Download Decreased Immunoreactivity for p27 Protein in Patients with Early

1591
Decreased Immunoreactivity for p27 Protein in
Patients with Early-Stage Breast Carcinoma Is
Correlated with HER-2/neu Overexpression and
with Benefit from One Course of Perioperative
Chemotherapy in Patients with Negative
Lymph Node Status
Results from International Breast Cancer Study Group Trial V
Vito J. Spataro, M.D.1,2
Heather Litman, M.S.3,4
Giuseppe Viale, M.D.5
Fausto Maffini, M.D.5
Michele Masullo, M.D.5
Rastko Golouh, M.D.6
Francisco J. Martinez-Tello, M.D.7
Piergioranni Grigolato, M.D.8,9
Keith B. Shilkin, M.B.B.S.10
Barry A. Gusterson, Ph.D.11,12
Monica Castiglione-Gertsch, M.D.13
Karen Price, B.S.3,14
Jurii Lindtner, M.D.15
Hernan Cortés-Funes, M.D., Ph.D.16
Edda Simoncini, M.D.17
Michael J. Byrne, M.B.B.S.18
John Collins, M.D.19
Richard D. Gelber, Ph.D.3,4,14
Alan S. Coates, M.D.20,21
Aron Goldhirsch, M.D.22,23 for the
International Breast Cancer Study
Group
1
BACKGROUND. The objective of this study was to clarify the prognostic and predictive value of immunoreactivity for the cyclin-dependent kinase inhibitor
p27(Kip1) in patients with early-stage breast carcinoma and to investigate its
relation with clinicopathologic features and other markers.
METHODS. Immunoreactivity for p27 protein was analyzed on tumor slides from
461 patients who were enrolled in the International Breast Cancer Study Group
(IBCSG) Trial V (median follow-up, 13 years), including 198 patients with lymph
node negative disease and 263 patients with lymph node positive disease. Tumors
with ⬍ 50% immunoreactive neoplastic cells were considered low expressors.
Immunoreactivity for p27 was correlated with several clinicopathologic characteristics. Disease free survival (DFS) and overall survival were analyzed according to
p27 immunoreactivity and treatment group.
RESULTS. In the lymph node negative population, decreased p27 immunoreactivity
was associated with higher tumor grade (P ⫽ 0.001) and HER-2/neu overexpression
(P ⫽ 0.04). In the lymph node positive population, low p27 expression was associated with higher tumor grade (P ⫽ 0.01), low expression of thymidylate synthase
(P ⫽ 0.001), and higher Ki-67 expression (P ⫽ 0.007). DFS was not significantly
different according to p27 status in either lymph node negative patients (10-year
DFS: low p27 expression, 53% ⫾ 5%; high p27 expression, 55% ⫾ 5%) or in lymph
node positive patients (10 year DFS: low p27 expression, 33% ⫾ 4%; high p27
expression, 32% ⫾ 4%). However, in the lymph node negative population, the
5
Department of Pathology and Laboratory Medicine, European Institute of Oncology, Milan, Italy.
10
The Western Australia Centre for Pathology and
Medical Research, Nedlands, Western Australia,
Australia.
Department of Medical Oncology, Ospedale San
Giovanni, Bellinzona, Switzerland.
6
Department of Pathology, Institute of Oncology,
Ljubljana, Slovenia.
11
2
International Breast Cancer Study Group (IBCSG)
Protocols Working Group, Bellinzona, Switzerland.
7
Department of Anatomic Pathology, Hospital Universitario 12 de Octubre, Madrid, Spain.
12
3
8
International Breast Cancer Study Group (IBCSG)
Pathology Centre, Glasglow, United Kingdom.
Department of Pathology, University of Glasglow,
Glasglow, United Kingdom.
International Breast Cancer Study Group (IBCSG)
Statistical Center, Boston, Massachusetts.
Anatomica Patologica, Spedali Civili, Brescia, Italy.
13
4
9
14
Harvard School of Public Health, Boston, Massachusetts.
© 2003 American Cancer Society
Anatomica Patologica, Universita degli Studi di
Brescia, Brescia, Italy.
International Breast Cancer Study Group (IBCSG)
Coordinating Center, Bern, Switzerland.
Frontier Science and Technology Research
Foundation, Boston, Massachusetts.
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CANCER April 1, 2003 / Volume 97 / Number 7
benefit of one course of perioperative chemotherapy with cyclophosphamide,
methotrexate, and 5-fluorouracil was confined exclusively to patients with tumors
that showed reduced p27 immunoreactivity (P ⫽ 0.03; test for interaction).
CONCLUSIONS. This analysis indicates that p27 immunoreactivity has little if any
prognostic value in patients with early-stage breast carcinoma. However, these
findings suggest that, in patients with breast carcinoma who have negative lymph
node status, reduced p27 immunoreactivity is associated with HER-2/neu overexpression and may be predictive of a benefit from the early administration of
adjuvant chemotherapy. Cancer 2003;97:1591– 600.
© 2003 American Cancer Society.
DOI 10.1002/cncr.11224
KEYWORDS: p27, breast carcinoma, HER-2/neu, Ki-67, adjuvant chemotherapy,
predictive factors.
F
or the vast majority of patients with early-stage
breast carcinoma, it has been shown that several
forms of adjuvant treatment provide a benefit in terms
of disease free survival (DFS) and overall survival (OS).
However, there remains considerable controversy
over how to tailor the most beneficial treatment to
each patient according to the individual characteristics of the patients and their tumors. Despite the variety of new tumor markers that have been investi-
gated in the last 10 –15 years, only a few provide
clinically useful information in addition to lymph
node status, hormone receptor status, and tumor
grade, and there remains a substantial need for clinically useful tumor markers.1
Various kinases called cyclin dependent kinases
(CDKs) are the key regulators of cell cycle progression
and are under the positive control of cyclins and the
negative control of CDK inhibitors. The p27 protein
15
Department of Surgery, The Institute of Oncology, Ljubljana, Slovenia.
16
Division of Medical Oncology, Hospital Universitario 12 de Octubre, Madrid, Spain.
17
Oncologia Medica, Spedali Civili, Brescia, Italy.
18
Department of Medical Oncology, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia.
19
Department of Surgery, Royal Melbourne Hospital, Melbourne, Victoria, Australia.
20
The Cancer Council Australia, Sydney, Australia.
21
School of Public Health, University of Sydney, Sydney, Australia.
22
Division of Oncology, European Institute of Oncology, Milan, Italy.
23
Oncology Institute of Southern Switzerland, Lugano, Switzerland.
Supported in part by Grant CA-75362 from the National Cancer Institute and in part by Grant AKT 302 from the Krebsforschung Schweiz, Breakthrough Breast Cancer.
The authors gratefully acknowledge the initial support provided by the Ludwig Institute for Cancer Research, the Cancer League of Ticino, and the Swiss Cancer
League. They further acknowledge the continuing support for central coordination, data management, and statistics provided by the Swedish Cancer League, the
Cancer Council Australia, the Australian-New Zealand Breast Cancer Trials Group, the Frontier Science and Technology Research Foundation, the Swiss Group for
Clinical Cancer Research, and the United States National Cancer Institute.
The authors thank the patients, physicians, nurses, and data managers who participate in the International Breast Cancer Study Group trials. The skillful technical
assistance of Miss Alessandra Cavallon in the immunocytochemical experiments also is recognized.
The authors acknowledge the following participants in this study of International Breast Cancer Study Group (IBCSG) Trial V: IBCSG Central Pathology Review (B.
Davis, W. Hartmann, R. Bettelheim, A. and M. Neville); IBCSG Data Management (M. Isley, R. Hinkle, and L. Blacher); The Institute of Oncology, Ljubljana, Slovenia
(J. Lamovec, J. Jancar, J. Nowak, D. Erzen, M. Naglas, M. Sencar, J. Cervak, O. Cerar, B. Stabuc, S. Sebek, and T. Cufer); Madrid Breast Cancer Group, Madrid,
Spain (C. Mendiola, F. Cruz-Caro, M. L. Larrodera, F. Calero, A. Suarez, F. Pastrana, S. Cruchaga, C. Guzman, and B. Rodriguez); Spedali Civili and Fondazione
Beretta, Brescia, Italy (L. Morassi, G. Marini, P. Marcpicati, U. Sartori, A. Barni, D. DiLorenzo, A. Albertini, G. Marione, and M. Zorzi); and Sir Charles Gairdner Hospital,
Nedlands, West Australia, Australia (P. M. Reynolds, H. J. Sheiner, S. Levitt, D. Kermode, R. Hahnel, and G. Van Hazel).
Address for reprints: Vito Spataro, M.D., International Breast Cancer Study Group Biological Protocol Working Group, Oncology Institute of Southern Switzerland,
Ospedale San Giovanni, CH-6500 Bellinzona, Switzerland; Fax: 011 (41) 918209044; E-mail: [email protected]
Received September 18, 2002; accepted November 15, 2002.
p27 and HER-2neu in Early Breast Carcinoma/Spataro et al.
belongs to the family of the Kip inhibitors, which has
a broad spectrum of inhibitory activity on different
CDKs and acts as a tumor-suppressor protein.2 A large
body of studies has shown that p27 activity is regulated almost exclusively posttranslationally at the level
of protein degradation3 and that decreased p27 protein levels are common in several tumor types.4
Several retrospective studies have shown that reduced immunoreactivity of the p27 protein in patients
with early-stage breast carcinoma was associated with
unfavorable prognostic factors and a significant increased risk of disease recurrence or death.5–7 Downregulated p27 was an independent, unfavorable prognostic factor in multivariate analysis, although it was
correlated with high tumor grade in one study5 and
with negative estrogen receptor (ER) status in two
studies.5,6 Tan et al. reported that low p27 expression
was an independent prognostic marker in patients
with small (T1a,b) lymph node negative tumors, suggesting the value of p27 in selecting patients with
small, lymph node negative breast carcinoma who
may benefit from adjuvant therapy. Compared with
those early reports, other studies found that low p27
expression was correlated strongly with tumor grade
and had no prognostic value in multivariate analyses.8 –10 Unfortunately, those studies used different
definitions for p27 status or different cut-off values
when p27 scoring was based on the percentage of
immunoreactive cells. Furthermore, the cohorts of patients investigated were not treated in randomized
clinical trials. Consequently, the interaction between
p27 status and the benefit from adjuvant treatment
usually could not be addressed. We are aware of only
a single report11 that addressed the question of the
possible predictive value of p27 protein levels on the
benefit of adjuvant chemotherapy. Those authors reported an improvement in DFS and OS for patients
with lower p27 immunoreactivity but not for patients
with high p27 immunoreactivity.
The current study was planned to assess the prognostic and predictive value of p27 immunoreactivity in
a large population of patients with early-stage breast
carcinoma who were enrolled in a randomized clinical
trial and to investigate the correlation of p27 status
with established markers.
MATERIALS AND METHODS
Patients
The material used in this study was from a cohort of
patients who were enrolled in the International Breast
Cancer Study Group (IBCSG) Ludwig Trial V.12–14 In
1981, the IBCSG (formerly the Ludwig Breast Cancer
Study Group) began a randomized clinical study to
assess the effect of early commencement of adjuvant
1593
cyclophosphamide, methotrexate, and 5-fluorouracil
(CMF) chemotherapy in patients with lymph node
negative and lymph node positive breast carcinoma. A
total of 2504 eligible patients were entered on the trial
between 1981 and 1985. All patients had their breast
tumors removed by either total mastectomy with axillary clearance or modified radical mastectomy. To be
eligible, patients had to have disease of unilateral clinical stage T1, T2, or T3a; N0 or N1; and M0 according
to the tumor-lymph node-metastasis classification
system. Patients with lymph node negative breast carcinoma (n ⫽ 1275 women) were randomized to receive either a single cycle of perioperative chemotherapy (PeCT) or no adjuvant chemotherapy. Lymph
node positive patients (n ⫽ 1229 women) were assigned to one of three treatments: PeCT, conventionally timed chemotherapy (ConCT), or both. The PeCT
regimen was a combination of intravenous cyclophosphamide, methotrexate, and fluorouracil given on
Days 1 and 8 and commencing within 36 hours of
mastectomy. The ConCT regimen was cyclophosphamide, methotrexate, and fluorouracil (cyclophosphamide was given orally) plus low-dose, continuous
prednisone started 25–36 days after mastectomy and
continuing for 6 cycles, every 28 days. Postmenopausal patients with positive lymph node status who
were assigned to receive ConCT also received concurrent tamoxifen for 6 months. The trial and the clinical
results have been described in detail elsewhere.12–15
The PeCT ⫹ ConCT group (seven courses) and the
ConCT group (six courses) were combined and labeled
the prolonged treatment group in this analysis, because no difference was found between the two
groups in the published trial report.12
In 1993, the IBCSG established a tissue bank of
tumor blocks from a subset of Trial V patients. All
breast tumors were assessed by conventional histopathologic assessment and were classified histologically according to the World Health Organization criteria. ER and progesterone receptor (PgR) status were
measured by standard biochemical methods according to guidelines of the central laboratory, and levels
ⱖ 10 fmol/mg cytosol protein were classified as positive. These pathologic parameters have been merged
with the clinical data base that was begun in 1981 and
is updated annually for survival and disease status.
The histologic material from the trial was used previously to investigate the relevance of HER-2/neu overexpression16 and of thymidylate synthase (TS) expression17 in patients with early-stage breast carcinoma,
and these features were available in the data base.
HER-2/neu expression was positive in 14 of 198 patients who were tested, because the antibody against
HER-2/neu was titrated to stain only tumors that had
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CANCER April 1, 2003 / Volume 97 / Number 7
gene amplification.16 The expression of p27 protein
was analyzed by immunostaining available material
from 461 patients: 198 patients with negative lymph
nodes and 263 patients with positive lymph nodes.
Low p27 immunoreactivity was defined as staining in
⬍ 50% of tumor cells, whereas high p27 immunoreactivity was defined as staining in ⱖ 50% of neoplastic
cells. This cut-off value was determined prior to data
analysis and was selected because it had been used by
other investigators.5,7 All analyses were done separately according to lymph node status. The median
follow-up was 13.3 years for lymph node negative
patients and 13.6 years for lymph node positive patients as of the data base update in November 1999.
curred first. OS was defined as the time from randomization to death from any cause.
Hazards ratios, 95% confidence intervals (95%CIs)
for the hazards ratios, and associated P values were
calculated based on Cox proportional hazards regression models with p27 immunoreactivity as a single
covariate as well as treatment as a single covariate.21
The models were created for each lymph node status
group and used both DFS and OS as endpoints.
Kaplan–Meier plots were created to graphically show
differences in DFS and OS according to p27 immunoreactivity by lymph node status as well as by treatment
group.22
RESULTS
Immunocytochemistry
All histopathologic tumor parameters were recorded
in the data base, and the current investigators were
unaware of any of the clinical or histopathologic parameters when p27 was assessed, because all specimens were defined by a randomization number. Formalin fixed, paraffin embedded tissue sections were
immunostained for p27 using a 1:200 dilution of the
specific monoclonal antibody (clone 57; Transduction
Laboratories, Lexington, KY) and a standard peroxidase labeled streptavidin-biotin staining procedure.18
Peroxidase activity was developed with diaminobenzidine as a chromogenic substrate.
The results were evaluated independently by two
pathologists, taking into account the percentage of
neoplastic cells that showed nuclear staining of the
same or greater intensity compared with the staining
in normal cell counterparts or in stromal lymphocytes,
which served as internal positive controls for immunostaining. At least 2000 neoplastic cells were evaluated in different randomly chosen high-power fields
(magnification, ⫻ 400) from the tissue sections.
Statistical Analyses
Fisher exact tests were performed to assess correlations between p27 expression (low or high) and patient characteristics.19 Patient characteristics of interest included pathologic tumor size, ER status, PgR
status, tumor grade, histology, vessel invasion, HER2/neu overexpression, TS level, treatment group,
menopausal status, and Ki-67 expression. Wilcoxon
rank-sum tests for comparing the medians of two independent samples were computed to detect a difference in age by p27 expression category.20 There was
no attempt to correct for multiple comparisons.
DFS was defined as the time from randomization
to recurrence, metastasis, appearance of a second primary tumor, or death from any cause, whichever oc-
In all samples, the vast majority of epithelial cells from
normal or hyperplastic ducts and lobules entrapped
within the tumor or surrounding it consistently displayed intense nuclear staining, as did some stromal
cells and most infiltrating lymphocytes. Overall, 201
tumors (44%) exhibited p27 nuclear immunoreactivity
in ⬍ 50% of neoplastic cells. The prevalence of p27
down-regulation was similar in both lymph node negative patients (43%) and lymph node positive patients
(44%).
Clinicopathologic Correlates of p27 Immunoreactivity
In patients with lymph node negative tumors, no significant associations were detected between p27 immunoreactivity and patient age, patient menopausal
status, pathologic tumor size or type, peritumoral vascular invasion, Ki-67 labeling index, or treatment;
whereas there was a statistically significant association of p27 down-regulation with high tumor grade (P
⫽ 0.001) and HER-2/neu overexpression (P ⫽ 0.04)
(Table 1). In addition, patients who had tumors with
negative ER status or negative PgR status and patients
with low TS expression more frequently had low p27
levels. The associations of p27 status with ER, PgR, and
TS status did not reach the level of statistical significance. With regard to histologic features, invasive lobular histology was associated less commonly with low
p27 levels. Within the group of patients with lymph
node positive disease, low p27 immunoreactivity was
associated significantly with high tumor grade (P
⫽ 0.01), low TS immunostaining (P ⫽ 0.001), and high
Ki-67 labeling index (defined as ⱖ 8% cells immunostained for Ki-67) (P ⫽ 0.007) (Table 2).
Given the importance of the correlation between
low p27 levels and HER-2/neu overexpression, we specifically analyzed the degree of decreased p27 immunoreactivity in relation to HER-2/neu overexpression.
It was very interesting to note that, among the 21
patients who had lymph node negative tumors with
p27 and HER-2neu in Early Breast Carcinoma/Spataro et al.
TABLE 1
Characteristics of Lymph Node Negative Patients with p27 Expression
Data Available
TABLE 2
Characteristics of Lymph Node Positive Patients with p27 Expression
Data Available
p27 expression
level
Characteristics
All lymph node negative patients
with p27 data available
Pathologic tumor size (cm)
ⱕ2
⬎2
ER status
Negative
Positive
Unknown
PgR status
Negative
Positive
Unknown
Tumor grade
1
2
3
Unknown
Histology
Limited invasion
Invasive ductal
Invasive lobular
Pure special features
Invasive ductal and lobular
Unknown
Vessel invasion
No
Yes
Unknown
HER-2/neu expression
Negative
Positive
Unknown
Thymidylate synthase expression
Low
High
Unknown
Treatment
PeCT
No PeCT
Menopausal status
Premenopausal
Postmenopausal
Ki-67 (%)
⬍8
ⱖ8
Unknown
Age (yrs)
Median
Range
1595
p27 expression
level
Low
High
% Low
P value
86
112
43
—
18
68
24
88
43
44
1.00
—
39
39
8
36
63
13
52
38
38
0.09a
—
—
44
26
16
43
39
30
51
40
35
0.25a
—
—
7
26
45
8
15
58
32
7
32
31
58
53
0.001a
—
—
—
2
67
5
4
0
8
3
77
15
4
6
7
40
47
25
50
0
53
0.07a
—
—
—
—
—
40
37
9
53
50
9
43
43
50
1.00a
—
—
72
14
0
103
7
2
41
67
0
0.04a
—
—
29
57
0
27
83
2
52
41
0
0.20a
—
—
58
28
74
38
44
42
0.88
—
48
38
60
52
44
42
0.78
—
40
40
6
57
50
5
41
44
55
0.77a
—
—
52
27–65
51
28–64
—
—
0.80b
—
ER: estrogen receptor; PgR: progesteron receptor; PeCT: perioperative chemotherapy.
a
Patients with unknown data were excluded from the calculation of P values.
b
P values were determined with a Wilcoxon rank-sum test for comparing the medians of two independent samples.
Characteristic
All lymph node positive patients
with p27 data available
Pathologic tumor size (cm)
ⱕ2
⬎2
ER status
Negative
Positive
Unknown
PgR status
Negative
Positive
Unknown
Tumor grade
1
2
3
Unknown
Histology
Limited invasion
Invasive ductal
Invasive lobular
Pure special features
Invasive ductal and lobular
Unknown
Vessel invasion
No
Yes
Unknown
HER-2/neu expression
Negative
Positive
Thymidylate synthase expression
Low
High
Unknown
Treatment
PeCT and ConCT
ConCT
PeCT
Menopausal status
Premenopausal
Postmenopausal
Ki-67 (%)
⬍8
ⱖ8
Unknown
Age (yrs)
Median
Range
Low
High
% Low
P value
115
148
44
—
17
98
22
126
44
44
1.00
—
44
55
16
47
84
17
48
40
48
0.22a
—
—
46
46
23
64
56
28
42
45
45
0.22a
—
—
8
40
61
6
20
68
53
7
29
37
54
46
0.01a
—
—
—
5
95
5
1
3
6
3
125
9
0
4
7
63
43
36
100
43
46
0.61a
—
—
—
—
—
28
81
6
30
111
7
48
42
46
0.45a
—
—
89
26
123
25
42
51
0.27
—
43
69
3
29
116
3
60
37
50
0.001a
—
—
33
34
48
53
47
48
38
42
50
0.26
—
—
74
41
103
45
42
48
0.43
—
41
63
11
81
60
7
34
51
61
0.007a
—
—
48
30–64
49
25–65
—
—
0.33b
—
ER: estrogen receptor; PgR: progesteron receptor; PeCT: perioperative chemotherapy; ConCT: conventionally timed chemotherapy.
a
Patients with unknown data were excluded from the calculation of P values.
b
P values were determined with a Wilcoxon rank-sum test for comparing the medians of two independent samples.
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CANCER April 1, 2003 / Volume 97 / Number 7
FIGURE 1. Kaplan–Meier plot of disease free survival (DFS) according to p27
immunoreactivity in patients with early-stage breast carcinoma who had
negative lymph node status. SE: standard error.
positive HER-2/neu overexpression, 8 patients (38%)
had very low levels of p27 immunoreactivity (⬍ 20%
positive cells); whereas only 18% of patients who had
lymph node negative tumors that did not overexpress
HER2/neu had such low levels of p27 immunoreactivity (P ⫽ 0.04), indicating that the correlation between
HER-2/neu overexpression and down-regulation of
p27 is particularly robust. In contrast, among the patients with lymph node negative, histologic Grade 3
tumors, which also are correlated significantly with
low p27 immunoreactivity, only a small proportion
(23% of all patients with lymph node negative, Grade
3 tumors) had very low p27 immunoreactivity (⬍ 20%
positive cells).
Analysis of Survival
No correlation was found between DFS or OS and p27
status in patients with lymph node negative breast
carcinoma (Fig. 1; Tables 3, 4). The same was true for
patients with lymph node positive breast carcinoma
(Fig. 2; Tables 3, 4). Although, for patients with lymph
node negative tumors, there was an apparent shift
between the survival curves for high and low p27
expression in the first 2 years of follow-up, there was
no statistically significant difference in DFS at any of
the time points.
Association of p27 Immunoreactivity with Outcome of
Adjuvant Chemotherapy
DFS and OS patterns of patients with lymph node
negative and lymph node positive breast carcinoma
were analyzed to determine whether p27 immunoreactivity predicted for outcome of CMF chemotherapy.
Patients with lymph node positive breast carcinoma
demonstrated a benefit from prolonged CMF chemotherapy compared with one cycle of perioperative chemotherapy, irrespective of their p27 status. There was
no difference in the magnitude of the benefit in patients with tumors that showed high or low p27 immunoreactivity (Tables 3, 4; Fig. 3A,B). In contrast,
patients with lymph node negative tumors and decreased p27 immunoreactivity had a greater benefit
from one course of perioperative chemotherapy compared with patients who had higher p27 levels (10-year
DFS for patients with low p27: 58% ⫾ 7% for perioperative chemotherapy; 43% ⫾ 9% without perioperative chemotherapy; hazard ratio, 0.58; 95%CI, 0.33–
1.03; P ⫽ 0.06). Conversely, patients with lymph node
negative tumors and high p27 immunoreactivity
showed an increasing risk in the perioperative chemotherapy group, although this effect was not significant
(Tables 3, 4; Fig. 4A,B). Based on a Cox proportional
hazards model, it was found that the interaction between p27 status and treatment in patients with
lymph node negative disease was significant (P
⫽ 0.03).
DISCUSSION
In this study, we examined the clinicopathologic correlates and the clinical implications of p27 immunoreactivity as a prognostic marker of disease recurrence
and survival and as a predictor of outcome to chemotherapy in patients with early-stage breast carcinoma.
We showed that slightly less than 50% of tumors had
decreased immunoreactivity for p27, regardless of tumor size or lymph node status. The prevalence of
reduced p27 immunoreactivity is consistent with that
reported in previous studies, ranging from 31% to 69%
of patients.5–10 Down-regulation of p27 likely is an
early event in breast carcinoma progression, because
we detected it with the same prevalence in small
lymph node negative tumors with limited invasion
and in larger, lymph node positive tumors. Overall,
our findings suggest that decreased levels of p27 are
more common in tumors with an aggressive phenotype, such as those with ER negative status and PgR
negative status, high histologic grade, HER-2/neu
overexpression, high Ki-67 labeling indexes, and low
TS expression. These observations also are consistent
with the recent report of a correlation between p27
down-regulation and BRCA1 germ-line mutations.23
To our knowledge, this is one of the few studies in
which p27 immunoreactivity and HER-2/neu overexpression have been investigated in the same tumors
from a large cohort of patients with early-stage breast
carcinoma.6,7 In prior published reports, data on the
relation between HER-2/neu and p27 immunoreactivity were not shown in detail; HER-2/neu overexpres-
p27 and HER-2neu in Early Breast Carcinoma/Spataro et al.
1597
TABLE 3
Disease Free Survival According to p27 Status: Overall and by Treatment Group
DFS (% ⴞ SE)
p27 status
Lymph node negative patients
p27 expression
Low
High
PeCT
Low
High
No PeCT
Low
High
Lymph node positive patients
p27 expression
Low
High
Prolonged treatment
Low
High
PeCT
Low
High
No. of
patients
No. of
failures
5-yr
10-yr
HRa
95% CIa
P valuea
86
112
49
56
61 ⫾ 5
67 ⫾ 4
53 ⫾ 5
55 ⫾ 5
1.21
—
0.83–1.78
—
0.32
—
58
74
29
39
71 ⫾ 6
63 ⫾ 6
58 ⫾ 7
50 ⫾ 6
0.92
—
0.57–1.48
—
0.72
—
28
38
20
17
43 ⫾ 9
74 ⫾ 7
43 ⫾ 9
66 ⫾ 8
2.26
—
1.17–4.37
—
0.02
—
115
148
83
102
51 ⫾ 5
47 ⫾ 4
33 ⫾ 4
32 ⫾ 4
0.99
—
0.74–1.32
—
0.92
—
67
100
45
64
55 ⫾ 6
49 ⫾ 5
38 ⫾ 6
38 ⫾ 5
0.98
—
0.66–1.44
—
0.90
—
48
48
38
38
46 ⫾ 7
43 ⫾ 7
26 ⫾ 6
19 ⫾ 6
0.83
—
0.52–1.31
—
0.42
—
DFS: disease free survival; SE: standard error; HR: hazard ratio; 95% CI: 95% confidence interval; PeCT: perioperative chemotherapy.
a
The hazard ratio (p27 low: p27 high), 95% confidence intervals, and P values were calculated based on Cox proportional hazards regression models with p27 expression used as a single covariate.
TABLE 4
Overall Survival According to p27 Status: Overall and by Treatment Group
OS (% ⴞ SE)
p27 status
Lymph node negative patients
p27
Low
High
PeCT
Low
High
No PeCT
Low
High
Lymph node positive patients
p27
Low
High
Prolonged treatment
Low
High
PeCT
Low
High
No. of
patients
No. of
failures
5-yr
10-yr
HRa
95% CIa
P valuea
86
112
35
37
77 ⫾ 5
81 ⫾ 4
64 ⫾ 5
73 ⫾ 4
1.33
—
0.84–2.11
—
0.23
—
58
74
21
25
81 ⫾ 5
80 ⫾ 5
69 ⫾ 6
70 ⫾ 5
1.09
—
0.61–1.95
—
0.76
—
28
38
14
12
68 ⫾ 9
84 ⫾ 6
51 ⫾ 10
79 ⫾ 7
1.95
—
0.90–4.22
—
0.09
—
115
148
67
87
63 ⫾ 4
69 ⫾ 4
49 ⫾ 5
43 ⫾ 4
0.96
—
0.69–1.32
—
0.78
—
67
100
34
57
70 ⫾ 6
73 ⫾ 4
59 ⫾ 6
43 ⫾ 5
0.80
—
0.52–1.24
—
0.32
—
48
48
33
30
54 ⫾ 7
62 ⫾ 7
35 ⫾ 7
42 ⫾ 7
1.06
—
0.64–1.76
—
0.82
—
OS: overall survival; SE: standard error; HR: hazard ratio; 95% CI: 95% confidence interval; PeCT: perioperative chemotherapy.
a
The hazard ratio (p27 low:p27 high), 95% confidence intervals, and P values were calculated based on Cox proportional-hazards regression models with p27 expression used as a single covariate.
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CANCER April 1, 2003 / Volume 97 / Number 7
FIGURE 2. Kaplan–Meier plot of disease free survival (DFS) according to p27
immunoreactivity in patients with early-stage breast carcinoma who had
positive lymph node status. SE: standard error.
FIGURE 4. Kaplan–Meier plots of disease free survival (DFS) in lymph node
negative patients with early-stage breast carcinoma according to treatment
arm among those with low p27 immunoreactivity (A) and high p27 immunoreactivity (B). SE: standard error; PeCT: perioperative chemotherapy.
FIGURE 3. Kaplan–Meier plots of disease free survival (DFS) in lymph node
positive patients with early-stage breast carcinoma according to treatment arm
among those low p27 immunoreactivity (A) and high p27 immunoreactivity (B).
SE: standard error; PeCT: perioperative chemotherapy.
sion and low p27 immunoreactivity, however, were
considered independent prognostic factors for reduced survival in a multivariate analysis reported by
Porter et al.6 Low p27 immunoreactivity also was an
independent prognostic factor for reduced survival in
patients with small tumors (T1a,b) in a multivariate
analysis by Tan et al.,7 who took into account several
biologic parameters, including HER-2/neu overexpression. In the current study, we have documented
that lymph node negative tumors with HER-2/neu
overexpression are more likely to show reduced p27
immunoreactivity. Similar findings were reported recently in a cohort of 51 patients with breast carcinoma
in which 41% of tumors overexpressed HER-2/neu and
92% of tumors had decreased p27 immunoreactivity;
all HER-2/neu overexpressing tumors in that cohort
exhibited down-regulation of p27 and increasing HER2/neu activity correlated with decreasing p27 immunostaining.24 The data described above are supported
p27 and HER-2neu in Early Breast Carcinoma/Spataro et al.
by the current study, in which a very stringent definition of HER-2/neu overexpression was applied, in that
the immunostaining experiments were titrated to detect only tumors with definite gene amplification
(more than three copies of the HER-2/neu gene).16
The observation of a correlation between p27 and
HER-2/neu is of particular relevance, because it confirms laboratory data showing that one of the downstream effects of HER-2/neu overexpression is p27
down-regulation.25 Furthermore, preclinical work also
showed that inhibition of the epidermal growth factor
(EGF)/ErbB1 receptor led to up-regulation of p27 in a
model of breast carcinoma tumorigenesis,26 and analyses of tissue samples from patients who were treated
with inhibitors of the EGF receptors has revealed upregulation of p27. Taken together, these data suggest
that p27 is a common downstream target of several
signaling pathways, including not only HER-2/neu but
also other receptors of the EGFR/Her family.
In the current study, p27 immunoreactivity did
not show any prognostic value in terms of disease
recurrence or survival, issues that have been debated
in the recent literature.5–10 The current study is the
first to evaluate patients who were enrolled in a randomized clinical trial, a population that has received
long and careful follow-up. This has the important
advantage of reducing the risk of a selection bias;
furthermore, although adjuvant treatment, in theory,
can obscure the power of a prognostic marker in an
untreated population, half of the population under
investigation (patients with lymph node negative tumors) either received no adjuvant systemic treatment
or received a single course of perioperative chemotherapy, making it possible to evaluate a prognostic
effect in the absence of adjuvant treatment. IBCSG
Trial V enrolled a large population of premenopausal
and postmenopausal patients with lymph node negative tumors and lymph node positive tumors. Because
only 461 patients were available for this investigation,
the number of patients with defined features, such as
lymph node negative status, was insufficient to detect
prognostic effects of small magnitude. However, the
size of the population investigated was larger than in
most previously reported studies on p27 and was adequate to detect large prognostic effects. The current
cohort was a subset of the patient cohort reported
previously by Gusterson et al.,16 in which it was found
that HER-2/neu overexpression was significant prognostically for patients with lymph node positive tumors, but not for patients with lymph node negative
tumors. Our finding of a correlation between low p27
expression and HER-2/neu overexpression in patients
with lymph node negative tumors and the lack of
1599
prognostic value of decreased p27 immunoreactivity
are consistent with previous findings.
The current study is the first to address the influence of p27 immunoreactivity on the outcome of adjuvant treatment. We found that low p27 immunoreactivity predicted a benefit from a single course of
perioperative chemotherapy in patients with negative
lymph node status, whereas it had no influence on the
magnitude of the benefit of prolonged treatment with
CMF-based chemotherapy in patients with positive
lymph node status. It is possible that the effect observed in the lymph node negative patients may have
been related to the timing, and not to the type, of
chemotherapy (CMF). We previously showed that
perioperative CMF was more effective for patients
who had ER negative tumors compared with patients
who had ER positive tumors, more effective for patients who had histologic Grade 2 and 3 tumors compared with patients who had histologic Grade 1 tumors, and more effective for patients in whom no
axillary micrometastases were found after serial sectioning.27,28 The current results, therefore, are consistent with these previous observations. There is no
suggestion that low p27 levels may be predictive of a
particular benefit from CMF-based chemotherapy,
given the lack of an interaction between p27 status
and the magnitude of effect of prolonged CMF treatment in patients with lymph node positive disease.
However, given its inverse correlation with HER-2/neu
overexpression, low p27 immunoreactivity may be
predictive of a benefit from anthracycline-based adjuvant chemotherapy, which has been suggested for the
treatment of patients with tumors that overexpress
HER-2/neu. An analysis of patients who are treated on
clinical trials that randomize patients to CMF versus
anthracycline-containing chemotherapy may be of interest in this regard.
The results of the current study found that decreased p27 immunoreactivity was not an independent prognostic factor in patients with early-stage
breast carcinoma and was correlated with known
prognostic factors, such as high histologic grade. In
addition, low p27 levels were associated with HER-2/
neu overexpression in patients with negative lymph
node status and with higher Ki-67 labeling indices in
patients with positive lymph node status. We also
found that p27 immunoreactivity was not predictive of
a benefit from CMF-based chemotherapy per se but
may be predictive of an important benefit from starting (perioperative) adjuvant chemotherapy very early.
In addition, this is the first study in a large population
of patients who were treated on a randomized clinical
trial that has addressed the correlation between HER2/neu overexpression and p27 immunoreactivity, and
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CANCER April 1, 2003 / Volume 97 / Number 7
our findings substantiate in vivo a biologic model in
which p27 down-regulation is an important event in
cells that overexpress HER-2/neu. These findings suggest that p27 may become a useful adjunct to HER-2/
neu status for predicting response to drugs that act on
EGF/Her pathways, such as trastuzumab and receptor
tyrosin-kinase inhibitors, which target other members
of the EGFR/Her family. Further studies clearly are
warranted to investigate the usefulness of p27 in the
selection of adjuvant treatments for patients with early-stage breast carcinoma.
13.
14.
15.
16.
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