Cyclooxygenase-2 Overexpression

Is Related to Polypoid Growth and

K-ras Gene Mutation in T1
Colorectal Carcinomas
Takuya Okawa, M.D.,
1
Keigo Yoshinaga, M.D.,
2
Hiroyuki Uetake, M.D.,
1
Takanobu Sato, M.D.,
1
Tetsuro Higuchi, M.D.,
1
Tetsuo Nemoto, M.D.,
3
Kenichi Sugihara, M.D.
1
1
Department of Digestive Surgery, Tokyo Medical and Dental University Graduate School, Tokyo, Japan
2
Department of Surgery, National Printing Bureau Tokyo Hospital, Tokyo, Japan
3
Department of Human Pathology, Tokyo Medical and Dental University Graduate School, Tokyo, Japan
PURPOSE: Cyclooxygenase-2 is thought to play a role in the
development of intestinal tumors and levels are elevated in
approximately 80 to 90 percent of human colorectal carci-
nomas. To clarify the role that cyclooxygenase-2 plays in the
development of colorectal carcinoma, we studied the rela-
tionship between cyclooxygenase-2 expression and tumor
morphology and that between cyclooxygenase-2 expres-
sion and K-ras mutation. METHODS: We classified 48 T1
colorectal carcinomas as polypoid or nonpolypoid and ana-
lyzed the clinicopathologic features. The expression of cy-
clooxygenase-2 was determined immunohistochemically,
and nested polymerase chain reaction-restriction fragment
length polymorphism detected a K-ras codon 12 mutation.
RESULTS: Cyclooxygenase-2 expression was higher in pol-
ypoid carcinomas than in nonpolypoid carcinomas (P <
0.001). The K-ras codon 12 mutation was associated with
higher levels of cyclooxygenase-2 expression compared
with carcinomas without this mutation (P = 0.028). CON-
CLUSIONS: Polypoid growth and K-ras gene mutation are
both associated with increased levels of cyclooxygenase-2
expression in T1 tumors. [Key words: Morphology; Polyp-
oid growth carcinoma; Nonpolypoid growth carcinoma; Co-
lorectal cancer; Cyclooxygenase-2 overexpression; K-ras
mutation]
S
everal epidemiologic studies have identified a 40
percent to 50 percent decrease in mortality from
colorectal carcinoma in patients who use nonsteroidal
anti-inflammatory drugs (NSAIDs).
1,2
Sulindac and ce-
lecoxib reduce the number and size of colorectal pol-
yps in familial adenomatous polyposis.
3,4
Further-
more, NSAIDs inhibit cyclooxygenase (COX) activity,
which catalyzes the conversion of arachidonic acid to
prostaglandin G
2
. Two isoenzymes of COX have been
identified. Cyclooxygenase-1 (COX-1) is constitu-
tively expressed in most tissues, whereas cyclooxy-
genase-2 (COX-2) is induced by pathologic stimuli,
cytokines, growth factors, and mitogens.
57
The ex-
pression of COX-2 mRNA and protein is elevated in
approximately 80 percent to 90 percent of human co-
lorectal carcinomas.
8,9
COX-2 expression is increased
in colorectal adenoma compared with normal mu-
cosa
1012
and the intensity of the expression correlates
with the size of adenoma
13
and grade of dysplasia.
14
These observations indicate that COX-2 plays an im-
portant role in colorectal carcinogenesis.
Clinical and histopathologic
15,16
and molecular
17
studies indicate that the adenomacarcinoma se-
quence is the main pathway of colorectal carcinogen-
Supported in part by grants from the Ministry of Education, Sci-
ence, Sports, and Culture of Japan.
Correspondence to: Takuya Okawa, M.D., Department of Diges-
tive Surgery, Tokyo Medical and Dental University Graduate
School, 1-5-45 Yushima, 113-8519, Tokyo, Bunkyo-ku, Japan,
e-mail: k-sugi.srg2@tmd.ac.jp
Dis Colon Rectum 2004; 47: 19151921
DOI: 10.1007/s10350-004-0684-y
The American Society of Colon and Rectal Surgeons
Published online: 11 October 2004
1915
esis. In 1985, Muto et al.
18
identified flat adenoma.
This tumor has a different configuration from polyp-
oid adenoma, and it constitutes a significant portion
of colorectal adenoma in Japan and in other coun-
tries.
1926
Flat adenoma might also follow the ad-
enomacarcinoma sequence and have high malignant
potential with early invasion even in small tumors
without polypoid growth.
2729
Shimoda et al.
30
clas-
sified colorectal carcinomas morphologically as pol-
ypoid and nonpolypoid. These investigators also sug-
gested that the polypoid type arises from the
adenomacarcinoma sequence, whereas the nonpol-
ypoid type arises de novo. One important difference
in oncological changes between polypoid and non-
polypoid carcinoma is the incidence of K-ras gene
mutation, the rate of which is lower in nonpolypoid
than in polypoid carcinoma. The K-ras gene is be-
lieved to determine the macroscopic appearance of
colorectal carcinoma.
31,32
We investigated the relationship between COX-2
expression and morphology and that between COX-2
expression and K-ras mutation in T1 colorectal carci-
nomas in which nonpolypoid and polypoid carcino-
mas can be discriminated.
MATERIALS AND METHODS
Human Colorectal Cancer Samples
Between 1988 and 1999, 483 colorectal carcinomas
were resected surgically at the Department of Diges-
tive Surgery, Tokyo Medical and Dental University. Of
the 483 colorectal carcinomas, 54 were T1 colorectal
carcinomas. Six samples were excluded because one
sample was obtained from a patient with familial ad-
enomatous polyposis and five samples were too small
for pathologic investigation. The remaining 48
samples were studied. Mean patient age was 62
(range, 3584) years. The ratio of men to women was
34:14. Thirty-two tumors had been located in the co-
lon and 16 tumors had been in the rectum. All speci-
mens were fixed in 10 percent formalin and embed-
ded in paraffin.
Histologic Classification
Following hematoxylin and eosin staining, the tu-
mors were grouped according to Shimodas classifi-
cation.
30
Colorectal carcinoma specimens with and
without intramucosal proliferation were classified as
polypoid (PG-Ca, Fig. 1A) and nonpolypoid (NPG-
Ca) (Fig. 1B) growth carcinoma. The maximal tumor
diameter, degree of histologic differentiation, lym-
phatic invasion, vascular invasion, lymph node me-
tastasis, and presence or absence an adenomatous
component were recorded.
Immunohistochemical Staining
Formalin-fixed, paraffin-embedded specimens
were cut in 3-m-thick sections, and were deparaf-
finized and rehydrated. After microwave heating to
maximize antigen retrieval, endogenous peroxidase
activity was blocked with 0.3 percent hydrogen per-
oxide in 100 percent methanol for 30 minutes. Non-
specific binding sites were blocked with 10 percent
normal goat serum and 10 percent stock blocking re-
agent

(Boehringer Mannheim, Mannheim, Ger-

many) for 60 minutes. The primary antibody used for
immunostaining was mouse monoclonal anti-human
Figure 1. Growth types of T1 colorectal carcinomas. A.
Macroscopic appearance of resected specimen of polyp-
oid growth carcinoma. Tumor tissue stands out from nor-
mal colonic mucosa. B. Macroscopic appearance of re-
sected specimen of nonpolypoid growth carcinoma.
Tumor tissue is covered with normal colonic mucosa.
1916 OKAWA ET AL Dis Colon Rectum, November 2004
COX-2 IgG

(IBL, Gunma, Japan) at a 1:100 dilution

applied overnight at 4C. The secondary antibody was
biotinylated goat anti-mouse IgG

(Vector Laborato-
ries, Burlingame, CA) at a 1:200 dilution. The sections
were incubated with streptavidin/biotin-horseradish
peroxidase complex

(Vectastain ABC Kit, Vector

Laboratories) at a dilution of 1:100 for 30 minutes.
Finally, the sections were incubated in 3,3-
diaminobenzidine tetrahydrochloride and were coun-
terstained with hematoxylin (Fig. 2). Nonspecific
mouse serum was the negative control, and human
appendicitis tissue was the positive control. The
specimens immunostained for COX-2 were scored in-
dependently by two investigators blinded to other in-
formation about the case. If there was a discrepancy
between two investigators, a consensus was reached
after further evaluation. The staining intensity of can-
cer cells was scored on a scale of 0 to 3+ (0, no
staining; 1+, weak; 2+, moderate; 3+ intense). Staining
intensity scores of 0 and 1 were considered COX-2
negative, and 2 and 3 as COX-2 positive.
DNA Extraction
Formalin-fixed, paraffin-embedded specimens
were sliced into 10-m-thick sections and were depa-
raffinized and rehydrated.
Approximately 10-mm
2
areas, precisely corre-
sponding to the cancer tissues, were scraped from a
semidried section under a light microscope and
heated at 9C for ten minutes with enzyme reaction
solution

(DNA Isolator PS Kit; Wako Pure Chemical,

Osaka, Japan). The mixture was incubated overnight
with enzyme activators and protease. Thereafter, DNA
was precipitated by means of an accelerator and with
isopropanol dissolved in 50 l of distilled water.
Analysis of K-ras Mutation
The K-ras codon 12-point mutation was analyzed
by nested polymerase chain reaction-restriction frag-
ment length polymorphism (PCR-RFLP) as described
previously.
33,34
PCR amplification was performed by
use of the primers and reaction mixture described by
Ohshima et al.,
33
except that the mixture contained
0.4 M of each primer, 1.25 units of Taq DNA poly-
merase, and 2 l of template DNA. After one cycle of
94C for 5 minutes, 57C for 1.5 minutes, and 72C for
1 minute, DNA was amplified 30 cycles at 94C for 1
minute, 57C for 1.5 minutes, and 72C for 1 minute,
with a final extension at 72C for 7 minutes. The sec-
ond PCR proceeded under the same conditions, ex-
cept for the initial annealing at 55C for 1.5 minutes.
The three control reactions for each experiment con-
sisted of no template, normal human placental DNA
(normal control), and an AsPc1 pancreatic carcinoma
cell line (positive mutant control). Second PCR prod-
ucts were digested with Mva-I and resolved by elec-
trophoresis in 3 percent agarose gel. Cleaved wild-
type DNA yielded 86base pair fragments, whereas
mutant PCR products were not cleaved and yielded
106base pair fragments (Fig. 3).
Statistical Analysis
The Mann-Whitney U test, Fishers exact test, and
chi-squared test were used to determine statistical sig-
nificance. A P value below 0.05 was considered sta-
tistically significant.
Figure 2. Immunohistochemistry for COX-2 on human co-
lorectal carcinoma. A, B. Cytoplasm of polypoid and non-
polypoid colorectal carcinoma shows intense and nega-
tive cytoplasmic immunostaining for COX-2, respectively
(400).
1917 COX-2 AND K-RAS IN T1 COLORECTAL CANCER Vol. 47, No. 11
RESULTS
Histopathologic Findings
The pathologic features of the 48 T1 colorectal car-
cinomas are summarized in Table 1. Thirty tumors
were polypoid and 18 tumors were nonpolypoid. The
maximum tumor diameter, degree of histologic differ-
entiation, lymphatic invasion, vascular invasion, and
lymph node metastasis did not differ significantly be-
tween the two groups. None of the NPG-Ca speci-
mens contained an adenomatous component,
whereas 19 of 30 (63.3 percent) of the PG-Ca did.
COX-2 Expression
COX-2 immunoreactivity was positive in tumor,
mucosal epithelial, stromal, muscle, and vascular en-
dothelial cells and in Auerbachs myenteric plexus. In
contrast, COX-2 was weakly stained or absent in nor-
mal epithelium at a distance from the cancer. The
staining was uniform in cancer tissues. In cancer cells,
COX-2 expression was observed mainly in the cyto-
plasm and nuclear envelope (Fig. 2). Figure 4 shows
that the intensity of COX-2 expression of cancer cells
in PG-Ca was significantly greater than that in NPG-Ca
(average intensity score, 2.2 vs. 1.0; P < 0.001). Of the
18 NPG-Ca specimens, 12 (66.7 percent) were COX-2
negative, whereas 28 of 30 PG-Ca specimens (93.3
percent) were immunopositive.
K-ras Mutation
The K-ras mutation was found in 12 of 30 (40 per-
cent) PG-Ca and in none of 18 NPG-Ca sections (P =
0.002). Colorectal carcinomas with the K-ras mutation
expressed significantly more COX-2 than those with-
out it (Fig. 5, P = 0.028).
DISCUSSION
The present study demonstrated that colorectal car-
cinomas express more COX-2 when they have a pol-
ypoid feature. This result suggests a close relationship
between COX-2 expression and polypoid growth.
Many studies have noted that COX-2 is induced in
gastrointestinal neoplasms, including colorectal carci-
noma,
8,9
colorectal adenomas,
1012
gastric carci-
noma,
3537
and esophageal carcinoma.
38
COX-2 is ex-
pressed in 80 percent to 90 percent of human
colorectal carcinomas.
8,9
Colorectal carcinogenesis
might arise via the adenomacarcinoma sequence
and by a de novo process. Carcinoma originating de
novo might include carcinoma that develops within
flat adenoma as a nonpolypoid type during the early
stage.
27,28,39,40
The incidence of carcinoma that arises
via each pathway has not been clarified. Shimoda et
al.
30
and Ikegami et al.
41
estimated that NPG-Ca rep-
resents approximately 80 percent of all colorectal car-
cinomas.
Matsui et al.
42
reported that over 70 percent of co-
lorectal carcinoma is polypoid. If de novo origin were
the main route of colorectal carcinoma development,
less colorectal carcinoma would express COX-2 than
that reported. Because 93 percent of PG-Ca, 33 per-
cent of NPG-Ca, and 71 percent of colorectal carci-
noma express COX-2, approximately 63 percent of
colorectal carcinoma should develop through the ad-
enomacarcinoma sequence. The K-ras gene regu-
lates cell proliferation, angiogenesis, and apoptosis in
colorectal carcinoma.
4245
A leading molecular study
and extensive review by Vogelstein et al.
17
and Wa-
tanabe et al.
46
concluded that the K-ras gene mutation
is associated with the progression of small to large
adenomas in the colorectum. The K-ras mutation has
been detected predominantly in codon 12 in approxi-
mately 50 percent of colorectal cancers, 44 percent to
67 percent of polypoid carcinoma, and in 0 to 23
percent of nonpolypoid carcinoma.
31,32,46,47
The fre-
quency of K-ras mutation is lower in nonpolypoid
carcinoma than in polypoid carcinoma.
These observations suggest that the K-ras gene de-
Figure 3. Nested polymerase chain reaction-restriction
fragment length polymorphism. Size markers, lane M;
DNA samples from colorectal carcinoma show mutant
bands (106 base pairs) and wild-type bands (86 base
pairs), respectively, in lanes 3 and 4; DNA samples from
colorectal carcinoma show wild-type bands (86 base
pairs) in lanes 1, 2, and 5. Normal control (normal human
placental DNA), lane 6; positive mutant control (AsPc1),
lane 7.
1918 OKAWA ET AL Dis Colon Rectum, November 2004
termines the macroscopic configuration of colorectal
carcinoma. In the present study, none of the nonpol-
ypoid but 40 percent of the polypoid carcinomas had
the K-ras gene mutation.
In this study we found that COX-2 expression was
significantly higher in tumors with a K-ras gene mu-
tation than in those without it. Fujita et al.
48
also
found that K-ras gene mutation in colorectal adeno-
mas is associated with COX-2 expression and sus-
pected that COX-2 is expressed early in the adenoma
carcinoma sequence. These findings suggest that
K-ras gene mutations are associated with increased
levels of COX-2 expression.
CONCLUSION
Polypoid growth and a K-ras gene mutation in T1
tumors are associated with increased COX-2 expres-
sion. These findings suggest a role of COX-2 and K-
ras in the morphogenesis and tumorigenesis of colo-
rectal carcinomas.
ACKNOWLEDGMENT
The authors thank Yoichi Ajioka, M.D., First De-
partment of Pathology, Niigata University School of
Table 1.
Pathologic Features of 48 T1 Colorectal Carcinomas
a
PG-Ca NPG-Ca P Value
Case no. 30 18
Median tumor size, mm
(interquartile range)
23 (1529.5) 18.5 (1525) 0.244
Histologic differentiation 25 (83.3%) 14 (77.8%)
Moderate 4 (13.3%) 4 (22.2%)
Poor 1 (3.3%) 0 (0%) 0.555
Lymphatic permeation 9 (30%) 4 (22.2%)
21 (70%) 14 (77.8%) 0.740
Vascular permeation 9 (30%) 10 (55.6%)
21 (70%) 8 (44.4%) 0.148
Lymph node metastasis 2 (6.7%) 1 (5.6%)
28 (93.3%) 17 (94.4%) >0.999
Adenomatous component 19 (63.3%) 0 (0%)
11 (36.7%) 18 (100%) <0.001
Mutation of K-ras gene 12 (40%) 0 (0%)
18 (60%) 18 (100%) 0.002
Median COX-2 intensity score 2 1 <0.001
NPG-Ca = nonpolypoid growth carcinoma; PG-Ca = polypoid growth carcinoma. Tumor size is maximum diameter.
a
Tumor size is expressed as median (interquartile range).
Figure 4. Relationship between COX-2 expression and
morphology of colorectal carcinomas. Horizontal line rep-
resents median value of COX-2 expression. NPG-Ca =
nonpolypoid growth carcinoma; PG-Ca = polypoid growth
carcinoma.
Figure 5. Relationship between COX-2 expression and
K-ras mutation in colorectal carcinomas.
1919 COX-2 AND K-RAS IN T1 COLORECTAL CANCER Vol. 47, No. 11
Medicine, for advice regarding the molecular biology
techniques.
REFERENCES
1. Giovannucci E, Egan KM, Hunter DJ, et al. Aspirin and
the risk of colorectal cancer in women. N Engl J Med
1995;333:60914.
2. Thun MJ, Namboodiri MM, Heath CW. Aspirin use and
reduced risk of fatal colon cancer. N Engl J Med 1991;
325:15936.
3. Giardiello FM. NSAID-induced polyp regression in fa-
milial adenomatous polyposis patients. Gastroenterol
Clin North Am 1996;25:34961.
4. Steinbach G, Lynch PM, Phillips RK, et al. The effect of
celecoxib, a cyclooxygenase-2 inhibitor, in familial ad-
enomatous polyposis. N Engl J Med 2000;342:194652.
5. Smith WL, Garavito RM, DeWitt DL. Prostaglandin en-
doperoxide H synthases (cyclooxygenase)-1 and -2. J
Biol Chem 1996;271:3315760.
6. Williams CS, DuBois RN. Prostaglandin endoperoxide
synthase: why two isoforms? Am J Physiol 1996;270:
393400.
7. DuBois RN, Awad J, Morrow J, Roberts LJ, Bishop PR.
Regulation of eicosanoid production and mitogenesis in
rat intestinal epithelial cells by transforming growth fac-
tor-1 and phorbol ester. J Clin Invest 1994;93:4938.
8. Hao X, Bishop AE, Wallace M, et al. Early expression of
cyclooxygenase-2 during sporadic colorectal carcino-
genesis. J Pathol 1999;187:295301.
9. Chen WS, Wei SJ, Liu JM, Hsiao M, Lin JK, Yang WK.
Tumor invasiveness and liver metastasis of colon cancer
cells correlated with cyclooxygenase-2 (COX-2) expres-
sion and inhibited by a COX-2-selective inhibitor,
etodolac. Int J Cancer 2001;91:8949.
10. Eberhart CE, Coffey RJ, Radhika A, Giardiello FM, Fer-
renbach S, Dubois RN. Up-regulation of cyclooxygen-
ase 2 gene expression in human colorectal adenomas
and adenocarcinomas. Gastroenterology 1994;107:
11838.
11. Maekawa M, Sugano K, Sano H, et al. Increased expres-
sion of cyclooxygenase-2 to -1 in human colorectal can-
cers and adenomas, but not in hyperplastic polyps. Jpn
J Clin Oncol 1998;28:4216.
12. Chapple KS, Cartwright EJ, Hawcroft G, et al. Localiza-
tion of cyclooxygenase-2 in human sporadic colorectal
adenomas. Am J Pathol 2000;156:54553.
13. Hasegawa K, Ichikawa W, Fujita T, et al. Expression of
cyclooxygenase-2 (COX-2) mRNA in human colorectal
adenomas. Eur J Cancer 2001;37:146974.
14. Sato T, Yoshinaga K, Okabe S, et al. Cyclooxygenase-2
expression in colorectal adenomas. Dis Colon Rectum
2003;46:78692.
15. Jackman RJ, Mayo CW. The adenoma-carcinoma se-
quence in cancer of the colon. Surg Gynecol Obstet
1951;93:32730.
16. Muto T, Bussey HJ, Morson BC. The evolution of cancer
of the colon and rectum. Cancer 1975;36:225170.
17. Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic
alterations during colorectal tumor development. N
Engl J Med 1988;319:52532.
18. Muto T, Kamiya J, Sawada T, et al. Small flat adenoma
of the large bowel with special reference to its clinico-
pathologic features. Dis Colon Rectum 1985;28:84751.
19. Kuramoto S, Ihara O, Sakai S, Shimazu R, Kaminishi M,
Oohara T. Depressed adenoma in the large intestine:
endoscopic features. Dis Colon Rectum 1990;33:10812.
20. Adachi M, Muto T, Okinaga K, Morioka Y. Clinicopath-
ologic features of the flat adenoma. Dis Colon Rectum
1991;34:9816.
21. Wolber RA, Owe DA. Flat adenomas of the colon. Hum
Pathol 1991;22:704.
22. Jaramillo E, Watanabe M, Slezak P, Rubio C. Flat neo-
plastic lesions of the colon and rectum detected by
high-resolution video endoscopy and chromoscopy.
Gastrointest Endosc 1995;42:11422.
23. Rubio CA, Kumagai J, Kanamori T, Yanagisawa A, Na-
kamura K, Kato Y. Flat adenomas and flat adenocarci-
nomas of the colorectal mucosa in Japanese and Swed-
ish patients. Dis Colon Rectum 1995;38:10759.
24. Cairns A, Quirke P. Flat adenomas. Br J Surg 1999;86:
148990.
25. Hart AR, Kudo S, Mackay EH, Mayberry JF, Atkin WS.
Flat adenomas exist in asymptomatic people: important
implications for colorectal cancer screening pro-
grammes. Gut 1998;43:22931.
26. Saitoh Y, Waxman I, West AB, et al. Prevalence and
distinctive biologic features of flat colorectal adenomas
in a North American population. Gastroenterology
2001;120:165765.
27. Spratt JS, Ackerman LV, Louis S. Small primary adeno-
carcinomas of the colon and rectum. JAMA 1962;179:
33746.
28. Crawford BE, Stromeyer FW. Small nonpolypoid carci-
nomas of the large intestine. Cancer 1983;51:17603.
29. Watanabe T, Sawada T, Kubota Y, et al. Malignant po-
tential in flat elevations. Dis Colon Rectum 1993;36:548
53.
30. Shimoda T, Ikegami M, Fujisaki J, Matsui T, Aizawa S,
Ishikawa E. Early colorectal carcinoma with special ref-
erence to its development de novo. Cancer 1989;64:
113846.
31. Kaneko K, Fujii T, Kato S, et al. Growth patterns and
genetic changes of colorectal carcinoma. Jpn J Clin On-
col 1998;28:196201.
32. Hasegawa H, Ueda M, Watanabe M, Teramoto T, Mukai
M, Kitajima M. K-ras gene mutations in early colorectal
cancer: flat elevated vs. polypoid-forming cancer. On-
cogene 1995;10:14136.
1920 OKAWA ET AL Dis Colon Rectum, November 2004
33. Ohshima S, Shimizu Y, Takahara M. Detection of c-ki-
ras gene mutation in paraffin sections of adenocarci-
noma and atypical broncioloalveolar cell hyperplasia of
human lung. Virchows Arch 1994;424:12934.
34. Matsubayashi H, Watanabe H, Nishikura K, Ajioka Y,
Kijima H, Saito T. Determination of pancreatic ductal
carcinoma histogenesis by Analysis of mucous quality
and K-ras mutation. Cancer 1998;82:65160.
35. Ohno R, Yoshinaga K, Fujita T, et al. Depth of invasion
parallels increased cyclooxygenase-2 levels in patients
with gastric carcinoma. Cancer 2001;91:187681.
36. Uefuji K, Ichikura T, Mochizuki H, Shinomiya N. Ex-
pression of cyclooxygenase-2 protein in gastric adeno-
carcinoma. J Surg Oncol 1998;69:16872.
37. Joo YE, Oh WT, Rew JS, Park CS, Choi SK, Kim SJ.
Cyclooxygenase-2 expression is associated with well-
differentiated and intestinal-type pathways in gastric
carcinogenesis. Digestion 2002;66:2229.
38. Ratnasinghe D, Tangrea J, Roth MJ, et al. Expression of
cyclooxygenase-2 in Human squamous cell carcinoma
of the esophagus; an immunohistochemical survey. An-
ticancer Res 1999;19:1714.
39. Kuramoto S, Oohara T. Flat early cancers of the large
intestine. Cancer 1989;64:90555.
40. Herrera L, Hanna S, Castillo N, Petrelli NJ. Primary de
novo adenocarcinoma of the colon measuring 8 mm in
diameter with lymph node metastases. Dis Colon Rec-
tum 1991;34:2759.
41. Ikegami M. A pathological study on colorectal cancer
from de novo carcinoma to advanced carcinoma. Acta
Pathol Jpn 1987;37:2137.
42. Matsui T, Yao T, Iwashita A. Natural history of early
colorectal cancer. World J Surg 2000;24:10228.
43. Kobayashi M, Watanabe H, Ajioka Y, Honma T,
Asakura H. Effect of K-ras mutation on Morphogenesis
of colorectal adenomas and early cancers. Hum Pathol
1996;27:10429.
44. Rak J, Mitsuhashi Y, Bayko L, et al. Mutant ras onco-
genes upregulate vegf/vpf expression -implications for
induction and inhibition of the tumor angiogenesis.
Cancer Res 1995;55:457580.
45. Ward RL, Todd AV, Santiago F, OConnor T, Hawkins
NJ. Activation of the K-ras oncogene in colorectal neo-
plasms is associated with decreased apoptosis. Cancer
1997;79:110613.
46. Watanabe T, Muto T. Colorectal carcinogenesis based
on molecular biology of early colorectal cancer, with
special reference to nonpolypoid (superficial) lesions.
World J Surg 2000;24:10917.
47. Yamagata S, Muto T, Uchida Y, et al. Polypoid growth
and K-ras codon 12 mutation in colorectal cancer. Can-
cer 1995;75:9537.
48. Fujita M, Fukui H, Kusaka T, et al. Relationship between
cyclooxygenase-2 expression and K-ras gene mutation
in colorectal adenomas. J Gastroenterol Hepatol 2000;
15:127781.
1921 COX-2 AND K-RAS IN T1 COLORECTAL CANCER Vol. 47, No. 11