Deleted in Colorectal Cancer Protein

Expression as a Possible Predictor of

Response to Adjuvant Chemotherapy in
Colorectal Cancer Patients
Rivka Gal, M.D.,
1,4
Evgeny Sadikov, M.D.,
2
Jaqueline Sulkes, Ph.D.,
3,4
Baruch Klein, M.D.,
2,4
Rumelia Koren, M.D.
1,4
1
Department of Pathology, Hasharon Hospital, Petah Tikva, Israel
2
Department of Oncology, Hasharon Hospital, Petah Tikva, Israel
3
Epidemiology Unit, Rabin Medical Center, Petah Tikva, Israel
4
Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel
PURPOSE: The deleted in colorectal cancer (DCC) gene pre-
dicts a poor outcome for patients with colorectal carci-
noma. This study was designed to investigate whether the
expression of the DCC protein also can predict response to
adjuvant chemotherapy. METHODS: The expression of DCC
was evaluated immunohistochemically in 74 paraffin-
embedded tumor samples from patients with Stage II (n =
41) and Stage III (n = 33) colorectal carcinomas. Follow-up
time was at least 60 (median, 64) months. Follow-up was at
least five years for all patients who are alive. End points of
the study were recurrence of disease and death. Forty-eight
patients received adjuvant therapy of 5-fluorouracil + le-
vamisole; 28 were not treated. RESULTS: Fifty percent of
tumors were deleted in colorectal cancer-positive (DCC+).
Proportion of survival and disease-free survival were higher
in the DCC+ patients (83 percent) than in deleted in colo-
rectal cancer-negative (DCC; 54 percent). In the DCC+
group, adjuvant treatment was a strong positive predictive
factor for survival and disease-free survival. All DCC+ pa-
tients who received adjuvant chemotherapy (CHEMO+) are
alive with no evidence of disease, whereas without chemo-
therapy (CHEMO) only 54 percent are alive (P = 0.0001).
When stratification was performed by stage, patients in
Stage II who were DCC+/CHEMO+ had survival and disease-
free survival of 100 percent, whereas in DCC+/CHEMO
survival rate was 75 percent and disease-free survival rate 62
percent (P = 0.042). Patients in Stage III who were DCC+/
CHEMO+ had survival and disease-free survival of 100 per-
cent, whereas in DCC+/CHEMO both dropped to zero
(P = 0.0002). On the other hand, in the DCC tumors, there
was no statistical significant relationship between chemo-
therapy and survival or disease-free survival (DCC/
CHEMO had 57 percent survival; DCC/CHEMO+ had 52
percent survival). CONCLUSIONS: DCC is a prognostic fac-
tor for colorectal cancer. Positive expression of DCC iden-
tifies a subgroup of patients who respond favorably to ad-
juvant chemotherapy, which resulted in our cases, in 100
percent survival and disease-free survival rates. Without
treatment, the survival rate of DCC+ patients dropped sig-
nificantly. We suggest that DCC immunostaining should be
performed routinely. All DCC+ patients should receive ad-
juvant chemotherapy. For DCC tumors, a larger cohort of
patients should be studied before definitive conclusions can
be drawn; however, clinical trials of new drug combina-
tions should focus on DCC patients. [Key words: Colorec-
tal cancer; Chromosome 18q; Chemotherapy; Prognosis]
C
arcinoma of the colon is one of the most com-
mon malignant diseases of western civilization.
Usually, the tumor is resectable and the prognosis
depends on the stage of the disease. Adjuvant che-
motherapy of fluorouracil plus levamisole is a toler-
Reprints are not available.
Supported in part by a grant from the Medical Research Fund of
the Rabin Medical Center.
Presented in part at the 4th World Congress on Advances in
Oncology and 2nd International Symposium on Molecular Medi-
cine, Vouliagmeni, Athens, Greece, October 7 to 9, 1999.
Correspondence to: Rivka Gal, M.D., Pathology Department,
Hasharon Hospital, Petah Tikva, Israel, e-mail: rumelia@isdnmail.
co.il
Dis Colon Rectum 2004; 47: 12161224
DOI: 10.1007/s10350-004-0552-9
The American Society of Colon and Rectal Surgeons
Published online: 19 May 2004
1216
able regimen that increases survival for patients in
Stage III.
1,2
Approximately one-third of patients pre-
sent with Stage II or Astler-Coller Stage B2 (penetra-
tion of the tumor through the full-thickness of the
bowel wall but without lymph node metastases).
These patients have a five-year survival of 70 to 80
percent
3
; therefore, adjuvant therapy would benefit
only a small proportion of this population. None of
the clinical studies have yet demonstrated a benefit
for adjuvant chemotherapy in patients with Stage II
disease.
4,5
Examination of biologic characteristics of
the tumor may define a subset of Stage II patients who
might benefit from adjuvant treatment. DNA aneu-
ploidy and S-phase fraction could identify Stage II
patients in whom the prognosis was similar to node-
positive patients,
6
but response to treatment was not
investigated.
Deletion involving chromosome 18q has been
identified in colon carcinoma
7
and was termed de-
leted in colorectal cancer (DCC) gene. Allelic loss of
chromosome 18q has been shown to be a negative
predictor of prognosis in colon cancer. Jen et al.
8
found that the survival rate of Stage II patients whose
cancer had allelic loss of chromosome 18q was 54
percent, which is similar to patients in Stage III,
whereas those with no evidence of allelic loss had 93
percent survival rate, which is comparable to patients
in Stage I. These results were confirmed by others.
9,10
Allelic loss of chromosome 18q also was associated
with metastatic potential of colorectal carcinoma.
11
Several other studies
1216
evaluated the expression of
DCC by immunohistochemistry and found that it was
a strong prognostic factor in all stages of colorectal
carcinoma. Shibata et al.
12
concluded that further un-
derstanding of DCC might improve the usefulness of
this marker in selecting patients for adjuvant therapy.
Jessup and Loda
17
analyzed the current state of sev-
eral molecular markers, including DCC, and con-
cluded that assessing the expression of molecules
within a primary cancer may predict the response to
therapy and outcome. This study was designed to ex-
amine the expression of DCC protein in Stages II and
III colorectal cancer by immunohistochemistry and to
assess its predictive value for the prognosis and for
the efficacy of 5-fluorouracil (5-FU)-levammisole ad-
juvant chemotherapy.
PATIENTS AND METHODS
Seventy-four consecutive patients with colorectal
carcinoma operated on in 1995 were studied. Patients
with rectal cancer underwent low anterior resection
(10 patients) or abdominoperineal resection (12 pa-
tients), and patients with colon cancer underwent ab-
dominal resection (52 patients).
There were 42 patients with Stage II and 32 with
Stage III. Forty-six patients received adjuvant chemo-
therapy with 5-FU-levamisole for one year. Loading
dose of 5-FU was 450 mg/m
2
on Days 1 to 5 for the
first month. After the first cycle, treatment was
switched to 450 mg/m
2
weekly and Levamisole 150
mg per day 3 days, biweekly, for one year. Patients
with rectal cancer also received radiotherapy.
Follow-up time was 60 to 96 (median, 64) months
(except for patients who died). Follow-up was at least
five years for all patients who are alive. All patients
were followed up every three months during the first
two years and every six months during subsequent
years. In addition to physical examination, colonos-
copy was performed yearly and hepatic ultrasonog-
raphy every six months. Blood chemistry, including
carcinoembryonic antigen and white blood cell count,
were performed every three months.
Tumor Specimens
Formalin-fixed, paraffin-embedded samples were
obtained from the archives of our department. All the
slides were reviewed, grade (according to the TNM
system
18
) and stage were recorded, and a represen-
tative paraffin block that contained tumor and adja-
cent mucosa was chosen for immunohistochemistry.
The sections that contained both normal mucosa and
adjacent tumor were selected for immunostaining.
Immunohistochemistry
Four-micron tissue sections were mounted on poly-
lysine coated slides. They were deparaffinized and
rehydrated. Antigen was retrieved by heating the tis-
sue sections to 90C for 20 minutes in a microwave
oven in 10 nM of citric acid monophosphate buffer
(pH 6.0). Immunohistochemical staining was per-
formed manually using the mouse antihuman DCC
monoclonal antibody (clone G97-449, Pharmingen,
San Diego, CA) at a dilution of 1:100 for 60 minutes.
A broad-spectrum secondary antibody (The Histo-
stain Plus Bulk Kit 2nd generation, Zymed Lab Inc.,
San Francisco, CA) was used according to the manu-
facturers instructions. Slides were counterstained
with Mayers hematoxylin, rehydrated, and mounted
with glycerol gelatin. The normal mucosa served as
positive control, and omission of the antihuman DCC
1217 DCC AND CHEMOTHERAPY IN COLORECTAL CANCER Vol. 47, No. 7
antibody served as a negative control. Two surgical
pathologists, who were blinded to the clinical data of
the patients, assessed DCC protein expression.
Statistical Analysis
To analyze statistically significant relationships in
the distribution of categorical variables (e.g., DCC,
stage, treatment), chi-squared test was performed or
Fishers exact test if appropriate. To analyze statisti-
cally significant differences in the distribution of con-
tinuous variable (e.g., age) between two groups of
patients (i.e., with or without DCC), Students t-test
was performed. Pearson correlation coefficients (r)
and significance (P) were calculated between the vari-
ables. Two major end points were studied: survival
and disease-free survival (DFS) interval. Survival was
measured from surgery to death or end of the study
(April 1999). DFS was measured from surgery until a
local recurrence or a metastatic disease was found or
to the end of the study. The prognostic factors ana-
lyzed in terms of survival or DFS were as follows: age,
gender, stage, grade, treatment, etc. The product limit
method of Kaplan-Meier
19
was used to estimate sur-
vival and DFS. To compare survival or DFS curves,
log-rank test was used. The Cox proportional hazard
model
20
was used to assess the joint effect of the
prognostic factors on survival or DFS. P 0.05 was
considered statistically significant.
RESULTS
Immunohistochemical Staining
Strong staining was observed in the crypts and lu-
minal epithelial cells of the normal mucosa. This pro-
vided a positive internal control. The tumor cells
showed a strong cytoplasmatic staining throughout all
the cells, which was considered positive (Fig. 1), or a
very faint staining, which was less than that of the
normal mucosa, and was considered negative (Fig. 2).
Usually, it was an all or nothing phenomenon, except
for one case that showed small areas of intense stain-
ing intermingled with areas of faint staining (this case
was considered negative for the statistical analysis).
Clinical Characteristics of Patients and
DCC Status
Table 1 shows the relevant clinical characteristics of
the patients by DCC status. There were 74 patients (47
males; 63.5 percent; mean age, 69.12 + 9.27 years).
Fifty percent of tumors were deleted in colorectal can-
cer-positive (DCC+) and 50 percent were deleted in
colorectal cancer-negative (DCC). There were no
significant differences between DCC status and age,
gender, tumor location, stage, grade, or proportion of
patients who received chemotherapy. Twenty-four
patients (32.4 percent) died of disease, 8 are alive
with metastases or local recurrence, and 42 are alive
with no evidence of disease. Eighty-two percent of
DCC+ patients are alive compared with 54 percent of
those with DCC tumors (P = 0.126).
Variables that Influence Prognosis
The overall five-year survival rate was 85 percent
for Stage II and 45 percent for Stage III. Statistically
Figure 1. Immunohistochemical stain of deleted in colo-
rectal cancer-positive protein expression (hematoxylin
and eosin; 200). Strong and homogeneous expression in
the cytoplasm of the tumor cells.
Figure 2. Immunohistochemical stain of deleted in colo-
rectal cancer (DCC)-negative protein expression (hema-
toxylin and eosin; 200). Tumor cells that have lost the
DCC protein expression show only very faint brown cyto-
plasmatic staining.
1218 GAL ET AL Dis Colon Rectum, July 2004
significant prognostic factors for survival were stage,
DCC status, and chemotherapy, both in the univariate
and multivariate analysis (Table 2). For DFS, only
DCC was a prognostic factor both in the univariate
and multivariate analysis (Table 2).
Kaplan-Meier curves for all patients showed a sig-
nificantly better survival for DCC+ patients compared
with DCC patients (P = 0.0112; for DFS, P = 0.0001;
Fig. 3). When survival was plotted in relation to stage
and DCC status, the differences were still statistically
significant (P = 0.0152).
Survival and DFS in Relation to
Chemotherapy and DCC Status
Table 3 outlines the outcome of patients according
to stage, by DCC status and by chemotherapy. In all
patients with DCC+ tumors, chemotherapy had sig-
nificant influence on the outcome. All 24 patients (100
percent) who received chemotherapy (CHEMO+)
are alive and well with no evidence of disease,
whereas of 13 DCC+ patients without chemotherapy
(CHEMO), only 5 (38 percent) are alive and well; 1
is alive with metastasis, and 7 are dead of disease.
This is shown in the Kaplan-Meier plot (Fig. 4). There
is a significantly better survival for DCC+/ CHEMO+
patients compared with DCC+/CHEMO patients (P =
0.0001). Similar results were observed for DFS (P =
0.0001; data not shown).
Conversely, in DCC patients, there was no signifi-
cant difference in survival (Fig. 5) between those who
received chemotherapy and those who did not (P =
0.457). Similarly, no significance was seen in DFS (P =
0.196; data not shown).
When analyzing survival and DFS by stage and che-
motherapy, in Stage II, of 13 DCC+/CHEMO+ pa-
Table 2.
Univariate and Multivariate Analysis by Prognostic Factors of DFS and Survival in Colorectal Cancer
DFS Survival
Prognostic Factor Univariate Multivariate Univariate Multivariate
Stage 0.59 0.192 0.037 0.012
DCC 0.0013 0.005 0.01 0.038
Chemotherapy 0.1768 0.089 0.037 0.012
Grade NS NS NS NS
Age NS NS NS NS
Gender NS NS NS NS
DFS = disease-free survival; DCC = deleted in colorectal cancer; NS = not statistically significant
Data are P values.
Table 1.
Clinical Characteristics of Patients by DCC Status
DCC-Positive (n = 37) DCC-Negative (n = 37) P Value
Age (yr) 69.12 9.27 62.32 70.9 0.11
Male/female ratio 47/27 22/15 25/12 NS
Site
Rectum 22 9 13 NS
Colon 52 28 24
Stage
II 41 21 20 NS
III 33 16 17
Grade
1 21 11 10 NS
2 48 25 23
3 5 1 4
Chemotherapy
Yes 47 24 23 NS
No 27 13 14
Vital status
Alive 50 30 (81.8%) 20 (54.1%) 0.0126
Dead 24 7 (18.9%) 17 (45.9%)
DCC = deleted in colorectal cancer; NS = not statistically significant.
1219 DCC AND CHEMOTHERAPY IN COLORECTAL CANCER Vol. 47, No. 7
tients, all are alive and well compared with 5 of 7
DCC+/CHEMO patients. This difference was not sta-
tistically significant (P = 0.13). However, a statistically
significant relationship was found between the distri-
bution of DFS with chemotherapy (P = 0.042; data not
shown). In DCC patients, both with and without
chemotherapy, four of ten patients are alive.
In Stage III, DCC+/CHEMO+ patients had signifi-
cantly better survival (11/11) compared with DCC+/
CHEMO (0/5; P = 0.0001). In the DCC group, che-
motherapy did not affect survival (P = 0.457) or DFS
(P = 0.196).
DISCUSSION
Adjuvant therapy is clearly of great benefit for cer-
tain patients with colorectal carcinoma. However,
these therapies have side effects, and thus they should
be given to those who really would benefit from
them.
21
It is important to further define this subgroup
of patients, because even a small improvement in the
outcome of colon carcinoma patients may translate
into substantial numbers of survivors. On the other
hand, it is also important to exclude therapy from
patients who are unlikely to respond. The results of
our study indicate that DCC protein expression may
be a helpful predictor for selecting patients who are
likely to benefit from adjuvant chemotherapy.
In 1997, a specific region of chromosome 18 was
found to be deleted in 73 percent of colorectal carci-
nomas
22
and was identified as a candidate for a tu-
mor-suppressor gene, termed DCC. Allelic loss of
chromosome 18q, or decreased expression of the
DCC protein, have been found in different types of
malignant tumors, such as colorectal,
8,10,12,13,23
gas-
tric,
24
endometrial,
25
breast,
26
and other tumors, and it
was reported to correlate with tumor progression and
metastatic potential. Shibata et al.
12
found that pa-
tients with Stage II/DCC+ colon carcinoma had a sig-
nificantly better prognosis than patients with Stage
II/DCC (P < 0.01). Carthers et al.
27
and Laurent-Puig
et al.
28
did not confirm that loss of heterozygosity for
18q had a significant prognostication power.
Most of our results are similar to those reported by
Shibata et al.
12
We also found that Stage II/DCC tu-
mors had a poor prognosis comparable to those with
Stage III and that patients with DCC+ tumors had
lower recurrence and death rates than DCC patients.
In contrast, unlike all other previous studies, we have
shown that in patients with DCC+ tumors, adjuvant
chemotherapy had a significant beneficial effect,
whereas DCC patients in our cohort did not seem to
benefit much from adjuvant therapy. In the study by
Shibata et al.,
12
the use of adjuvant therapy did not
prove to be a significant independent prognostic in-
dicator. However, only 21 of 66 of their DCC+ patients
received adjuvant therapy, and the protocol is not
given. Jen et al.
8
also did not find that adjuvant
therapy had an effect on outcome; however, only five
patients without 18q loss received adjuvant chemo-
therapy. Therefore, we cannot compare these results
to ours.
The most significant result of our study is that all the
DCC+ patients who received adjuvant therapy were
alive and free of recurrence at the end of the study
(median follow-up, 64 months). This suggests that all
DCC+ patients, irrespective of stage, should receive
adjuvant chemotherapy if they can tolerate it. On the
other hand, our results do not indicate that adjuvant
therapy influences the outcome of patients with
DCC tumors in both stages examined.
The observation that DCC patients do not seem to
benefit from chemotherapy is unexpected, because it
is conceivable to assume that patients with DCC tu-
mors, who have a worse prognosis, would probably
benefit from adjuvant chemotherapy more than DCC+
patients, who have a better prognosis.
29
What could be the possible explanation for the high
sensitivity of DCC+ cells to 5-FU-levamisole treat-
Figure 3. Kaplan-Meier life-table analysis of the overall
disease-free survival (DFS) of patients with colorectal
cancer according to deleted in colorectal cancer (DCC)
expression. Patients with positive DCC expression had a
significantly better survival than DCC patients (P =
0.0112). DCC+ = deleted in colorectal cancer positive;
DCC = deleted in colorectal cancer negative.
1220 GAL ET AL Dis Colon Rectum, July 2004
ment? It was reported that the DCC gene product in-
duces apoptosis in settings in which the ligand, net-
rin-1 is unavailable, for example, during metastases or
tumor growth beyond local blood supply.
30
It also
was shown that 5-FU induced apoptosis in gastric car-
cinoma, in vivo,
31
in human colon carcinoma cell
lines
32,33
and that 5-FU-induced apoptosis correlated
with its the efficacy against human gastric and colon
cancer xenografts in nude mice.
34
Therefore, it is pos-
sible that the DCC protein and 5-FU have a synergistic
effect on apoptosis, which allows the eradication of
cancer cells that are still viable after the resection of
the primary tumor. Another explanation is that le-
vamisole, which is an immunopotentiator in cancer
Table 3.
Outcome of Patients According to Stage, DCC Status, and Chemotherapy
Colon Rectum
Chemo+ Chemo Chemo+ Chemo
DCC+/Stage II
Alive and well 8 5 5 0
Alive with recurrence 0 1 0 0
Dead of disease 0 1 0 1
DCC+/Stage III
Alive and well 9 0 2 0
Alive with recurrence 0 0 0 0
Dead of disease 0 4 0 1
DCC/Stage II
Alive and well 0 3 4 1
Alive with recurrence 3 1 0 1
Dead of disease 1 2 2 2
DCC/Stage III
Alive and well 3 2 0 0
Alive with recurrence 2 0 0 0
Dead of disease 6 1 2 1
DCC = deleted in colorectal cancer; DCC+ = deleted in colorectal cancer positive; DCC = deleted in colorectal cancer
negative; CHEMO+ = patients who received chemotherapy; CHEMO = patients who did not receive chemotherapy.
Figure 4. Kaplan-Meier life-table analysis of deleted in
colorectal cancer-positive (DCC+) patients according to
chemotherapy. Patients who received chemotherapy had
statistically significantly better prognosis than those with-
out therapy (P = 0.001). CHEMO+ = patients who re-
ceived adjuvant chemotherapy; CHEMO = patients with-
out chemotherapy.
Figure 5. Kaplan-Meier life-table analysis of deleted in
colorectal cancer-negative (DCC) patients according to
chemotherapy. There was no statistical difference in sur-
vival between patients who received or did not receive
chemotherapy. CHEMO+ = patients who received adju-
vant chemotherapy; CHEMO = patients without chemo-
therapy.
1221 DCC AND CHEMOTHERAPY IN COLORECTAL CANCER Vol. 47, No. 7
therapy, induces apoptosis of the tumoral vascula-
ture.
35
As for the DCC tumor cells, which seem to be
resistant to 5-FU, generally, tumor resistance to drugs
is multifactorial
36
and includes protection from apo-
ptosis and mutational status of regulators of apopto-
sis.
37,38
The allelic loss of chromosome 18q might act
through these mechanisms.
In reviewing the literature for other markers that
could predict the response to adjuvant chemotherapy,
we found that high thymidilate synthase expression
and p53 overexpression were associated with high
recurrence rate
39
and that these markers predict the
resistance to 5-FU treatment.
4042
However, DCC ex-
pression by immunohistochemistry seems to be a bet-
ter predictor for chemotherapy efficacy; the antigen is
commercially available, and the examination can be
easily performed in every pathology department.
As for the treatment of rectal cancer patients, in our
study, there was no difference between colon and
rectal tumors. 5-FU-levamisole and x-ray therapy
proved to be a useful treatment for DCC+ patients,
resulting in 100 percent cure rate. Therefore, it is pos-
sible that DCC expression could predict which patient
would be a better candidate for sphincter preserva-
tion operation with postoperative chemotherapy.
43
These issues should be further investigated in a larger
cohort of patients and in a prospective study.
Patients with DCC tumors did not benefit from
5-FU-levamisole treatment, therefore, the search for
better therapeutic modalities should focus on this
group of patients. One candidate for an alternative
treatment is the combination of 5-FU + leucovorin
and CPT11, a topoisomerase inhibitor that has shown
consistent activity in carcinoma refractory to 5-FU.
44,45
Another possibility is the combination of 5-FU with
oxaliplatin, which has a proven efficacy in meta-
static colon cancer.
46
Other combinations of drugs,
especially those that are known to be effective in
multidrug-resistant tumors, also should be investi-
gated.
47
CONCLUSIONS
DCC protein expression is not only a prognostic
factor but also can predict the response to adjuvant
chemotherapy. Therefore, we suggest that DCC im-
munohistochemistry should be performed routinely
in colorectal cancers. 5-FU-based adjuvant treatment
should be administered, if possible, to all DCC+ pa-
tients, irrespective of the stage, because they have the
potential for prolonging survival and DFS. In our
study, the five-year survival and DFS rates were 100
percent. As for DCC patients, although we did not
find statistical benefit for adjuvant therapy, a larger
cohort of patients should be examined before chang-
ing the therapeutic recommendations for this group.
However, clinical trials of new drug combinations
should focus on DCC patients.
ACKNOWLEDGMENTS
The authors thank Hannah Gertzman for the excel-
lent technical assistance, and Miriam Kleiner and
Yossi Sadovnic for photographs.
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