentiated
Sample site2
primary
metastasis
Mutated n (%)
OR
[95% CI]
p value
[0.14–6.91]
[0.11–16.39]
[0.21–2.89]
0.9601
51
30 (58.8)
30 (58.8)
5 (9.8)
3 (5.9)
13 (25.5)
18 (60.0)
3 (60.0)
2 (66.7)
7 (53.8)
1
1.00
1.33
0.78
4 (8.0)
46 (92.0)
0
29 (63.0)
1
15.173
[0.77–299]
0.0261
4 (9.1)
27 (61.4)
13 (29.5)
4 (100)
15 (55.6)
9 (69.2)
1
0.143
0.233
[0.01–2.81]
[0.01–5.36]
0.2551
0.7014
26 (57.8)
19 (42.2)
14 (53.8)
15 (78.9)
1
3.21
[0.84–12.3]
0.0825
Percentages are shown in parentheses.
1Fisher’s exact test.–2Ductal-type EPC only.–3Woolf-Haldane’s correction.–4Mantel-Haenszel x2 test for
linear trend.–5Pearson’s x2.
MALATS ET AL.
664
TABLE III – PATIENT, CLINICAL AND TOXICOLOGICAL CHARACTERISTICS AND DISTRIBUTION OF Ki-ras MUTATIONS
Total n (%)
Total
Gender of patients
Female
Male
Age (years)
,56.4
56.4–63.2
63.3–73.2
.73.2
Hospital3
HSD
HM
Stage
I
II
III
IV
Treatment intention
radical
palliative-symptomatic
Survival (months)
median
Diagnostic certainty
lower
higher
Tobacco
No
Yes
Unspecified
Alcohol
No/light
Moderate/heavy
Unspecified
Alcohol
No
Light
Moderate
Heavy
Mutated n (%)
OR
[95% CI]
p value
51
30 (58.8)
20 (39.2)
31 (60.8)
13 (65.0)
17 (54.8)
1.0
0.65
[0.20–2.08]
0.4721
13 (25.5)
12 (23.5)
13 (25.5)
13 (25.5)
10 (76.9)
6 (50.0)
7 (53.8)
7 (53.8)
1.0
0.30
0.35
0.35
[0.05–1.67]
[0.06–1.89]
[0.06–1.89]
0.4921
0.2832
18 (35.3)
33 (64.7)
8 (44.4)
22 (66.7)
1.0
2.50
[0.77–8.12]
0.1231
17 (33.3)
3 (5.9)
8 (15.7)
23 (45.1)
11 (64.7)
1 (33.3)
4 (50.0)
14 (60.9)
1.0
0.27
0.55
0.85
[0.02–3.67]
[0.10–3.00]
[0.23–3.11]
0.7534
0.8712
10 (19.6)
41 (80.4)
7 (70.0)
23 (56.1)
1.0
0.55
[0.12–2.42]
0.4954
2.83
3.16
22 (44.9)
26 (54.2)
13 (59.1)
15 (57.7)
1.0
0.94
[0.30–2.99]
0.9221
21 (41.2)
24 (47.0)
6 (11.8)
12 (57.1)
16 (66.7)
2 (33.3)
1.0
1.50
[0.45–5.04]
0.5111
19 (37.2)
26 (52.0)
6 (11.8)
9 (47.4)
19 (73.1)
2 (33.3)
1.0
3.02
[0.86–10.5]
0.0791
15 (33.3)
4 (8.9)
16 (35.6)
10 (22.2)
7 (46.7)
2 (50.0)
11 (68.8)
8 (80.0)
1.0
1.14
2.51
4.57
[0.13–10.4]
[0.58–10.9]
[0.72–29.1]
0.3364
0.0712
0.1165
Percentages are shown in parentheses. OR, odds ratio (an OR of 1 denotes the reference category).
1Pearson’s x2.–2Mantel-Haenszel x2 test for trend.–3HSD, Hospital Son Dureta, Palma de Mallorca; HM,
Hospital del Mar, Barcelona.–4Fisher’s exact test.–5Log-rank test.
TABLE IV – ASSOCIATION BETWEEN Ki-ras MUTATIONS AND ALCOHOL
AND TOBACCO CONSUMPTION IN PATIENTS WITH EXOCRINE
PANCREATIC CANCER (N 5 45)
Alcohol
Tobacco
No/light
No/light
Moderate/heavy
Moderate/heavy
No
Yes
No
Yes
Mutations
Yes
No
4
5
8
11
8
2
1
6
OR
p value1
[95% CI]
1
5.00 0.170 [0.65–38.15]
16.00 0.024 [1.45–176.45]
3.67 0.102 [0.77–17.43]
OR, Crude (unadjusted) odds ratio.
exact test.
1Fisher’s
which was also a study based on hospital tumor registries. Of the
124 patients with available tissue, Hruban et al. (1993) were able to
determine Ki-ras mutations in 82 (66%), a proportion only slightly
above our own.
FIGURE 1 – Kaplan-Meier survival curves for mutated and wild-type
exocrine-pancreas-cancer cases.
selection of patients. Unfortunately, seldom has this been specified
in the area of Ki-ras and EPC. An exception is provided by Hruban
et al. (1993), who found no significant differences between cases
with and without molecular results; this finding agrees with ours,
On the prevalence and spectrum of Ki-ras mutations
The overall prevalence of Ki-ras mutations found in this series
was 59%, a figure that is in the lower range of the literature (Caldas
and Kern, 1995). This result was essentially unaltered when the
single acinar-type tumor was excluded from the analysis (60%).
When 3 additional anaplastic carcinomas were excluded, the
mutation frequency was 63%. Several facts could explain the
slightly lower prevalence found in our study: the inclusion of a
wide spectrum of cases, in contrast to other more selected case
Ki-ras MUTATIONS IN EXOCRINE PANCREATIC CANCER
series; geographical variation (Scarpa et al., 1994), especially if we
consider that the hospital from which cases showed the lowest
frequency of mutations (44%) is located in an island (Mallorca);
and less sensitive mutation-detection techniques. The latter is an
unlikely factor, since the sensitivity of the RFLP technique was
high (1%) and, when applied to the study of bile-system cancer, this
method yielded a mutation prevalence higher than previously
described in these neoplasms (Malats et al., 1995).
The mutation spectrum found was similar to that described in
other studies (Caldas and Kern, 1995). Asp and Val were the most
frequent amino-acid substitutions. A bi-mutational pattern was
detected in 5 tumors, and it was not associated with clinical or
pathological characteristics. In 3 of them, the 2 mutations were
present in a single tissue section. Because there is currently no
technique available for detecting Ki-ras mutations in situ, microdissection will be necessary to establish whether both mutations occur
in the same cell population or not. Other authors have described
more than one mutation in samples from the same individual with
EPC (Grünewald et al., 1989; Mariyama et al., 1989; Nagata et al.,
1990; Motojima et al., 1993; Schaeffer et al., 1994; Caldas et al.,
1994; Iguchi et al., 1996), but this pattern appears to be more
prevalent in our series. In this regard, it is noteworthy that a Val
substitution was identified in the 5 cases with a bi-mutational
pattern. Similarly, a review of the literature shows that 14 of the 17
cases in which 2 mutations were present harbored a Val, a
proportion well above that observed in cases with a single mutation
(approximately 30%). The biological significance of this finding is
currently unknown, although it has been proposed that different
Ki-ras mutations may be associated with lesions of distinct
malignant potential (Tada et al., 1996; Berrozpe et al., 1994). If Val
mutations confer on cells a low capacity of progression, additional
mutations in Ki-ras could provide a selective growth advantage.
On the association of Ki-ras mutations with pathological and
clinical characteristics
This analysis yielded 2 significant associations. First, mutations
were not detected in the 4 non-ductal-type tumors. There is now
ample evidence that pancreatic acinar- and islet-cell tumors do not
harbour Ki-ras mutations (Hoorens et al., 1993), but little is known
about anaplastic tumors. Second, the probability of having a
mutated sample when specimens were obtained from metastases
was 3-fold that of primary tumors, in agreement with the results of
Finkelstein et al. (1994). If confirmed, this finding could be
clinically relevant, because it has been proposed that Ki-ras
mutation detection might aid in the diagnosis of EPC (Urrutia and
Dimagno, 1996), and metastases are often biopsied to avoid
complications due to normal pancreatic-tissue damage. Ki-ras
mutations have been reported in ductal proliferative lesions in
patients with and without EPC (Yanagisawa et al., 1993a,b; Tabata
et al., 1993; Tada et al., 1996), in small or pre-invasive EPC
(Schaeffer et al., 1994), and in cystadenomas (Berrozpe et al.,
1994), suggesting that they are an early event in pancreatic
carcinogenesis. These findings are not necessarily in conflict with
our results, since Ki-ras activation might play a role both early and
late in the course of tumor progression. It is surprising that very
little information is available in the literature for comparison: few
studies provide details on Ki-ras mutations in primary and
metastatic tumors; primary and metastatic lesions from a given
patient have been studied only in a very small number of cases
(Almoguera et al., 1988; Mariyama et al., 1989). The analysis of
multiple tissue samples from the same patient, including preneoplastic ductal lesions, primary tumor and metastasis, remains a
priority. Of even greater interest and complexity is the analysis of
serial tissue samples obtained from the same individual at different
time points.
In agreement with other reports (Grünewald et al., 1989; Shibata
et al., 1990; Nagata et al., 1990; Hruban et al., 1993), Ki-ras
mutations were weakly or not at all associated with the clinical
characteristics of the patients, or with survival. The hypothesis of a
665
higher frequency of mutations among cases with higher diagnostic
certainty (Porta et al., 1994) was not confirmed here. Probably, this
was due to the fact that all patients with available tissue had a solid
diagnostic basis.
On the association of Ki-ras mutations with tobacco
and alcohol consumption
Only 2 studies have previously assessed the relationship between
tobacco and Ki-ras mutations in EPC. Nagata et al. (1990)
observed a negative association, with 86% of mutated cases among
ever-smokers and 96% among never-smokers (OR, 0.25; p 5 0.289).
Conversely, Hruban et al. (1993) found a positive association, with
88% of mutated cases among ever-smokers and 68% among
never-smokers (OR, 3.53; p 5 0.046). In our series, the prevalence
of mutated cases was 67% in smokers and 57% in non-smokers. ras
mutations have been described in other tobacco-related neoplasms
such as lung, upper aerodigestive tract, and bladder cancer,
although the prevalence and ras gene affected in each of these
locations differ. In lung cancer, Ki-ras mutations appear to be
strongly associated with smoking, and a large proportion are
G-to-T transversions (Husgafvel-Pursiainen et al., 1993). This
nucleotide substitution is consistently induced by benzopyrene, a
tobacco carcinogen, in experimental studies (Fujji et al., 1990). We
found no significant association between tobacco and mutation
spectrum, regardless of alcohol consumption (data not shown).
Little is known concerning Ki-ras mutations and alcohol drinking. The prevalence of ras mutations in alcohol-related cancers
(stomach, liver, and upper-aero-digestive-tract tumors) is reportedly low. We here report an association between Ki-ras mutations
and alcohol consumption. Our results suggested an interaction
between tobacco and alcohol. The possibility of such interaction
has received little attention until now. A recent study by Silverman
et al. (1995) indicated that moderate alcohol drinking does not
increase the risk of EPC. However, black non-smokers who had
heavy alcohol consumption did have an increased risk; such
increase was not observed among smokers, suggesting that a
detrimental effect of alcohol drinking might be stronger among
non-smokers than among smokers.
There is currently no information on a possible interaction
among tobacco, alcohol consumption and Ki-ras mutations at any
tumor site in humans. Two studies have described the relationship
between tobacco, alcohol and mutations in the p53 gene in patients
with upper-aero-digestive-tract cancers. Brennan et al. (1995)
observed a higher risk for p53 mutations in patients who smoked
and drank in comparison with those who only smoked, suggesting a
synergistic interaction. Franceschi et al. (1995) did not find such
interaction, although in their study only immunohistochemical
techniques were used. The only report on p53 alterations and
tobacco in EPC showed that nuclear accumulation of p53 was more
common among non-smokers than among smokers; alcohol drinking was not assessed (Lee et al., 1995).
The antagonistic interaction between tobacco and alcohol consumption suggested by our results must be interpreted cautiously. If
it is confirmed by subsequent studies, a number of theoretical
issues would need to be addressed: what is the role of the pancreas
in the metabolism of carcinogens involved in EPC? can alcohol
consumption modify the metabolism of tobacco-derived carcinogens potentially involved in the development of EPC? do tobacco
and alcohol compounds compete for the same metabolic pathways?
Foster et al. (1993) found higher levels of P450 enzymes in
pancreas and liver tissue from patients with chronic pancreatitis
and pancreas cancer than in organ donors. Recent work has
confirmed that CYP2E1, which is alcohol-inducible, and CYP1A1,
which is involved in the metabolism of aromatic hydrocarbons, are
present in the exocrine pancreas (Chassagne et al., 1995).
Limitations and concluding remarks
This study is an example of how the molecular classification of
tumors may be of help to better assess risk estimates for specific
666
MALATS ET AL.
exposures. Nevertheless, several study limitations must be noted.
First, although this is one of the largest published series on Ki-ras
mutations in EPC, the number of cases analyzed is small, and risk
estimates lack precision. Second, information on tobacco and
alcohol consumption was obtained from medical records and could
be subject to a misclassification bias leading to underestimation of
the association; however, the interaction between alcohol and
tobacco was observed regardless of the cut-off used to categorize
alcohol drinking. Finally, the interaction could be an effect of the
statistical model used. Yet, the calibration and discrimination tests
of the multiplicative model, including the interaction term, indicated that the model fitted the data well. Moreover, similar results
were obtained using an additive model (data not shown).
Whereas our findings need to be confirmed in larger, preferably
prospective studies of unselected groups of patients, they provide
new leads on the clinico-pathological correlates of Ki-ras mutations, and on the role of tobacco and alcohol in pancreatic diseases.
ACKNOWLEDGEMENTS
The study was partly supported by the Fondo de Investigación
Sanitaria (FIS) (grants 91/0595 and 92/0007), by Fundación Salud
2.000 (grant Serono), by the Institut Municipal d’Investigació
Mèdica (IMIM) (grant 875138/9), and by the Comissió Interdepartamental de Recerca i Innovació Tecnològica (CIRIT) (grants
GRQ93-9301 and SGR 434). We are grateful to Dr. C. Balagué, Dr.
G. Berrozpe, Ms. E. Carrillo, Mr. D.J. MacFarlane, Dr. A. Salas,
Ms. A. Serrat, Dr. T. Thomson and Dr. M. Torà for valuable
contributions, and to Mr. L. Español, Ms. P. Barbas and Ms. H.
Martinez for secretarial assistance.
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