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(CANCER RESEARCH 55. 5038-5042,
November 1, 1995]
Aberrant p53 Expression Predicts Clinical Resistance to Cisplatin-based
Chemotherapy in Locally Advanced Non-Small Cell Lung Cancer
Valerie Rusch,1 David Klimstra, Ennapadam Venkatraman,
Julie Oliver, Nael Martini, Richard Gralla,2 Mark Kris,
and Ethan Dmitrovsky
Division of Thoracic Surgen; Departments of Surgery ¡V.R., N. M.I and Pathology ID. K., J. O.J; Bioslarislics Ser\'ice, Department of Epidemiolog\ and Bioslatistics [E. V.};
Division of Solid Tumor Oncology ¡R.G., M. K., E. D.I and Laboratory of Molecular Medicine ¡V.R.. E. D.I, Department of Medicine: and Molecular Pharmacology and
Therapeutics Program /£. D.I. Sloan-Kette ring Institute. Memorial Sloan-Kettering Cancer Center. New York. New York 10021
ABSTRACT
The development of cisplatin-based induction chemotherapy followed
by surgical resection or radiation has improved the poor prognosis of
stage III non-small cell lung cancer (NSCLC). In vitro studies indicate that
p53 can modulate cisplatin-induced cytotoxicity, but the molecular genetic
features determining response or resistance to cisplatin in vivo must be
defined. For this reason, tumor specimens from 52 patients with stage IIIA
NSCLC entered in a prospective clinical trial of cisplatin-based induction
chemotherapy followed by surgical resection were examined for p53
expression by immunohistochemical
staining before and after induction
chemotherapy. p53 expression was correlated with clinical and patholog
ical response using Fisher's exact test. No correlation was established
between p53 expression and clinical response because 47 of the 52 patients
studied had a major response. However, a significant association was
observed between aberrant p53 expression and resistance to chemother
apy as assessed by pathological response. Only 3 of the 20 patients whose
tumors exhibited a high level ( + + to + + + + ) of p53 staining experienced
a major ( + + + to + + + + ) pathological response to chemotherapy. Only
7 of 52 cases examined before and after chemotherapy treatment exhibited
a change in the level of p53 expression after cisplatin-based chemotherapy.
These results indicate that cisplatin alters p53 expression infrequently and
suggest a direct link between aberrant p53 expression and resistance to
cisplatin-based chemotherapy in NSCLC.
INTRODUCTION
Lung cancer is a formidable problem. There are more than 170,000
cases of lung cancer each year in the United States, and it is the most
common cause of cancer-related deaths in both men and women. Each
year, approximately 40,000 patients with NSCLC3 present with lo
cally advanced tumors (stage IIIA or IIIB). Traditional treatment of
stage HI tumors with surgical resection or radiation alone yielded
5-year survival rates of only 5-10% (1). Recently, multimodality
treatment using induction chemotherapy or chemoradiotherapy fol
lowed by surgical resection or chemotherapy followed by radiation
has significantly improved these poor survival rates (2-8). The treat
ment common to these neoadjuvant therapy regimens is the use of
high-dose cisplatin-based induction chemotherapy. However, at least
one-third of all patients do not achieve an appreciable clinical re
sponse to cisplatin-based induction chemotherapy. Long-term survival
is linked directly to the degree of clinical response to induction
chemotherapy. Therefore, it is important to understand why some
patients with stage III NSCLC respond to cisplatin-based chemother
apy, and others do not. Defining those molecular genetic features that
determine response or resistance to cisplatin-based chemotherapy has
important implications for this clinical setting.
Received 4/24/95; accepted 8/23/95.
The costs of publication of this article were defrayed in part by Ihe payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom requests for reprints should be addressed, at Memorial Sloan-Kettering
Cancer Center. 1275 York Avenue, New York, NY 10021. Phone: (212) 639-5873; Fax:
(212) 639-28(17.
2 Current address: Section on Hematology Medical Oncology, Department of Internal
Medicine, The Ochsner Clinic, New Orleans, Louisiana 70121.
1The abbreviation used is: NSCLC, non-small cell lung cancer.
In this regard, it is notable that abnormal expression or structure of
p53 is one of the most frequent genetic abnormalities in NSCLC,
occurring in approximately 60% of tumors (9). In vitro studies,
conducted primarily in fibroblast cell lines, show that p53-dependent
apoptosis mechanisms appear to be involved in the cytotoxicity in
duced by ionizing radiation and by several anticancer agents including
5-fluorouracil, etoposide, and doxorubicin (10-12). In addition, adenovirus-mediated transfer of the wild-type p53 gene into monolayer or
multicellular spheroid tumor cultures of a human NSCLC cell line
with a homozygous deletion of p53 markedly increased the sensitivity
of the cells to cisplatin (13). This suggests a direct link between
wild-type p53 expression and cisplatin-mediated cytotoxicity in lung
cancer. Additional clinical evidence for a relationship between wildtype p53 and exquisite sensitivity to chemotherapy is found in another
solid tumor, germ cell cancer. Germ cell tumors rarely exhibit p53
mutations and are quite sensitive to cisplatin-based chemotherapy
(14).
Taken together, these findings indicate that p53 might regulate
cisplatin response in NSCLC. Although this hypothesis is consistent
with the available in vitro data, to our knowledge, this possibility has
not as yet been tested in the in vivo setting. A large clinical trial at
Memorial Sloan-Kettering Cancer Center (New York, NY) evaluating
the use of induction cisplatin-based chemotherapy in stage IIIA
NSCLC permitted us to explore the relationship between p53 expres
sion and response to cisplatin chemotherapy. The patients who par
ticipated in this single-institution trial were carefully staged and were
a clinically homogeneous group. Almost all patients had an initial
mediastinoscopy, which made tissue available for immunohistochem
ical staining before chemotherapy treatment. The induction regimen
consisted of high-dose cisplatin-based chemotherapy without concur
rent radiation therapy. The surgical management of patients who
responded to chemotherapy was uniform and included thorough intraoperative staging. The clinical and histopathological evaluations of
response and the long-term outcome of patients enrolled in the study
have been analyzed and reported previously (7). This study reports
findings indicating that aberrant p53 expression is linked to clinical
resistance to cisplatin-based chemotherapy in these NSCLC patients.
MATERIALS
AND METHODS
Clinicopathological
Features. The tumors analyzed in this study were
obtained from patients enrolled in a prospective clinical trial of neoadjuvant
therapy for stage IIIA NSCLC. The details of this trial, performed from 1984
to 1991, were described previously (7). In brief, Ihe patients enrolled in this
trial had stage IIIA NSCLC thai was confirmed pathologically and untreated
previously. These patients received two or three cycles of an induction chem
otherapy regimen containing cisplatin (120 mg/nr) and mitomycin (8 mg/nr)
on days 1, 29, and 71 and vinblastine (4-4.5 mg/m2) or vindesine (3 mg/M')
on days 1, 8, 15, 22, and 29 and subsequently every 2 weeks. Patients
experiencing a partial or complete radiographie response after induction ther
apy underwent thoracotomy to resect residual tumor 4-6 weeks after the final
dose of cisplatin. Of 136 patients entered in this study, 105 (77%) experienced
a major radiographie response, 98 (72%) underwent thoracotomy, and 82
(60%) had a complete resection. With a minimum follow up of 12 months, the
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p53 AND CHEMOTHERAPY
RESPONSE
median survival for all 136 patients was 19 months, and the overall survival at
5 years was 17%. The results of this trial represent a distinct improvement in
survival compared with historical experience with surgical resection alone in
similarly staged patients. This finding has helped make neoadjuvant therapy an
accepted treatment for stage I11A NSCLC.
Cases were selected for this study only when tumor specimens were avail
able both before and after chemotherapy. This criterion permitted both corre
lation of the pathological response with p53 expression before chemotherapy
and a determination
of whether induction chemotherapy
altered p53
expression.
Histopathological
Methods. Five-/xm sections were cut from archival
paraffin blocks and placed on superfrost/Plus microscope slides. The slides
were baked at 60°Cfor 2 h immediately before staining. The sections were
then dcparaffinized in xylene and rehydrated through graded alcohol steps
to distilled water. The sections were placed in a citrate buffer solution (2.1
g of citric acid in 1 liter of distilled water, buffered to pH 6.0 with NaOH)
in a microwavable
container. The slides were microwaved in a citrate
buffer at 560 W for two 5-min cycles, and distilled water was added if
needed to correct for evaporation. The sections were cooled in the solution
for 20 min at room temperature, rinsed in distilled water, and transferred to
a PBS bath. The sections were then placed in a 0.05% BSA/PBS bath for
1 min, and normal horse suppressor serum diluted 1:20 in 2% BSA/PBS
was added for 10 min. The normal horse suppressor scrum was removed,
and the sections were incubated overnight with the primary antibody
diluted in 2% BSA/PBS at 4°C.The following day the primary antibody
was removed from the slides by rinsing in three changes of PBS. The
secondary antibody (hiotinylated horse anti-mouse IgG diluted 1:500 in 1%
BSA/PBS) was applied for 60 min. The slides were rinsed in three changes
of PBS, and peroxidase-conjugated
streptavidin was applied for 60 min
(diluted 1:500 in 1% BSA/PBS). Diaminobenzidine
was used as the chro-
\A). Aggregates of foamy histiocytes,
hcmosiderin-laden
macrophages,
and
inflammatory cells were present within the more densely fibrotic areas, as were
nests of viable carcinoma in the tumors showing partial response. In tumors
exhibiting a focal bronchioloalvcolar growth pattern, interstitial fibrosis and
chronic inflammation were seen in response to therapy. Residual tumor cells
often showed cytomegaly and nuclear pleomorphism that exceeded that found
in the pretherapy biopsies (Fig. Iß).
The pathological response in the primary tumor was scored as follows:
none = no evidence of treatment effect; + = treatment effect involving up to
one third of the gross tumor mass; + + = effect involving one third to two
thirds of the gross tumor mass; + + + = treatment effect in more than two
thirds of the gross tumor mass; + + + + = treatment effect of the entire tumor
with no viable carcinoma identified. Some primary tumors with a + + + +
response exhibited residual viable tumor in lymph node métastasesthat were
used for the postchemotherapy p53 staining. The pathological response was
scored by a single pathologist (D. K.), who was unaware of the clinical
response to induction chemotherapy.
The data on clinical response were obtained from the analysis of the trial
that was reported previously. A complete response was defined as the resolu
tion of all radiographie findings after chemotherapy, and a partial response was
scored when the product of two diameters of the tumor was reduced by one
half or more. The total number of major responses was the combination of all
complete and partial responses.
Microscopic examination for the p53 nuclear reaction product was scored as
follows: negative ( —¿)
= <5% of cells staining; + = 5-20% of cells staining:
+ + = 20-50% of cells staining; + + + = 50-80% of cells staining;
+ + + + = >80% of cells staining.
Statistical Analysis. The patients were cross-classified by p53 expression
before and after chemotherapy and by clinical and pathological responses to
chemotherapy. The resultant tables were analyzed for association between p53
staining before chemotherapy and clinical or pathological response using
Fisher's exact test. P < 0.05 was considered significant. The relationship
mogen. The slides were counterstained with hematoxylin. dehydrated, and
coverslipped. The mAb used in this study was pAhlXOl (Oncogene, Uniondale, NY) at a dilution of 1:500.
A negative control for the antibody was performed using nonimmune serum
instead of the primary antibody. The positive control consisted of a pulmonary
squamous cell carcinoma known to exhibit nuclear p53 expression using the
p53 mAb.
Assessment of Histopathological
Response. Pathological changes in the
resected posttherapy tumors varied from case to case and between different
regions of individual tumors. Changes regarded as reflecting a response to
therapy included evidence of necrosis and fibrosis. Foci of necrotic tumor were
noted less frequently and were occasionally difficult to distinguish from tumor
necrosis, which is often encountered in untreated high-grade NSCI.C. The
areas of fibrotic tumor were distinct. Pauciccllular regions of hyalinized tissue
with abundant small vessels and sparse inflammatory cells were found (Fig.
between p53 expression before and after chemotherapy was studied by clas
sifying the results into three groups: (a) a decrease in the level of p53
expression; (/;) no change in p53 expression; and (c) an increase in the level
of p53 expression.
RESULTS
Adequate pathological material for this study was available from
52 of the 82 patients who underwent resection of their tumor after
induction chemotherapy. The 30 cases viewed as unsuitable for
this study either had inadequate archival material for analysis or
had outside slides of mediastinal nodal biopsies that were unuvail-
.-S
~-
Fig. 1. a, b, and c, histológica! appearance of
NSCLC after prcoperative chemotherapy, a. tu
mors showing near-complete response consist
largely of paucicellubr fibrous tissue with numer
ous small vessels and scattered lymphocytes. A
focus of residual viable adcnocarcinoma is present
(arrows), b, in tumors with minimal treatment ef
fect, there is focal inflammation and fibrosis only:
the tumor cells show cytomegaly and nuclear en
largement (Ã-Ã-mw.v),
which is better seen at higher
magnification (c; X 40).
IN LUNG CANCER
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5039
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p53 AND CHEMOTHERAPY
RESPONSE
Table 1 Correlation between partial or complete clinical response and p53
immunohistochemical staining before chemotherapy
responseImmunohistochemical
Patients experiencing major
staining
before chemotherapy01+2+3+4+TotalComplete
response2111(15Partial
response20756442
IN LUNG CANCER
able because they were returned to the referring hospitals. Of the
52 specimens obtained before chemotherapy, 5 were primary tu
mors, and the remainder were lymph nodes biopsied at mediastinoscopy.
The correlation between pre-p53 expression and clinical response is
shown in Table 1. A total of 47 patients had a major objective
response, but the response was partial in 42 of these patients. Of the
47 patients, absent p53 staining was found in 22 specimens and
positive staining was observed in 8 specimens before chemotherapy.
Seventeen specimens revealed + + to + + + + staining.
The correlation between pre-p53 expression and pathological re
sponse is shown in Table 2. A total of
response. This was scored as + to + +
+ + + + in 17 patients. Because of the
levels were collapsed before a Fisher's
Table 2 Correlation between pathological response and p53 immunohistochemical
staining
response091510161
before chemotherapy01+2+3+4+Total0-12-4Pathological
+1233312Pathological0-218172+500117Response"3+530T0103-41434+421007
" When tumor specimens are grouped together into high and low categories only 3 of
the 17 patients with a +++ or ++++ pathological response had a high level (++ to ++++)
of p53 staining before chemotherapy.
Table 3 Comparison between level of p53 immunohisiochemical
after chemotherapy"
staining before and
chemotherapyÜ1831101+133212+003203+100114+00102
Beforechemotherapy01+2+3-f4+After
" Cases in which there were complete pathological responses after induction chemo
therapy could not be assessed because there was no residua] viable tumor on which to
perform immunohistochcmical staining.
36 patients had a pathological
in 19 patients and as + + + to
small numbers of patients, the
exact test was used to test for
association. The 20% cutpoint in the percentage of cells staining was
chosen by the pathologist involved in this study (D. K.) as the level at
which staining could be consistently defined as positive across all
samples examined. No association between p53 expression and clin
ical response was found because of the large proportion of clinical
responses which were partial. A statistically significant association
was found when p53 expression and pathological response were
grouped into the low and high categories. Only 3 of the 20 patients
who had tumors exhibiting a high level (++to+
+ + +)of p53 tumor
staining before chemotherapy had a + + + to + + + + pathological
response (P = 0.04).
The comparison between p53 expression before and after chemo
therapy is shown in Table 3. This comparison could not be made in
cases in which a major pathological response to chemotherapy offered
little or no viable tumor on which to perform staining for p53. In 27
of the 44 évaluablespecimens, there was no change in the level of p53
staining (Fig. 2). In 14 cases, the level of p53 staining decreased, and
in 3 cases it increased. However, in only 7 cases did the level of p53
positivity change by more than one level from the prechemotherapy to
the postchemotherapy specimen. Therefore, most of the changes ob
served were small enough to be caused by technical factors, such as
variations in fixation or variable sampling in tumors with heterogenous p53 expression.
Fig. 2. a, and b, immunohistochemical staining
for p53. Both pretreatmen! tumor from mediastinal
lymph nodes (a) and posttrcatment tumor (b) show
intense nuclear positivity. The surrounding lym
phocytes and stromal cells are negative.
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p53 AND CHEMOTHERAPY
RESPONSE
DISCUSSION
Patients with stage III NSCLC constitute a large and important
segment of the lung cancer population, representing approximately
40,000 cases annually in the United States. Previously, treatment with
radiation therapy or surgical resection alone offered most patients
with stage III disease only a 10% or lower chance of survival at 5
years. These poor survival rates are related to the locally advanced
nature of the primary tumor and the presence of micrometastatic
disease. Recently, combined modality regimens using chemotherapy
as the induction therapy have significantly improved the prognosis of
stage III NSCLC patients (2-7). The chemotherapy drug common to
all of these regimens is high-dose cisplatin. A complete pathological
response to induction chemotherapy identifies those patients with the
most favorable long-term survival (7). However, only 20% or fewer of
patients develop a complete pathological response, and 20-40% of
patients do not respond to induction chemotherapy. Understanding the
molecular genetic features that determine response or resistance to
cisplatin-based chemotherapy should permit 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 stage III NSCLC patients most likely to respond.
This could enhance development of innovative treatment for patients
most likely to be refractory to cisplatin-based chemotherapy.
The precise mechanisms responsible for cisplatin-mediated toxicity
are not fully understood. In vitro studies indicate cisplatin inhibits
DNA synthesis by causing double-strand breaks (15), leading to
apoptosis at the O2-M transition (16). In vitro studies also indicate a
link between wild-type p53 and chemotherapy-induced apoptosis. A
similar relationship is reported for cisplatin in NSCLC cell lines (13),
esophageal adenocarcinoma, and gastric carcinoma cell lines (17).
Related in vitro studies suggest that wild-type p53 is involved in
growth suppression of malignant cell lines induced by radiation and
several chemotherapeutic agents thought to function through apopto
sis (11, 12, 18-21). The hypothesis that p53 is an important regulator
of the clinical response to cisplatin is supported by the observation
that germ cell cancer, a solid tumor that is highly sensitive to cisplatin,
almost never exhibits p53 mutations (14). This report extends previ
ous in vitro work by exploring the relationship between p53 expres
sion in tumors and clinical treatment response to cisplatin-based
chemotherapy in stage III NSCLC.
The results of this study reveal that the absence of p53 immunostaining before chemotherapy does not correlate statistically with
pathological response to induction with cisplatin-based chemotherapy
in this clinical setting. However, aberrant p53 expression was signif
icantly associated with a lack of pathological response to cisplatinbased chemotherapy. It is recognized that there often is a discrepancy
among clinical, radiographie, and pathological responses after induc
tion therapy of NSCLC. Pathological response provides the most
precise measure of the clinical effects of induction treatment (1,5, 8).
Although cisplatin may act through p53-independent pathways (2225), aberrant p53 expression predicts resistance to cisplatin treatment
of the lung cancer patients examined in this study. The number of
cases available for analysis in this study is too small to allow statis
tically valid correlations between the immunohistochemical results
and overall survival. However, the previous analysis of the clinical
trial from which these samples were drawn showed a significant
correlation between complete pathological response and overall sur
vival (7). Therefore, our results have important clinical implications.
Some factors could complicate the interpretation of the findings of
this study. Most of the prechemotherapy specimens available for
analysis were mediastinal nodal métastases.It is conceivable that p53
expression in nodal métastasesdoes not fully reflect the pattern of p53
IN LUNG CANCER
expression found in the primary tumor. Arguing against this possibil
ity are previous reports indicating that p53 mutations found in a
primary lung cancer are conserved in the métastasesfrom that tumor
(26). Therefore, the specimens used in this study should be useful for
analysis.
A larger prospective study is needed to confirm the findings of this
report. It is notable that the induction regimen in this trial included a
Vinca alkaloid and mitomycin. How this combination regimen modulates
the interactions between cisplatin and p53 remains to be determined.
However, the finding of a correlation between p53 expression and a
response to cisplatin chemotherapy reported in this study is more likely to
be accurate than is an analysis performed in other neoadjuvant trials using
concurrent cisplatin and radiation for induction therapy. A chemotherapyonly induction regimen prevents the potentially complicating effect of
radiation, which also is thought to inhibit tumor cell growth via p53dependent apoptosis mechanisms (12).
Finally, immunohistochemical staining does not distinguish be
tween p53 expression that is abnormal because of p53 mutations and
expression that is abnormal because of deregulated expression of a
structurally normal p53 protein (27). Several mechanisms can lead to
p53 protein overexpression in primary NSCLC, and the concordance
rate between mutations and overexpression as assessed by immuno
histochemical staining is reported to be only 67% (28). It is viewed
that aberrant p53 protein expression as assessed by immunohisto
chemical techniques provides a clinically useful measure of deregu
lated p53 function in NSCLC.
In summary, the results reported here show a statistically significant
link between aberrant p53 expression and cisplatin-based chemotherapy
resistance. This link is not yet strong enough to dictate clinical treatment
decisions in stage III NSCLC. These findings also indicate that this
cisplatin-based treatment infrequently alters p53 expression, and that the
absence of p53 staining does not predict increased clinical sensitivity to
this combination chemotherapy regimen. Which other cell cycle regula
tors act in concert with or independent of p53 to elicit these beneficial
clinical responses remains to be determined. The role of Rb, cyclindependent kinases, or inhibitors in mediating these clinical responses
warrants study. Additional work is needed to identify those molecular
pathways that mediate major clinical chemotherapy responses in NSCLC,
particularly in the setting of wild-type p53.
ACKNOWLEDGMENTS
The authors thank Melody Owens for expert assistance in the preparation of
the manuscript. The authors acknowledge the other physicians in the Depart
ments of Surgery, Medicine, and Radiology at Memorial Sloan-Kettering
Cancer Center who participated in the clinical trial from which the specimens
for this study were drawn: Drs. Manjit S. Bains, Michael E. Burt, Robert J.
Ginsberg, Robert Heelan, Patricia M. McCormack, Katherine M. W. Pisters,
and James R. Rigas.
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Downloaded from cancerres.aacrjournals.org on April 14, 2017. © 1995 American Association for Cancer Research.
Aberrant p53 Expression Predicts Clinical Resistance to
Cisplatin-based Chemotherapy in Locally Advanced Non-Small
Cell Lung Cancer
Valerie Rusch, David Klimstra, Ennapadam Venkatraman, et al.
Cancer Res 1995;55:5038-5042.
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