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A comparison of patterns of care of nonsmall cell lung carcinoma patients in a younger and Medigap commercially insured cohort

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Expression of p53 Protein and Resistance to
Preoperative Chemotherapy in Locally Advanced
Gastric Carcinoma
Stefano Cascinu, M.D.1,2
Francesco Graziano, M.D.1,2
Elena Del Ferro, Sc.D.1,2
Maria P. Staccioli, Sc.D.3
Marco Ligi, Sc.D.1,2
Andrea Carnevali, M.D.3
Pietro Muretto, M.D.3
Giuseppina Catalano, M.D.2
Section of Experimental Oncology, Azienda Ospedaliera S. Salvatore, Pesaro, Italy.
Division of Medical Oncology, Department of Hematology/Oncology, Azienda Ospedaliera S. Salvatore, Pesaro, Italy.
Department of Pathology; Azienda Ospedaliera S.
Salvatore, Pesaro. Italy.
BACKGROUND. Inactivation of the p53 gene has been reported to be associated with
resistance to chemotherapy. The authors evaluated the significance of p53 status to
the clinical outcomes of patients with locally advanced, unresectable gastric carcinoma (LAGC) who received chemotherapy.
METHODS. Thirty chemotherapy-naive patients with LAGC received weekly administration of cisplatin 40 mg/m2, epi-doxorubicin 35 mg/m2, 5-fluorouracil 500
mg/m2, 6S-leucovorin 250 mg/m2, and glutathione 1500 mg/m2. After eight administrations of these agents, patients were assessed for response. Biopsy specimens of primary tumors were analyzed for p53 status using monoclonal antibody
RESULTS. Characteristics of patients were as follows: The median age was 66 years
(range, 44 –70 years); 18 were males and 12 were females. Eastern Cooperative
Oncology Group performance status was 0 for 14 patients and 1 for 16. Histology
was intestinal for 13 patients; for 17, it was diffuse. The site of the primary tumor
was the cardia in 8 patients, the body of the stomach in 13, and the antrum in 9.
The response rate (assessed with CT scan and endoscopy) for patients with p53
negative tumors was significantly higher than for those with overexpression of p53
(71% vs. 12%, P ⫽ 0.004).
CONCLUSIONS. p53 status analyzed before chemotherapy seems to be associated
with response to treatment in patients with LAGC. This may provide a useful guide
to selecting neoadjuvant chemotherapy for these patients. Cancer 1998;83:
1917–22. © 1998 American Cancer Society.
KEYWORDS: p53, locally advanced gastric carcinoma, chemotherapy.
Address for reprints: Stefano Cascinu, M.D., Section of Experimental Oncology, Department of Hematology/Oncology, Azienda Ospedaliera S. Salvatore. v. Lombroso, 61100 Pesaro. Italy.
Received December 23, 1997; revision received
April 9, 1998; accepted April 9, 1998.
© 1998 American Cancer Society
lthough gastric carcinoma is declining in incidence, it remains a
significant cause of mortality.1 Many patients present with locally
advanced disease; and in more than two-thirds of cases, local extension prevents curative resection. Unfortunately, resectability is one of
the main prognostic factors in patients with gastric carcinoma, and
survival is longer when tumors are completely removed.2 The use of
neoadjuvant chemotherapy is an attractive idea for increasing curative resection. Although this approach determined about 50 – 60% of
clinical responses and allowed radical surgery in 40 –50% of patients
with previously unresectable tumors,3– 8 chemotherapeutic regimens
currently administered to gastric carcinoma patients have substantial
toxicities, so that identification of responsive patients appears worthwhile to avoid the side effects associated with chemotherapy in unresponsive patients. Currently, the use of clinical parameters cannot
accurately predict which patients may be best served by preoperative
CANCER November 1, 1998 / Volume 83 / Number 9
Patient Characteristics
No. of patients
Total no. of patients
Age (yrs)
Site of primary tumor
Body of stomach
Histologic type
ECOG performance status
ECOG: Eastern Cooperative Oncology Group.
The p53 protein is a pleiotropic molecule with
numerous functions. One of these functions involves
the pathway of programmed cell death induced by
DNA-damaging chemotherapeutic drugs.9 It has been
demonstrated that p53-dependent apoptosis modulates the cytotoxic effects of common antitumor
agents such as 5-fluorouracil, doxorubicin, and cisplatin.10 Cells lacking wild-type p53 are resistant to these
agents, and p53 assay could be used to predict the
response of cancer to chemotherapy.11,12 This association was recently found in nonsmall cell lung carcinoma and ovarian carcinoma.13,14
Nuclear overexpression of p53 protein determined by immunohistochemistry (IHC) occurs in
approximately 50% of gastric carcinomas and is an
independent prognostic factor for relapse and survival.15–18
To test the hypothesis that p53 alteration could
lead to resistance to cytotoxic therapy and also predict
responsiveness in gastric carcinoma patients, we examined the p53 status of patients with locally advanced, unresectable gastric carcinoma who received
preoperative chemotherapy including cisplatin, 5-fluorouracil (5-FU), and epi-doxorubicin (epi-ADR).
Patients’ Characteristics and Treatment
Table 1 shows the characteristics of the 30 patients included in this study. All patients had locally advanced,
unresectable gastric carcinoma confirmed pathologically. In 14 patients, the diagnosis was based on a computed tomography (CT) scan that evaluated for tumor
size, invasion of adjacent structures, or advanced locoregional lymph node involvement, whereas in the other 16
patients locally advanced disease was confirmed by laparotomy.
The chemotherapeutic regimen consisted of a
1-day weekly administration of cisplatin (CDDP) 40
mg/m2 as a 30-minute infusion in 250 mL of normal
saline solution, 5-FU 500 mg/m2 as a 15-minute infusion in 100 mL of normal saline solution, and epi-ADR
35 mg/m2 by intravenous bolus. 6S-stereoisomer of
leucovorin was administered at a dose of 250 mg/m2
diluted in 250 mL of normal saline solution in a 4-hour
infusion concurrent with hydration. Glutathione was
given at a dose of 1.5 g/m2 in 100 mL of normal saline
over 15 minutes immediately before each CDDP administration.
Standard intravenous hydration was used: 2 hours
before initiation of the CDDP infusion, patients were
hydrated with 1500 mL of 0.9% sodium chloride, to
which 20 mEq of potassium chloride and 15 mEq of
magnesium sulfate were added. Posthydration was
continued for 2 hours with 1000 mL of normal saline
solution. From the day after to the day before each
chemotherapy administration, filgastrim was administered by subcutaneous injection at a dose of 5 g/kg.
Evaluation of response was performed after eight
weekly treatments. Patients were required to have CT
scan and endoscopic evaluation with biopsy if tumor
was visible. Partial response (PR) was defined as evidence on both CT scan and endoscopy of a ⬎50%
reduction in the visible tumor or complete disappearance of tumor but positive histology on biopsy of the
previously involved area. Complete response (CR) was
defined as a normal-appearing stomach on CT scan
with a complete resolution of the endoscopically visible tumor and a negative biopsy of the original site of
the tumor.
Tumor Specimen Selection
For each patient, three endoscopic samples were collected and examined. Tumor sampling was supervised
by a pathologist. Sections of the samples were examined by conventional histology and were used for immunohistochemistry.
Formalin fixed, paraffin embedded tissue sections
from the tumors were analyzed immunohistochemically for altered patterns of p53 expression, using a
standard avidin-biotin technique. Sections (4 ␮m
thick) were deparaffinized in xylene, rehydrated in a
graded ethanol series, and incubated in 3% hydrogen
peroxide for 20 minutes. Specimens were placed in a
plastic Coplin jar containing citric buffer and heated
p53 in Locally Advanced Gastric Carcinoma/Cascinu et al.
FIGURE 1. Positive (A) and negative (B) immunohistochemical staining with monoclonal antibody Bp53–12 are shown.
4 ⫻ 2.5 minutes in a microwave processor at 95°C.
After the microwave processing, sections were left in
the Coplin jar at room temperature for 30 minutes.
Specimens were covered with normal goat serum for
15 minutes to reduce nonspecific staining and incubated with a 1:100 dilution of primary antibody
Bp53–12 (Biogenex, San Ramon, CA) at room temperature for 2 hours. The sections were washed with
Tris-buffered saline, incubated with a 1:100 dilution of
biotinylated goat antimouse immunoglobulin G at
room temperature for 30 minutes, and then covered
with a 1:100 dilution of streptavidin-biotin-peroxidase
complex at room temperature for 30 minutes. The
antibody was localized with 3,3⬘-diaminobenzidine
tetrahydrochloride (DAB). Tissue sections were counterstained with light hematoxylin, dehydrated with
ethanol, and mounted under a coverslip.19 Immunohistochemical staining of tumors with this antibody
shows primarily a nuclear localization of p53 protein.
Bp53–12 reacts with both wild-type and mutant p53
The staining results were interpreted independently by one investigator who was unaware of the
clinical outcome. In each case, the entire section was
systemically examined on high-power fields (⫻40) for
p53 immunoreactivity. Among all immunoreactive
nuclei, only those clearly immunostained were recorded as being p53 positive.
The level of immunoreactivity was expressed as
the percentage of p53 positive cancer cell nuclei. For
analysis, tumors were classified into p53 immunoreactivity categories of low level (negative or ⬍20% positive nuclei) and high level (⬎20% positive nuclei).
The patients were cross-classified by p53 expression and by clinical responses to chemotherapy. Fisher’s exact test was used to evaluate the statistical significance. P ⬍ 0.05 was considered significant.20
Adequate pathologic material for this study was available in at least 2 of the 3 endoscopic samples from all
of the 30 patients entered into the study of preoperative chemotherapy.
A high level of p53 immunoreactivity was seen in
16 of 30 patients (53%) (Fig. 1). Intratumor heterogeneity of p53 expression was systemically assessed in
the different endoscopic samples, without finding
variations able to modify the interpretation of p53
status in each tumor.
Correlation of p53 status with other histologic and
CANCER November 1, 1998 / Volume 83 / Number 9
p53 Protein Expression and Clinicopathologic Findings
Age (yrs)
ECOG performance status
Site of primary
Body of stomach
Tumor size (cm)
Clinical outcome
No response
% positive
P value
ECOG: Eastern Cooperative Oncology Group; ns: not statistically significant.
clinical variables was reported in Table 2. No association was seen among p53 immunoreactivity and any
of variables analyzed, apart from clinical response.
Among the 30 patients who received chemotherapy
and had an assessment of p53 immunoreactivity, 12
patients achieved an objective response (3 CRs) (40%),
13 had stable disease, and 5 experienced disease progression during therapy. Ten of 12 patients who
showed a clinical response had p53 negative tumors,
whereas 14 of 18 nonresponders had p53 positive tumors. The 3 patients who achieved a CR had the
lowest levels of p53 (⬍5%, ⬍10%, and ⬍10%, respectively).
Eleven patients (36%) underwent radical surgery.
Nine of them are alive and disease free at a median
follow-up of 20 months. Two relapsed (peritoneum
and liver) and died 8 and 10 months after surgery,
respectively. In these two patients, p53 expression was
20% and 50%, respectively.
In nine of the patients who underwent surgery,
p53 expression status was assessed in the resection
specimens; in two cases it was not performed because
of the pathologic CR status of the patients. A high level
of p53 immunoreactivity (ⱖ50%) was seen in 7 patients, whereas in the other 2 cases it was considered
lower than the cutoff.
Preoperative chemotherapy seems to be a logical approach to improving surgical resectability that is one
of the main prognostic factors in patients with gastric
carcinoma.2 However, only 40 –50% of patients take
advantage of this therapeutic modality, whereas approximately 30% of patients experience moderate-tosevere toxic effects.5– 8 Understanding the molecular
genetic features that determine response or resistance
to chemotherapy should permit the selection of the
most suitable patients for neoadjuvant therapy. This
would avoid exposing some patients to ineffective
treatment and could tailor chemotherapy treatment to
those cancer patients most likely to respond.
In vitro studies indicate that p53-dependent apoptosis modulates the cytotoxicity of chemotherapeutic
agents such as 5-FU, doxorubicin, and CDDP, and that
the absence of wild-type p53 function results in cellular resistance to chemotherapy in several cell lines,
including gastric carcinoma.10 –12,21 Overexpression of
p53 protein as the result of an altered gene has been
demonstrated in 40 – 60% of primary gastric carcinomas.15–18 Hamada et al. reported that p53 status
seemed to predict response to preoperative chemotherapy in three patients with gastric carcinoma.22
Similar data were also reported by Nakata et al. for 15
patients who received preoperative chemotherapy
with 5-FU and CDDP. Four of the six patients who
responded to chemotherapy were p53 negative,
whereas all of nonresponders overexpressed p53 protein.23
The current report extends previous works by exploring the correlation between p53 expression in tumors and clinical treatment response to chemotherapy in locally advanced gastric carcinoma. The results
of this study show clearly that the absence of p53
immunostaining before chemotherapy correlates with
clinical response to neoadjuvant chemotherapy in this
clinical setting.
Another finding seems to confirm indirectly that
inactivation of the p53 gene contributes to enhanced
cellular resistance to chemotherapy. In seven of nine
resected specimens, determination of p53 levels demonstrated higher values than endoscopic specimens
and all of them were higher than the cutoff value,
suggesting that chemotherapy kills mainly p53 negative cells.
However, some caution is required in the interpretation of these data, because some factors could
result in misinterpretation. Immunohistochemical
staining does not distinguish p53 expression that is
abnormal because of p53 gene mutations from expression that is abnormal because of deregulated expres-
p53 in Locally Advanced Gastric Carcinoma/Cascinu et al.
sion of a structurally normal p53 protein.24 Although
molecular studies have confirmed point mutations in
the gene to be the most usual cause of p53 accumulation,25,26 other events can also result in overexpression of the protein (i.e., changes in the cellular environment resulting from DNA-damaging events).27
Nevertheless, p53 protein accumulation, whether it is
dependent on gene alteration or not, could be the
main prognostic factor affecting response to chemotherapy, as recently found for ovarian carcinoma.14 In
fact, as stabilization of a mutant form of p53 abrogates
the normal function of the protein as an inducer of
apoptosis, overexpression of a wild-type p53 could
increase the repair efficiency of the cell, thus reducing
the cytotoxic effects of the DNA-damaging agents.14
The choice of Bp53–12 antibody was unlikely to
influence results of p53 analysis. In fact, although for
gastric carcinoma there are no data, Baas et al. showed
that six different antibodies (including Bp53–12) gave
the same results in colorectal carcinoma.28
A critical point in this study could be the choice
of a higher cutoff value (20%) than that generally
used to analyze the prognostic implications of p53
overexpression in gastric carcinoma (10%).17,29 In
our opinion, because the aim of preoperative chemotherapy for patients with gastric carcinoma is
to reduce tumor size in order to increase the chance
of a curative resection, focally positive areas that
result from a small subclone of cells with mutations
and are potentially unresponsive to chemotherapy
should not preclude the effectiveness of a preoperative chemotherapeutic program. However, a similar cutoff value was used by Hamada et al. to assess
the role of p53 expression as a determinant of chemosensitivity in gastric and colorectal carcinoma.22
Furthermore, for other tumors, such as nonsmall
cell lung carcinoma and bladder carcinoma, in
which the correlation between p53 overexpression
and preoperative chemotherapy was investigated,
the cutoff value was similarly defined as 20% of
positive cells.13,30
In conclusion, the results of the current study
are consistent with the idea that p53 is a direct
determinant of the chemosensitivity of gastric carcinoma. These data may have clinical relevance for
implications in the treatment of locally advanced
gastric carcinoma. Additional studies are needed to
identify other cell cycle regulators that act in concert with or independently from p53 (such as Rb and
cyclin-dependent kinase) and may contribute to
the modulation of clinical response to chemotherapy.
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