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© 2003 American Society for Clinical Oncology HER-2 Expression and Cell Proliferation: Prognostic Markers in Patients With Node-Negative Breast Cancer
From the Department of Medical Oncology, Pierantoni Hospital; Istituto Oncologico Romagnolo, Forlì; Department of Medical Oncology, Infermi Hospital, Rimini; Clinical Experimental Oncology Laboratory, National Oncology Institute; Breast Unit, National Oncology Institute, Bari; and Department of Medical Oncology, S. Maria delle Croci Hospital, Ravenna, Italy. Address reprint requests to Dino Amadori, MD, Dept of Medical Oncology, Pierantoni Hospital, via Forlanini 34, 47100 Forlì, Italy; email: segronco{at}ausl.fo.it or a.volpi{at}ausl.fo.it.
Purpose: We analyzed the clinical relevance of HER-2 expression, widely investigated in breast cancer but with contradictory results, in the largest case series of node-negative breast cancer patients investigated to date. Patients and Methods: The pure prognostic value of HER-2 expression was investigated in 529 patients treated with locoregional therapy alone until early relapse. Proliferative activity was evaluated as [3H]thymidine labeling index and HER-2 expression by immunohistochemistry. All biologic determinations were conducted within the context of an intra- and interlaboratory National Quality Control Program.
Results: HER-2 expression was not related to relapse-free survival in the overall series but was a significant discriminant of prognosis in the subgroup of patients with rapidly proliferating tumors. Six-year rate of relapse was 40% for patients with highly ( Conclusion: HER-2 expression in association with proliferative activity identifies a subgroup of node-negative breast cancer patients with the worst prognosis, who are candidates for specific intensive adjuvant therapy.
A LONG-TERM interest in a research topic does not necessarily lead to conclusive results. An emblematic example is the overexpression of HER-2 and its clinical relevance. This proto-oncogene, which encodes for a 185-kd transmembrane glycoprotein receptor that is structurally similar to the epidermal growth factor receptor, has been widely investigated as a prognostic indicator, a predictor of response to systemic hormonal or chemical treatment, or as a target of biologic therapy. This last objective has been definitively assessed, whereas the first two, which have long been studied, have produced contradictory and inconclusive results. Whereas some important works on the prognostic role of HER-2 evaluated its molecular alterations,1–3 the majority of studies on large case series determined HER-2 overexpression using an immunohistochemical approach. A large number of studies have been published, some reporting positive results and others reporting negative results, as summarized in various reviews.4–8 Despite this, HER-2 expression is sometimes used as an indicator of risk to identify patients with node-negative breast cancer as candidates for standard adjuvant chemotherapy. The disagreement in results can be attributed to different factors, including the absence of quality controls in the preanalytic and analytic steps of HER-2 overexpression assay; the use of different monoclonal antibodies and cutoff values; the lack of prospective planning of the determinations; the heterogeneity of often underpowered case series; and the sometimes not rigorous follow-up in old case series. Furthermore, few studies have investigated the pure prognostic value of this biomarker in breast cancer patients not submitted to any kind of systemic therapy, and results are compromised by the relatively small-sized samples and limited follow-up. Moreover, HER-2 overexpression has only occasionally been studied in relation to cell proliferation and, in particular, to thymidine-labeling index (TLI),9 which has been shown to be a consistent and powerful prognostic factor over time. In the present study, we aimed to define the prognostic relevance of these two biologic markers; TLI, which has attained the highest level of evidence (LOE 1) for clinical application,10,11 and HER-2 expression, which has aroused great scientific interest for its potential clinical usefulness, as shown in a large consecutive case series of node-negative breast cancer patients. To provide reliable results, we tried to avoid preanalytic and analytic biases by participating in National Quality Control Programs for both markers.
Patient Characteristics and Follow-Up The prognostic relevance of HER-2 expression was studied on 529 node-negative breast cancer patients treated with locoregional therapy alone (surgery ± radiotherapy [RT]) until early relapse, recruited between 1989 and 1994 by the Oncology Institutes of Romagna (IOR) and Bari (Istituto di Ricovero e Cura a Carattere Scientifico [IRCCS] Bari). A diagnosis of infiltrating breast cancer and the absence of axillary lymph node involvement (at least 10 lymph nodes examined) were histologically confirmed.
Clinical and biologic characteristics of the case series are listed in Table 1
In the studies aimed at evaluating the prognostic relevance of HER-2 expression, all patients had close follow-up. Clinical, hematologic, and biochemical assessment was carried out for each patient at 3-month intervals for the first 2 years, every 6 months during the third, fourth, and fifth year, and once a year thereafter up to the tenth year. During the first year, a biannual liver scan and chest x-ray were carried out; during the second year, a chest x-ray was performed at the sixth and twelfth month, as well as a liver scan at the twelfth month. Subsequently, both examinations were carried out once a year. Furthermore, all patients underwent an annual mammography and bone scan.
Biologic Determinations All the determinations of HER-2 expression were carried out in the Forlì laboratory only. TLI was determined in both the Forlì and Bari laboratories, and ER and PgR were assessed in each recruiting center.
HER-2 Expression
TLI
Steroid Receptor Content
Statistical analysis The role of HER-2 overexpression as a prognostic marker was investigated in terms of probability of relapse. The crude cumulative incidence was computed starting from the time of surgery by the Kaplan and Meier product limit method.16 The comparison of cumulative incidence relapse curves was performed by log-rank test.17 The first occurrence of locoregional relapse, distant metastasis, contralateral breast cancer, or death without evidence of disease was considered an event. Median follow-up was 79 months. The 6-year crude cumulative incidence and its 95% confidence interval (95% CI) were calculated.
The prognostic effect of HER-2 expression was evaluated in univariate and multivariate analyses using a Cox regression model18 that included other known prognostic variables such as ER, PgR, tumor size, and TLI. The variables were categorized as follows: tumor size more than 2 cm and In the regression model, HER-2 was considered a continuous or dichotomous variable. Three-knots restricted cubic spline regression19 was used to model the relationship between the logarithm of the hazard ratio and the percentage of HER-2 overexpressing cells. Considering the lack of significant prognostic relevance of any HER-2 immunopositive level, as emerged from the continuous variable analysis, and the heterogeneity of cutoff values used in other studies, we chose a value of 30% immunopositive cells, which, in exploratory analysis, is the value that maximally, albeit not significantly, identified subgroups at different risk of relapse. The relative risk increase of cumulative incidence of relapse for high compared to low HER-2–expressing cases was also reported. In view of the exploratory intent of the analysis, P values were not adjusted for the multiplicity of the performed tests. Statistical analyses were carried out by S-plus (Version 3.3, Statistical Sciences, Seattle, WA) and SAS (SAS/STAT User’s Guide, Version 6. Cary, NC, SAS Institute, 1990).
Of the overall series of patients, the number of HER-2-expressing cells ranged from 0% to 100%, with a median value of 5%. Two hundred and seventy (51%) tumors had at least one positive cell, and 151 (28.5%) had more than 30% positive cells, which is the value subsequently used for dichotomous analyses. HER-2 expression was indirectly related to ER (rs -0.14; P = .002) and PgR (rs -0.19; P < .0001) and directly related, albeit weakly, to tumor size and not related to TLI or patient age.
The prognostic relevance of HER-2 expression was analyzed as a continuous or dichotomous variable on the consecutive series of 529 node-negative breast cancer patients. For the former analysis, the results obtained by the application of the Cox model showed that HER-2 expression is not related to relapse-free survival (RFS; Fig 1
In the latter analysis, the relevance of HER-2 expression, considered as a dichotomous variable, was evaluated in the overall series and in subsets of patients defined according to clinical, pathologic, and biologic features (Table 2  ). The 6-year relapse-free survival for the overall series was 75% (95% CI, 71% to 78%) and was not statistically different for patients with high or low HER-2-expressing tumors, even when the most discriminating cutoff of 30% was used. The somewhat higher, but not significantly different, cumulative incidence of relapse for patients with high HER-2-expressing tumors (Fig 2) compared with that of patients with low HER-2-expressing tumors was observed immediately after surgery and remained constant throughout the entire observation period. Furthermore, HER-2 expression was not a prognostic discriminant within the subsets of patients categorized according to clinical, pathologic, and biologic characteristics, except for cell proliferation (Table 2). In fact, HER-2 expression was not a discriminant in patients with slowly proliferating tumors (20% v 21%; P = .854) but was in those with rapidly proliferating tumors (40% v 26%; P = .039). As shown in Figure 3, the clinical outcome was substantially worse for the subset of patients with tumors in which a high proliferative activity was coupled with high HER-2 expression, compared with that of patients with weak expressing HER-2 or highly HER-2 expressing but slowly proliferating tumors (P = .002). For this subset of patients the cumulative relapse incidence was up to twice as high as that observed for the other three subgroups.
In multivariate analysis, including tumor size and all the biologic variables evaluated, only TLI and ER showed an independent prognostic relevance (Table 3 ).
Pathologic HER-2 expression, so-called overexpression, means that thousands of HER-2 receptor molecules are present together on the cell surface and that a growth and proliferative signal is continuously being transduced to the nucleus, even in the absence of ligands. This is the biologic description, but from a clinical point of view, some shadowy areas have yet to be clarified. The molecular mechanism of HER-2 overexpression could, in fact, account for a prognostic role of this biomark in breast cancer, but the abundant literature data do not unequivocally confirm this hypothesis. The problem is whether just a few cells showing overexpression make the difference or whether a threshold in the percentage of overexpressing tumor cells is needed to define positive and negative tumors. Researchers have not yet reached an agreement about this. Different scoring systems have been adopted and the cutoff values chosen for prognostic analysis vary from 10% to 50% or more. This results in an analysis of different-sized subgroups, also in studies based on large case series, and has obvious implications on statistical power. Another aspect of the problem is the sensitivity of the various antibodies used. It is generally accepted that monoclonal antibodies are more specific than polyclonal antibodies and the use of the latter may result in a higher false-positive rate. We tested the pure prognostic value of HER-2 overexpression, according to internationally proposed guidelines,10,11 on the largest case series of node-negative breast cancer patients studied up to now. We failed to find a relevance of any cutoff value of HER-2 overexpression on RFS. However, a breakdown analysis permitted us to attribute a discriminant prognostic power to HER-2 overexpression within the subgroup of patients with rapidly proliferating tumors. In particular, patients with tumors in which a high proliferative activity was associated with HER-2 overexpression showed a risk of relapse approximately two times higher than that observed for patients with rapidly proliferating but weak HER-2-expressing tumors, or for those with slowly proliferating tumors regardless of HER-2 expression. In conclusion, HER-2 expression and cell proliferation would seem to provide complementary prognostic information for node-negative breast cancer patients. Obviously, the clinical use of HER-2 expression requires confirmation of our results in large prospectively planned studies, such as those already performed for cell proliferation.9,20
The following participants contributed to this study: P. Serra, S. Bravaccini, Istituto Oncologico Romagnolo, Forlì, Italy; D. Casadei Giunchi, R. Maltoni, R. Ridolfi (Department of Medical Oncology), L. Saragoni (Department of Pathology), Pierantoni Hospital, Forlì, Italy; A. Tienghi, M. Marangolo, B. Vertogen, F. Zumaglini (Department of Medical Oncology), C. Morisi (Department of Pathology), S. Maria delle Croci Hospital, Ravenna; A. Ravaioli, G. Drudi (Department of Medical Oncology), P. Rinaldi, M. Ricci (Department of Pathology), Infermi Hospital, Rimini; A. Rossi, P. Turci (Medical Oncology Unit), F. Nuzzo (Department of Histopathology), Bufalini Hospital, Cesena, Italy; M. Aldi (Department of Histopathology), A. Gambi, L. Amaducci (Medical Oncology Unit), Degli Infermi Hospital, Faenza, Italy; G. Catalano, S. Luzi Fedeli (Department of Medical Oncology), P. Muretto (Department of Histopathology), S. Salvatore Hospital, Pesaro, Italy; C. Fallai (Oncology Day Hospital), Careggi Hospital, Florence, Italy; A. Becciolini, M. Balzi (Dept. of Clinical Physiopathology), S. Bianchi (Histopathology Institute), University of Florence, Florence, Italy; C. Salvatore (Histopathology and Cytodiagnostic Unit), B. Stea (Clinical Experimental Oncology Laboratory), D. Sambiasi (Dept. of Medical Oncology), National Cancer Institute, Bari, Italy.
We thank Rosella Silvestrini, PhD, for invaluable scientific contribution, Marcella Flora, BSc, for technical assistance, and Gráinne Tierney for editing the manuscript.
This study was funded by the National Research Council (CNR-Progetto Strategico MIUR, grant nos. 02.00452.ST97 and 02.00105.ST97), Rome, and by Istituto Oncologico Romagnolo, Forlì, Italy.
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
30%) positive tumors and 26% for those with weakly HER-2-expressing tumors (P = .039). 
2 cm in size. With regard to steroid receptor content, two thirds of patients had estrogen receptor- (ER) positive and half had progesterone receptor– (PgR-) positive lesions. Clinicopathologic and biologic characteristics were fairly similar for the two case series recruited by IOR and IRCCS Bari. 
