Download Specific codon 13 K-ras mutations are predictive of clinical outcome

Original article
Annals of Oncology 13: 1438–1446, 2002
DOI: 10.1093/annonc/mdf226
Specific codon 13 K-ras mutations are predictive of clinical
outcome in colorectal cancer patients, whereas codon 12 K-ras
mutations are associated with mucinous histotype
V. Bazan1†, M. Migliavacca1†, I. Zanna1, C. Tubiolo1, N. Grassi2, M. A. Latteri2, M. La Farina3,
I. Albanese3, G. Dardanoni4, S. Salerno5, R. M. Tomasino6, R. Labianca7, N. Gebbia5 & A. Russo1*
Department of Oncology, 1Section of Molecular Oncology, 5Section of Medical Oncology and 2Section of Oncology Surgery, Regional Reference Center for
the Biomolecular Characterization of Neoplasies and Genetic Screening of Hereditary Tumors, Palermo; 6Institute of Pathology, School of Medicine;
3
Department of Cellular and Development Biology, Biomolecular Section, University of Palermo, Palermo; 4Epidemiological Observatory,
Center of Sicilian Region, Palermo; 7Medical Oncology Unit, Ospedali Riuniti Bergamo, Italy
Received 9 November 2001; revised 12 February 2002; accepted 11 March 2002
Background: K-ras mutations, one of the earliest events observed in colorectal carcinogenesis, are
mostly found in codons 12 and 13, and less frequently in codon 61, all three of which are estimated to be
critical for the biological activity of the protein. Nevertheless the prognostic significance of such
mutations remains controversial. Our purpose was to assess whether any or specific K-ras mutations in
primary colorectal cancer had prognostic significance and were linked to clinico-pathological parameters.
Patients and methods: Paired tumor and normal tissue samples from a consecutive series of
160 untreated patients (median of follow up 71 months), undergoing resective surgery for primary colorectal carcinoma, were prospectively studied for K-ras mutations by PCR/single strand conformation
polymorphism sequencing.
Results: Seventy-four of the 160 (46%) primary colorectal carcinomas presented mutations in K-ras:
54% in codon 12, 42% in codon 13 (particularly G→A transition) and 4% in both. Codon 12 K-ras
mutations were associated with mucinous histotype (P <0.01), while codon 13 K-ras mutations were
associated with advanced Dukes’ stage (P <0.05), lymph-node metastasis (P <0.05) and high S-phase
fraction (P <0.05). Multivariate analysis showed that codon 13 K-ras mutations, but not any mutation,
were independently related to risk of relapse or death.
Conclusions: Our results suggest that codon 12 K-ras mutations may have a role in the mucinous
differentiation pathway, while codon 13 mutations have biological relevance in terms of colorectal
cancer clinical outcome.
Key words: colorectal carcinoma, DNA ploidy, K-ras mutations, prognosis
Introduction
The family of ras genes codes monomeric proteins of 21 kDa
(p21ras), which are able to bind and hydrolyze GTP. Three
different proteins, known as Harvey- (H-), Kirsten- (K-) and
N-RAS, with ∼90% homology [1], each of which is preferentially enriched in specific tissue types, act as mediators in the
transduction of extracellular signals to the cytoplasm and
nucleus. By interacting with a great number of regulators and
effectors, they control multiple pathways affecting cell
growth, differentiation and apoptosis [2]. Specific mutations
†V. Bazan and M. Migliavacca contributed equally to this work
*Correspondence to: Dr A. Russo, Via Veneto 5, 90144 Palermo, Italy.
Tel/Fax: +39-091-6554529; E-mail: [email protected]
© 2002 European Society for Medical Oncology
in the ras genes lead to the formation of constitutively active
(i.e. GTP-bound) proteins, which trigger the transduction of
proliferative and/or differentiative signals even in the absence
of extracellular stimuli [3]. Activating mutations in such genes
have been observed in ∼30% of human tumors, with an
extremely variable incidence according to the type of tumor
(colon, lung, thyroid, pancreas) and its site [4, 5]. In particular,
oncogenic mutations of K-ras are involved in 40% (20–50%)
of colorectal cancers (CRCs) [6–9]. Most of the mutations
(90%) are found in codons 12 and 13; less frequently, they
may affect also codon 61 [10–13]. These mutations are monallelic and appear early in carcinogenesis, mostly between the
stages of early and intermediate adenoma, maintaining a constant incidence in late adenomas and in carcinomas [14, 15].
Recent studies seem to suggest that specific ras gene
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mutations may not only be linked to dietary factors and/or
ethnic differences, but also characterize the different phases of
colorectal carcinogenesis [16–19]. So far, most of the studies
conducted to assess the prognostic significance of K-ras
mutations in CRCs have taken into account any mutations of
the gene [20–31]. The results thus obtained have proved to be
discordant, with only some of them indicating an influence of
K-ras mutations on clinical outcome [26–31]. It has recently
been suggested that some specific K-ras mutations among all
those that can alter codons 12 or 13, both of which are important for protein functionality, might have a stronger predictive
value [32–37]. The aim of this study, therefore, was to assess
the mutational status of the K-ras gene in 160 cases of CRCs
and to establish whether or not any or specific mutations of
the gene might have prognostic significance or be linked to
clinico-pathological parameters.
Patients and methods
Study design and clinicopathological data
A prospective study was performed on paired tumor and normal tissue
samples collected by the Molecular Oncology Section of the University of
Palermo from a consecutive series of 160 patients undergoing potentially
radical surgical resection for primary operable CRC at a single institution
(Department of Oncology, University of Palermo) from January 1988 to
December 1992.
Elegibility criteria used were: (i) electively resected primary CRC;
(ii) processing of fresh paired normal mucosa tumor samples within
30 min after tumor removal; (iii) available DNA from normal and tumor
tissue for biomolecular analyses; and (iv) access to accurate follow-up
information.
Briefly, the following exclusion criteria were used: (i) history of previous neoplasms; (ii) patients from families with familial adenomatous
polyposis or hereditary non-polyposis CRC with a highly penetrant
genetic predisposition to CRC; (iii) synchronous or metachronous CRC;
and (iv) chemotherapy or radiation therapy prior to surgery.
This series of patients comprised 84 females and 76 males with a
median age of 66 years (range 31–88 years). A resection of the primary
CRC was performed in all cases. A total of 137 patients was potentially
cured by means of radical surgical tumor resection with regional en bloc
lymphadenectomy proximally up to the origin of the vascular trunks.
Twenty-three patients had either non-radical surgery or distant metastases. In order to avoid evaluator variability in the patients, all resection
specimens and microscopic slides were meticulously examined by two
independent pathologists (R.M.T. and M.M.), who were not aware of the
original diagnosis or the results of the molecular analyses. The complete
excision of the primary tumor was histologically proven by examination
of the resected margins. All tumors were histologically confirmed to be
CRCs. In addition, the pathologists assessed tumor site (proximal or distal
tumors), tumor size, pathological stage, tumor grade (histological
differentiation), presence or absence of lymph node metastases, tumor
growth (expansive or infiltrative), tumor type [not otherwise specified
(NOS) or mucinous adenocarcinoma], presence or absence of vascular
and lymphatic invasion or tumor lymphocytic infiltrate.
Sixty of the tumors were ≤5 cm and 100 were >5 cm. This series
included 31 proximal tumors (from the caecum to the right-sided half of
the transverse colon), and 129 distal (from the left half of the trasverse
colon to the rectum). According to Turnbull’s modification of Dukes’
system [38], the tumors were staged from A to D as follows: 40 as A, 51 as
B, 41 as C and 28 as D. Assessment of the grade of differentiation of the
tumor showed that there were 23 well differentiated (G1), 104 moderately
differentiated (G2) and 33 poorly differentiated CRC. Node status was
known for all patients: 59 were node positive and 101 were node negative.
One hundred and forty cases were CRC with infiltrative growth and 20
were expansive. One hundred and thirty-seven tumors were classified as
NOS adenocarcinomas and 23 were considered as mucinous (presence of
mucin in >50% of the tumoral area as determined from the available histological sections). One hundred and fifteen tumors showed vascular and/or
lymphatic invasion. Moreover, 48 CRCs had a prominent tumor lymphocytic infiltrate. Finally, metastatic cases were identified by clinical
and histopathological analyses of neoplastic cells in organs such as the
lymph nodes, the liver and so on, along with the primary tumor. Postoperatively, all patients were checked at 3-monthly intervals for the first
2 years, at 6-monthly intervals for the next 2 years and annually thereafter.
The follow-up program included a clinical examination, blood tests
[including carcinoembryonic antigen (CEA) assay], annual chest radiography and endoscopy. Abdominopelvic computed tomography (CT)
scan was also performed each year for the first 2 years. Disease relapse
(local recurrence or distant metastases) was confirmed histologically
where possible.
Written informed consent was obtained from all patients included
in this study. Information on survival [disease-free survival (DFS) and
overall survival (OS)] was obtained directly from clinical charts and
through the Oncology Section at our Institution. Clinico-pathological
and follow-up data for all patients have been recorded prospectively in a
computerized registry database.
Patients with Dukes’ stage A and B CRC were treated with surgery
alone, whereas only 10 patients with Dukes’ stage C received adjuvant
chemotherapy with 5-fluorouracil (FU), leucovorin and levamisole, since
hardly any of the patients had received adjuvant treatment during the
period previous to 1991. Patients who had undergone non-radical surgery
and/or with distant metastases were treated by 5-FU and leucovorin.
The study end points were DFS (the interval between the day of
primary surgery and the date of the first recurrence or metastasis) and OS
(the time from the date of surgery to the date of death, from cancer-related
causes, or last follow-up). The median follow-up time in our study group
was 71 months (range 34–115 months). The median survival of the whole
group was 43 months.
Tissue handling
Multiple samples (6–10) of the primary tumor tissue were taken from different tumor areas (including the core and the invasive edge of the tumor).
The portion of primary tumor was obtained by superficial biopsy of either
the tumor bulk or the edge of the malignant ulcer for more infiltrative
cancer. All tissues were carefully trimmed to remove as much nonneoplastic tissue as possible, avoiding the non-viable areas. Furthermore,
multiple samples of normal mucosa (as confirmed by histology) were
taken from macroscopically uninvolved area 20–40 cm away from the
tumor site, to be used as control for biomolecular and flow cytometric
analysis. The tissues were bisected, one half of each sample was processed for pathological examination, and the remaining half of the sample
pool was immediately frozen and stored at –80°C until analyzed. The
adequacy of the material was checked on frozen tissue sections, and only
tissue samples with >80% tumor content were utilized in subsequent biomolecular and flow-cytometric analysis. Where present, areas with a high
content of non-neoplastic cells were removed from the frozen block with
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a scalpel. Evaluation of each biomolecular variable [K-ras alterations,
DNA ploidy and S-phase fraction (SPF)] was performed independently by
researchers who had no knowledge of the clinical data for the samples.
DNA extraction
High molecular weight genomic DNA was extracted from primary CRC
and normal colon specimens as described previously [39].
Detection of K-ras gene mutations
Mutations within the K-ras gene were detected by single strand conformation polymorphism (SSCP) analysis following PCR amplification
of exon 1, performed as described previously [40]. In every instance,
negative controls (DNA was replaced with water) were amplified by PCR
and included in the experiment. In all PCR assays, aerosol-resistant
pipette tips were used to avoid cross-contamination (Eppendorf, Egham,
Germany). The quality and the concentration of the amplification products were verified by 1.5% agarose gel electrophoresis and ethidium
bromide staining. Aliquots (100 ng) of the amplified DNA fragments,
purified and concentrated by filtration through Microcon 50 columns
(Amicon, Beverly, MA, USA) were denatured and analyzed by SSCP
analysis. PCR/SSCP analysis was repeated twice for each sample to minimize the possibility of artifacts due to contamination or polymerase
errors. Interpretation of SSCP analysis of DNA fragments was performed
by consensus of two investigators. DNA of normal colon tissue from each
patient was also amplified and run in parallel with matched tumoral DNA
samples on SSCP gels in order to evaluate the occurrence of non-somatic
mutations or polymorphisms.
Individual single-strand (ss) DNA fragments with shifted mobilities,
compared with normal control, were electroeluted from polyacrylamide
gel, re-amplified and sequenced as described previously [41].
Flow-cytometric analysis
DNA flow cytometry was performed on mechanically disaggregated
samples of frozen tumor tissue to determine DNA ploidy, DNA index and
SPF as previously reported [42].
Statistical analysis
Fisher’s exact test (StatXact Turbo, Cytel Software Corporation,
Cambrige, MA, USA) was used to evaluate the associations between biological variables. The relationship of different prognostic variables with
DFS and OS was assessed univariately using the Kaplan–Meier method.
Significant differences among survival curves were checked by the logrank test and Wilcoxon test, or a test for trend when appropriate. Multivariate analysis was carried out by means of Cox’s logistic regression
model, using a backward procedure [43]. P values <0.05 were considered
significant.
Results
Mutation analysis of the K-ras gene
Mutation analysis of exon 1 of the K-ras gene was performed
on genomic DNA from primary CRCs of 160 patients by the
PCR/SSCP technique. The absence of abnormal bands was
assessed in at least two independent PCR/SSCP analyses.
Figure 1. SSCP analyses of exon 1 of the K-ras gene, amplified from
CRC and mucosa genomic DNA of two patients. In each pair of lanes the
normal tissue DNA is on the right and the tumor DNA is on the left. The
extra bands visualized in lanes 1 and 3 correspond to ssDNA molecules
harboring mutations in codon 12 (GGT→TGT) and codon 13
(GGC→GAC), respectively, as confirmed by sequencing. Lane 5 shows
the negative control: DNA wild type.
Aberrantly migrating bands were found in 46% (74 of 160) of
the cases (Figure 1). Sequence analysis of the DNA fragments
with altered electrophoretic mobility made it possible to establish the exact site and nature of the genetic alteration in all
tumor samples. Overall, 80 k-ras2 mutations were identified
in 74 of the 160 screened CRCs. Of the 80 mutations, 57% (46
of 80) were found in codon 12 and 43% (34 of 80) in codon 13
(68 of 74 tumors presented single mutations, three of 74 a
double mutation in codon 12, and three of 74 a mutation in
codon 12 and one in codon 13). No mutations were detected at
any other site of the first exon. The most frequent mutation in
codon 12 was GGT to GAT (G→A), which was observed in
17 of 46 cases (37%). The other mutations observed in the
same codon resulted in replacement of glycine with valine
(GGT to GTT; 13 cases, 28%), cysteine (GGT to TGT; nine
cases, 20%), serine (GGT to AGT; five cases, 11%) or alanine
(GGT to GCT; two cases, 4%).
The most frequent mutation in codon 13 was GGC to GAC
(G→A), which was detected in 32 of 34 cases (94%). The
other mutation observed was GGC to TGC (G→C) in two of
34 cases (6%). The distribution of the 80 mutations identified
in the CRC DNA samples analyzed is summarized in Table 1.
Overall, transitions (70%; 56 of 80) were far more frequent
than transversions (30%), G:C to A:T mutations being the
most frequent type of transition represented (96%).
The most common type of mutation was a G:A transition at
the second base of codon 13, which occurred in 40% of all
k-ras mutations. In this same codon, no other type of transition, and only two (two of 34, 6%; two of 80, 2.5%) G:T
trasversions, in the first position, were detected in our group of
patients. In codon 12, the most frequent mutations were again
G:A transitions in the second position (17of 80, 21%; 17 of 46,
37%), but other mutations were also detected frequently (29 of
46 in all). No germ-line mutations were found, indicating that
in every case the change was somatic.
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Table 1. Distribution of the 80 mutations detected in the first exon of K-ras gene in 160 colorectal cancers
Mutated codon
Type of mutation
12
Transitions
Mutated sequence (amino acid)
No. of mutations (%)
(1st position)
AGT (Ser)
5 (11)
(2nd position)
GAT (Asp)
17 (37)
GCT (Ala)
2 (4)
(1st position)
TGT (Cys)
9 (20)
(2nd position)
GTT (Val)
13 (28)
GAC (Asp)
32 (94)
TGC (Cys)
2 (6)
Transversions
13
Transitions
(2nd position)
Transversions
(1st position)
Table 2. Significant relationships of specific K-ras gene mutations to clinico-pathological and biological variables in 160 colorectal
cancer patients
Variables
K-ras
P
No mutations (%)
Mutations in codon 12 (%)
Mutations in codon 13 (%)
A+B
54 (60)
24 (26)
13 (14)
C+D
32 (46)
16 (23)
21 (31)
Negative
60 (59)
27 (27)
14 (14)
Positive
26 (44)
13 (22)
20 (34)
≤18.3%
47 (58)
24 (30)
10 (12)
>18.3%
39 (50)
16 (20)
24 (30)
77 (56)
27 (20)
33 (24)
9 (39)
13 (57)
1 (4)
86 (54)
40 (25)
34 (21)
Dukes’ stage
<0.05
Node status
<0.05
SPF
<0.05
Tumor type
Not mucinous
Mucinous
Total
Cellular DNA content and SPF evaluation
<0.01
Adequate DNA histograms were obtained from all normal and
tumoral tissues by means of flow cytometry. The coefficients
of variation of the DNA diploid peak ranged from 2.5% to
4.8% (median 3.4%). DNA aneuploidy was found in 75% of
cases (120 of 160), and 18% of these (22 of 120) showed
multiclonality. The SPF ranged from 2.1% to 32.6% (median
18.3%, interquartile range 14.1–21.7%). The median SPF of
DNA aneuploid tumors was 19.2%, while that of the DNA
diploid tumors was 12.4% (P <0.01). By using the SPF
median value as cut-off point, tumors were accordingly
divided into low (≤18.3) and high (>18.3) SPF tumors.
codon 12 versus in codon 13, or Asp13 versus Asp12 versus
Val12 versus Cys12 versus Ser/Ala12, or trasversions versus
transitions) and the clinico-pathological and biological variables analyzed.
However, when all the patients were divided into three
groups according to their K-ras gene status (no mutation
versus codon 12 ras mutation versus codon 13 ras mutation), a
significant association (P <0.05) emerged between codon 13
mutations and advanced Dukes’ stage (C and D), lymph node
metastases and high SPF (>18.3%). Moreover, codon 12
mutations were significantly associated with mucinous histotype (Table 2).
Relationship between biomolecular indicators and
clinical data
Univariate and multivariate analysis of prognostic
factors
No significant relationship was seen between the presence
(any mutations) or type of K-ras mutations (mutations in
Upon univariate analysis, distal cancers, advanced Dukes’
stage, lymphohematic invasion, DNA aneuploidy, high SPF,
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Table 3. Kaplan–Meier DFS (n = 138) and OS (n = 160) analysis of biological variables in 160 colorectal cancer patients
No. of patients
5-year DFS (%)
No. of patients
5-year OS (%)
Diploid
38
75
40
77
Aneuploid monoclonal
81
34
Aneuploid multiclonal
19
10
98
33
22
9
≤18.3%
72
60
81
59
>18.3%
66
22
79
21
No mutations
77
50
Any mutations
61
32
86
47
74
33
No mutations
77
50
86
47
Mutation in codon 12
34
47
Mutation in codon 13
27
15
40
47
34
18
No mutations
77
50
86
47
Mutation Asp13
Mutation Asp12
25
9
32
14
14
50
15
49
Mutation Val
9
56
12
64
Mutation Cys
7
80
9
71
Mutation Ser/Ala
6
17
6
14
No mutations
77
50
86
47
G→A
43
24
51
25
G→T
16
64
21
67
G→C
2
–
2
–
DNA ploidy status
P
P
a
<0.01
<0.01
b
SPF
<0.01
<0.01
K-rasc
<0.05
<0.01
<0.01
<0.05
<0.01
<0.01
<0.01
<0.01
a
All DNA aneuploid subgroups are compared with patients with DNA diploid tumors.
All high SPF subgroups are compared with patients with low SPF tumors.
c
All mutation subgroups are compared with patients with no mutations (wild-type K-ras).
DFS, disease-free survival; OS, overall survival.
b
the presence and type of K-ras mutations (mutations in codon
13 versus in codon 12, or Asp13 versus Asp12 versus Val12
versus Cys12 versus Ser/Ala12, or trasversions versus transitions) proved to be significantly related to quicker relapse,
whereas these same factors, and in addition infiltrative tumor
growth, absence of lymphocytic infiltration and presence of
lymph node metastases, were significantly related to shorter
overall survival (Table 3). Figure 2 shows the probability of
DFS and OS according to K-ras mutations in codons 12 and
13. The significant variables at univariate analysis were
entered in a multivariate logistic regression model with backward elimination. The major significant predictors for both
disease relapse and death were advanced Dukes’ stage,
aneuploid tumors, high SPF and K-ras mutations in codon 13,
while lymphohematic invasion was an independent factor
only for relapse and non-curative resection for death (Table 4).
Discussion
Vogelstein was the first to suggest that K-ras mutations are
early events in colorectal carcinogenesis, found mostly in the
transition stage of a small benign adenoma into a larger,
dysplastic (more aggressive villous) adenoma [4, 44].
The frequency reported for such mutations in CRCs varies
from 20% to 50% [6–9]. Such a wide range might be due to
several factors, such as tumor storage method (fresh/frozen
tissue and paraffin-embedded blocks), the different techniques used for assessing the presence of mutations (SSCP,
denaturing gradient gel electrophoresis, temperature gradient
gel electrophoresis, direct sequencing), tumoral heterogeneity, or the specific features of the patient cohorts included
in the study, such as histopathological staging, grading, site of
the tumor and/or the production of biliary acids and feco-
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pentanes [17, 45]. At any rate, the fact that the K-ras mutation
rate does not exceed 50%, suggests that there may be an
alternative pathway in colorectal carcinogenesis.
In our own study, conducted on 160 CRCs, we found a
K-ras mutation rate in exon 1 of 46%: 57% in codon 12 and
43% in codon 13. This is hardly surprising since most of the
mutations found in human tumors involve these two codons,
coding for two adjacent glycines located in proximity of the
catalytic site of RAS. Any mutation resulting in the incorporation of a different amino acid at these positions leads, albeit to
a different degree, to a reduction of the intrinsic GTPase
activity of RAS [46].
All the mutations that we identified were missense and, as
several other authors have already observed, lead predominantly to the substitution of glycine for aspartate [17, 32, 33, 36,
37]. Our results also confirm that in CRCs the transition rate is
higher than the transversion rate, with a frequency of 70% and
30%, respectively. All the transitions were of the GC→AT
type.
Our results showed an association between the K-ras
mutations in codon 13 and advanced clinical stages, and the
presence of lymph node metastases, suggesting that they
might be directly involved in tumoral progression and aggression; alternatively, they might either occur later on, during
advanced stages of the disease, or be associated with a form of
disease that is already advanced at diagnosis.
Codon 13 mutations also proved to be associated with high
SPF, thus confirming a previously known fact, that is, that the
constitutively active RAS protein causes a deregulation of the
cell cycle with resulting hyperproliferation [47]. Quite interestingly, however, we found that K-ras mutations in codon 12,
which we expected would have an effect very similar to that of
mutations in codon 13 on the functionality of RAS, are instead
significantly associated with the mucinous histotype, suggest-
Figure 2. DFS (A) and OS (B) of 160 patients with colorectal cancer according to specific K-ras mutations.
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Table 4. Multivariate regression analysis to predict the hazard ratio of relapse or death in 160 CRC patients
Relapse (n = 138)
Death (n = 160)
HR (95% CI)
P
HR (95% CI)
P
3.07 (1.26–7.45)
<0.05
6.40 (2.68–15.3)
<0.01
3.96 (1.78–8.79)
<0.01
Dukes’ stage
D versus A
Surgery
Non-curative resection versus curative resection
Lymphohematic invasion
Present versus none
2.02 (1.12–3.63)
<0.05
Aneuploid monoclonal versus diploid
2.97 (1.43–6.16)
<0.01
2.46 (1.22–4.98)
<0.05
Aneuploid multiclonal versus diploid
6.21 (2.66–14.5)
<0.01
4.92 (2.20–11.0)
<0.01
2.49 (1.51–4.10)
<0.01
2.15 (1.36–3.39)
<0.01
1.79 (1.01–3.20)
<0.05
1.93 (1.17–3.18)
<0.05
DNA ploidy
SPF
>18.3% versus ≤18.3%
K-ras
Mutation in codon 13 versus no mutation
HR, hazard ratio; CI, confidence interval.
ing that they preferentially affect the signal transduction pathways involved in the regulation of mucin production in
colonic mucosa cells, whereas they do not prove to be associated with an altered rate of cell proliferation. Other authors
have already reported an association between the presence of
specific codon 12 K-ras mutations and mucinous histotype,
both in colorectal carcinomas [48–50] and in intestinal-type
ovarian carcinomas [51]. Current knowledge on the biological
consequences of different RAS mutations on cell behavior
does not allow the proposition of a clear explanation for this
unexpected finding. However, several reports indicate that at
least in some cell types a different outcome of RAS activation,
i.e. proliferation versus differentiation, is dependent on the
strength and the duration of the signal that activates RAS [52].
Since different amino acid substitutions in RAS alter to a
different extent the GTPase activity of this protein and/or its
ability to interact with its regulators and effectors [53, 54],
thus leading to a more or less persistant ‘active’ state of RAS,
it may not be unreasonable to suggest that some mutations
may stimulate cell proliferation more effectively than others.
Conversely, mucin production, a physiological property of
intestinal epithelial cells, as well as of several other epithelia,
and stimulated by a number of factors, among which are
TGF-α or other signal molecules whose receptors activate
RAS-mediated pathways, may be preferentially upregulated
in tumoral cells expressing RAS activated by a different
subset of mutations. Direct evidence for RAS-dependent
activation of the MUC2 gene, coding for one of the >13 types
of mucin identified so far [55, 56], in airway and colon epithelial cell lines has recently been reported by Li et al. [57],
and TGF-α upregulation of the MUC4 promoter in pancreatic
carcinoma cells has been observed by Perrais et al. [58].
Conflicting results have been reported in previous studies
on the prognostic significance of K-ras mutations in CRC,
probably due to some of the previously mentioned factors.
Several studies have, in fact, reported a reduced survival rate
in patients with K-ras mutated tumors [26–31], while others
have found no association with prognosis [20–25]. It has
recently been demonstrated that evaluation of the specific
nucleotide substitutions, and therefore amino acid changes, in
ras may provide more relevant information concerning the
aggressive potential of the tumor and the clinical outcome of
CRC patients [32–37].
Although earlier studies reported that patients with codon
12 K-ras mutated to GAT (Asp) or GTT (Val) had a worse
prognosis [9, 31, 37], several recently published reports also
suggest the importance of specific codon 13 mutations [31,
36]. In a large population-based study of 1413 individuals
with CRC, Samowitz et al. [36] observed that the codon 13
G→A mutation was associated with reduced survival rate.
We too found that codon 13 mutations, which in our case
were almost all G→A transitions, leading to the amino acid
substitution Gly13→Asp13, had a stronger predictive value
than any K-ras mutations. Multivariate analysis showed that
this specific codon 13 K-ras mutation was an independent
prognostic factor for DFS and OS, together with the biological
variables of DNA ploidy and SPF and staging, and lymphohematic invasion for DFS and the type of resection for the OS.
Since GGC to GAC was by far the most frequent mutation in
codon 13 found in our study, we are unable to discriminate
between the prognostic significance of codon 13 mutations in
general and that of this specific codon 13 mutation. Confirming some of the previously mentioned recent reports, however,
our data indicate quite clearly that at least this mutation is
1445
significantly linked to the biologically aggressive potential of
the tumor and the clinical outcome of patients affected by
CRCs. In contrast to several other reports [9, 31, 37], in our
study neither any nor a specific codon 12 mutation appeared to
have any prognostic relevance.
It is still, therefore, not clear whether both or only one of the
two codons are important for prognosis, and in what way the
different nucleotide substitution in these codons might lead to
variations in the biological activity of the oncogene.
According to Cerottini et al. [33], the two codons might both
be equally important, at least when affected by specific mutations; these authors found that CRCs haboring the codon 12
TGT mutation or codon 13 GAC mutation were significantly
associated with worse prognosis, if taken together. It cannot
be ruled out, however, that the differences found in the
frequency of specific mutations in K-ras, due to the genetic
and/or dietary differences between the various populations of
patients analyzed [16, 17, 19], might justify the different
codon 12 and 13 prognostic relevance reported in the literature. In fact, several studies showing that specific codon 12
K-ras mutations are associated with recurrence or poorer
prognosis found a lower proportion of codon 13 mutations in
their cases [9, 31]. On the contrary, our cases showed a more
similar mutation rate for the two codons (57% of mutations in
codon 12 versus 43% in codon 13).
In conclusion, our own results suggest that mutations affecting codon 13 may also have biological relevance in terms of
CRC clinical outcome and those of codon 12 may be specifically involved in the regulation of mucins. Since the protein
RAS is involved in various cell processes, it might be that
different mutations of the gene affect to a different extent the
numerous, diverse pathways regulated by this protein.
Further studies might clarify the predictive role of specific
K-ras mutations in CRCs and, moreover, might form the basis
for a more patient-specific adjuvant therapy and a more careful follow-up, above all in the case of intermediate tumors,
where the outcome might be more closely linked to the genetic
features of the tumor.
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