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© 2003 American Society for Clinical Oncology Invasion Factors uPA/PAI-1 and HER2 Status Provide Independent and Complementary Information on Patient Outcome in Node-Negative Breast Cancer
From the Frauenklinik and Institut für Allgemeine Pathologie und Pathologische Anatomie, Technische Universität, and Gemeinschaftspraxis Lachnerstrasse 2 für Pathologie und Zytologie, München, Germany; and Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, NY. Address reprint requests to Nadia Harbeck, MD, Frauenklinik und Poliklinik, Klinikum rechts der Isar, Technische Universität München, Ismaninger Strasse 22, D-81675 Munich, Germany; email: nadia.harbeck{at}lrz.tum.de.
Purpose: The independent clinical relevance of invasion factors urokinase-type plasminogen activator (uPA)/PAI-1 and HER2 status was evaluated in lymph node-negative breast cancer patients (N = 118) without adjuvant systemic therapy after long-term follow-up of more than 10 years (median, 126 months). Patients and Methods: Levels of uPA and its inhibitor PAI-1 were prospectively measured by enzyme-linked immunosorbent assay in primary tumor tissue extracts. HER2 gene amplification (HER2_AMP) was evaluated by fluorescence in situ hybridization (FISH; Ventana Medical Systems HER-2/neu probe; Tucson, AZ), and HER2 protein overexpression (HER2_EXP) was evaluated by immunohistochemistry (IHC; Oncogene Science antibody Ab-3; Cambridge, MA) on parallel-cut formalin-fixed paraffin-embedded tissue sections. Results: uPA/PAI-1 was high (either one or both factors were high) in 44% of the tumors. HER2_AMP was detected by FISH in 33% of the patients, and HER2_EXP was found by IHC in 44% of the patients. In a multivariate analysis of established and tumor-biologic prognostic factors, uPA/PAI-1 was the only independent prognostic factor for disease-free survival ([DFS]; P < .001; relative risk [RR], 8.3; 95% confidence interval [CI], 3.4 to 20.4). Although HER2_AMP and HER2_EXP did not reach significance for DFS, they were significant for overall survival (OS), even in multivariate analysis (HER2_AMP: P = .004; RR, 3.7; 95% CI, 1.5 to 9.2; HER2_EXP: P = .009; RR, 3.4; 95% CI, 1.4 to 8.7). Conclusion: After long-term follow-up, uPA/PAI-1 levels in primary tumor tissue reliably and strongly indicate an aggressive course of disease in lymph node-negative breast cancer independent of HER2 status. The particular prognostic effect of HER2 status on OS may reflect its ability to predict resistance to systemic therapy.
IN LYMPH NODE-NEGATIVE breast cancer, established histomorphologic and clinical factors, such as tumor size, tumor grade, steroid hormone receptor status, age, or menopausal status, are not sufficient for precise risk-group discrimination and subsequent individualized therapy decisions. Even though no more than one third of lymph node-negative patients will eventually develop metastases, current guidelines recommend adjuvant systemic therapy for more than 90% of all lymph node-negative breast cancer patients.1 To date, invasion markers urokinase-type plasminogen activator (UPA) and its inhibitor PAI-1 are the only novel prognostic factors that have reached the highest level of evidence for clinical utility in breast cancer according to the Tumor Marker Utility Grading System2,3 when measured by robust and quality-assured enzyme-linked immunosorbent assays (ELISAs). A randomized multicenter therapy trial validated the independent prognostic effect of uPA/PAI-1 in lymph node-negative breast cancer and showed benefit from adjuvant chemotherapy with cyclophosphamide, methotrexate, and fluorouracil chemotherapy in high-risk patients according to uPA/PAI-1.4 Moreover, a pooled analysis comprising more than 8,000 breast cancer patients has substantiated the independent prognostic effect.5 Finally, recent data have demonstrated that the combination of both factor uPA and factor PAI-1 (both low v either one or both high) is superior to either factor taken alone. uPA/PAI-1 outperforms established prognostic factors with regard to risk-group stratification and supports risk-adapted individualized therapeutic strategies in the adjuvant setting.6 Because of this compelling clinical data as well as the key role of uPA/PAI-1 in tumor cell invasion and metastasis,7 the uPA system represents an interesting target for tumor-biologic therapy.8 HER2 has gained increasing clinical importance over the last few years, particularly in breast cancer—not just as a potential prognostic or predictive factor but also as a target for tumor-biologic therapy.9,10 Unfortunately, only limited data are available on the clinical relevance of HER2 and uPA/PAI-1 within the same patient collective. However, it is important to determine whether assessment of both of these clinically relevant systems is required for optimal decision making in primary breast cancer. Our group originally showed that uPA/PAI-1 and HER2 give independent information on disease-free survival (DFS) in lymph node-negative breast cancer after a median follow-up of 7 years.11 Konecny et al12 substantiated this finding qualitatively in a large collective of 587 primary breast cancer patients after a short follow-up of less than 3 years, which did not permit analysis of overall survival (OS). The prognostic effect of tumor-biologic factors on DFS and OS generally reflects the effects of administered adjuvant systemic therapy.13 For uPA/PAI-1, there is recent evidence that patients with high levels in their primary tumor do respond well to adjuvant systemic therapy,4,6,13 and to chemotherapy in particular.14 Resistance of HER2-positive tumors to endocrine therapy and to certain types of chemotherapy has been suggested by several researchers.10 Thus, uPA/PAI-1 and HER2 seem to be prognostic and predictive factors at the same time. Therefore, to focus on the relationship between uPA/PAI-1, HER2, and tumor aggressiveness in breast cancer (ie, their purely prognostic effect), we consider only patients without any adjuvant systemic therapy. In addition, whereas analysis of DFS in these patients solely reflects prognostic effects, analysis of OS provides some clues about prediction of response/resistance in the palliative setting. In the clinically increasingly relevant group of lymph node-negative breast cancer patients with their rather good prognosis, only long-term follow-up data enable definite statements on the course of the disease. Thus, this investigation addresses the prognostic relevance of HER2 gene amplification (HER2_AMP) determined by fluorescence in situ hybridization (FISH), HER2 protein overexpression (HER2_EXP) measured by immunohistochemistry (IHC), and uPA/PAI-1 in a homogeneous collective of lymph node-negative breast cancer patients without adjuvant systemic therapy (N = 118) after a long-term median follow-up of more than 10 years.
Patients Tumor-biologic factors (HER2_AMP, HER2_EXP, and uPA/PAI-1) and established prognostic factors (tumor size, steroid hormone receptor status, histologic grade, and menopausal status) were evaluated in 118 lymph node-negative breast cancer patients (Table 1  ). This study consisted of consecutive lymph node-negative patients treated for primary breast cancer at the Department of Obstetrics and Gynecology (Frauenklinik) of the Technische Universität München, Munich, Germany, between 1987 and 1993. The patients had not received any adjuvant systemic therapy, and sufficient paraffin-embedded tissue was still available to perform HER2 analysis. This period was selected because subsequent participation in a clinical trial4 with stratification according to uPA/PAI-1 status was available to lymph node-negative patients in this clinic. Moreover, systemic treatment for lymph node-negative patients was becoming more common in Germany after this period.
Established factors were assessed as described previously.6 Patients underwent either a modified radical mastectomy (n = 74) or breast conserving surgery with subsequent breast irradiation (n = 44). In accordance with practice in Germany at the time, none of the patients received any adjuvant systemic therapy. The median age of all patients at diagnosis was 55 years (range, 36 to 82 years). At that time, no patient had clinical or radiologic evidence of distant metastases. Follow-up data were obtained on a regular basis.15 Median length of follow-up in patients still alive at time of analysis was 126 months (range, 22 to 176 months). Within the follow-up period, 33 patients (28%) suffered disease relapse, and 30 patients (25%) died.
HER2_AMP Determined by FISH
HER2_EXP Measured by IHC
uPA and PAI-1 Antigen Determination
Statistical Analysis
Distribution of Traditional and Tumor-Biologic Factors In a group of 118 lymph node-negative breast cancer patients without adjuvant systemic therapy, established histomorphologic and clinical factors (tumor size, grade, and steroid hormone receptor and menopausal status), tumor invasion markers (uPA/PAI-1), and HER2_AMP and HER2_EXP were determined (Table 1  ) and related to patient outcome. Sufficient tissue for assessment of uPA/PAI-1 was available in 115 patients, for HER2_AMP by FISH in 81 patients, and for determination of HER2_EXP in 113 patients. In 27 breast carcinomas (33% of the analyzed patients), HER2_AMP (median, three gene copies; range, two to 10.2 copies) was detected. In 50 tumors (44%), HER2_EXP (median, 0%; range, 0% to 90%) was seen. Correlations and associations between established and tumor-biologic factors have been reported previously.11,15,17
DFS
Because of the limited number of HER2_AMP results, the above multivariate analysis was performed in the group of 73 patients for whom all information was available. To control for possible selection bias, a composite variable HER2_STATUS (HER2_AMP and HER2_EXP negative v rest) was created, resulting in 110 patients being available for multivariate analysis. Accordingly, 49 patients (42%) had negative HER2_STATUS and 68 patients (58%) had positive HER2_STATUS. For DFS, the results of multivariate analysis using HER2_STATUS instead of the separate variables HER2_AMP and HER2_EXP essentially did not differ from the initial multivariate analysis shown in Table 2 ; uPA/PAI-1 remained the only significant factor for DFS (P < .001; RR, 7.9; 95% CI, 3.2 to 19.5).
Because of the long follow-up, it is interesting to consider possible deviations from proportional hazards. The factors of grade (P = .029; RR, 3.8; 95% CI, 1.1 to 12.7), pT stage (P = .047; RR, 4.2; 95% CI, 1.02 to 16.7), and steroid hormone receptor status (P = .011; RR, 4.6; 95% CI, 1.4 to 14.9), but not menopausal status, were significant in a univariate time-varying Cox model of logistic form F(T) = 1/{1+EXP[(T-30)/6]}, where T is the time in months. This functional form allows the effects of these factors to remain strong through about the first 2.5 years and then rapidly diminish toward zero with longer follow-up. This time variation is also reflected in the Kaplan-Meier curves shown in Fig 2
OS Univariate analysis for OS showed the significant effect of uPA/PAI-1, HER2_AMP, and HER2_EXP (Fig 3 ). Among established factors, only tumor grade was significant for OS. None of the patients with a grade 1 tumor has died so far, whereas 10-year OS for patients with grade 2 and grade 3 tumors was 80% and 53%, respectively (P = .0048). Whereas menopausal status showed borderline significance for OS, pT stage and steroid hormone receptor status failed to reach statistical significance. Univariate and multivariate results regarding DFS are listed in Table 3.
Again, to check for selection bias, multivariate analysis of OS was repeated, replacing HER2_AMP and HER2_EXP with HER2_STATUS (n = 111). This yielded slightly different results than the initial multivariate analysis using HER2_AMP and HER2_EXP as separate variables (n = 71). It is noteworthy that in the second analysis both uPA/PAI-1 and grade enter the model.
Risk-Group Assessment by uPA/PAI-1 and HER2
In 78 patients, data on uPA/PAI-1 and HER2_AMP were available. Of the 36 patients with high uPA/PAI-1, 14 were HER2 positive by FISH, and of the 42 patients with low uPA/PAI-1, 12 were HER2 positive. With regard to DFS, HER2_AMP did not have a significant prognostic effect in patient subgroups according to uPA/PAI-1. For OS, HER2_AMP was significant only in low-risk patients according to uPA/PAI-1 (P = .012; RR, 6.0; 95% CI, 1.5 to 24.0). These two trends are also seen in Kaplan-Meier curves for DFS and OS separated according to uPA/PAI-1 low/high and HER2_AMP negative/positive as shown in Fig. 4
Precise prognostic and predictive information at the time of primary therapy is the prerequisite for individualized therapy concepts in primary breast cancer. The tumor-biologic factors uPA/PAI-1 and HER2 have become increasingly clinically relevant in breast cancer management because of their ability to identify patients at risk, their predictive value regarding therapy response/resistance, and their potential as targets for directed therapy approaches. This study underlines the clinical use of determining both uPA/PAI-1 tumor antigen content and HER2 status in lymph node-negative breast cancer. This study shows for the first time that uPA/PAI-1 and HER2 both provide clinically important information but differ with regard to clinical effect and the consequences of this information. Because none of our patients received any adjuvant systemic therapy, their course of disease until first relapse (ie, DFS) entirely reflects the effect of factors on tumor aggressiveness (ie, their prognostic effect). After first relapse, palliative systemic therapy was administered. Palliative therapy is usually tailored according to patient symptoms and tumor response and tends to comprise several lines of systemic treatment.20 Thus, the effect of factors on OS reflects their association not only with tumor aggressiveness but also with therapy response/resistance (ie, their predictive effect). In our study, uPA/PAI-1 and HER2 status differ substantially in their effect on DFS and OS. Whereas uPA/PAI-1 is a significant factor for both DFS and OS, HER2 status is significantly associated with OS only. In our long-term follow-up, no qualitative difference is seen between HER2_AMP and HER2_EXP. Our finding that uPA/PAI-1 and HER2 characterize different risk groups is consistent with the results of Konecny et al,12 who found that the invasive phenotype associated with HER2 is not primarily a result of upregulation of uPA or PAI-1. The situation for uPA and PAI-1 as prognostic factors in breast cancer is quite unique for any tumor-biologic factors because researchers under a variety of demographic conditions (in Europe, the United States, and Japan) have unanimously reported a strong prognostic effect for uPA and PAI-1 when they are measured by biochemical assays in tumor tissue extracts. On the basis of the Chemo N0 trial4 and the pooled analysis,5 the clinical utility of uPA/PAI-1 in primary and, in particular, in lymph node-negative breast cancer has been validated at the highest level of evidence.2,3 In this article, we confirm this evidence for DFS and OS in a homogeneous collective of lymph node-negative patients without adjuvant therapy after a quite substantial follow-up period of more than 10 years. Lymph node-negative breast cancer patients with low uPA/PAI-1 levels in their primary tumor have a very good prognosis independent of established prognostic factors and are candidates for being spared the burden of adjuvant chemotherapy. In contrast, patients with high uPA/PAI-1 are at increased risk of relapse despite their negative axillary lymph nodes, a risk comparable with that of patients with three or more involved lymph nodes, and they definitely do need adjuvant systemic therapy, preferably including chemotherapy.
With regard to patient outcome, our cohort seems to be quite comparable with historic controls, with a relapse rate of 28% after a median of 10.5 years of follow-up.21 After this substantial follow-up period, we do not see a significant effect of HER2 on DFS in ordinary Cox analysis, which we had previously found, at least for HER2_AMP.11 Note that the earlier evaluation had a shorter follow-up period and a substantial percentage of patients with adjuvant systemic therapy (22%). In this report, the CI of the univariate proportional hazards model for HER2_AMP for DFS is consistent with the earlier evaluation. However, including time variation, but on a 96-month scale, we do see a borderline significant effect of HER2_AMP on DFS (P = .09; RR, 2.3; 95 CI, 0.86 to 6.0), despite omitting patients receiving adjuvant therapy. Hence, these results are consistent with the earlier evaluation and with the visual impression in the Kaplan-Meier curve for HER2_AMP (Fig 1 With the long follow-up of this analysis, we observe a strong effect of both HER2_AMP and HER2_EXP on OS, which has proved to be even stronger than in our earlier work. The strong effect is consistent with the idea of HER2 being a predictive rather than a prognostic marker. Retrospective analyses have associated HER2-positive tumors with resistance to tamoxifen therapy22 and to chemotherapy with cyclophosphamide, methotrexate, and fluorouracil23 and with response to anthracycline- or taxane-containing chemotherapy.24,25 In fact, the prognostic effect of HER2 in lymph node-positive breast cancer has been partly attributed to its potential role as a predictor of therapy response/resistance26,27 because the majority of these patients will receive some kind of adjuvant systemic therapy. This may also hold true for studies in lymph node-negative breast cancer that are not restricted to patients without systemic therapy.28 In conclusion, uPA/PAI-1 levels in primary tumor tissue reliably and strongly indicate an aggressive course of disease in node-negative breast cancer after long-term follow-up, independent of HER2. The particular prognostic strength of HER2 with regard to OS may reflect its ability to predict resistance to systemic therapy. Our data indicate that determination of uPA/PAI-1 and HER2 in lymph node-negative breast cancer may be quite complementary; uPA/PAI-1 can characterize a patient’s risk for relapse and indicate whether adjuvant (chemo) therapy may be beneficial. In addition, HER2 may be suitable for helping to decide what kind of systemic therapy is needed and whether the targeted trastuzumab therapy29 is appropriate. However, further prospective studies are needed to validate and specify this predictive effect to enable individualized therapy concepts.
We thank American Diagnostica Inc, Greenwich, CT, for their continuous technical support.
Supported in part by a grant to N.H. by the State of Bavaria (KKF Project no. 8756159), the Wilhelm-Sander Stiftung (2000.017.1), and Ventana Medical Systems, Inc, Gaithersburg, MD.
1. Goldhirsch A, Glick JH, Gelber RD, et al: Meeting highlights: International consensus panel on the treatment of primary breast cancer. J Natl Cancer Inst 90:1601–1608, 1998
2. Hayes DF, Bast RC, Desch CE, et al: Tumor marker utility grading system: A framework to evaluate clinical utility of tumor markers. J Natl Cancer Inst 88:1456–1466, 1996 3. Hayes D: Do we need better prognostic factors in node-negative breast cancer? Arbiter Eur J Cancer 36:302–306, 2000
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5. Look MP, van Putten WLJ, Duffy MJ, et al: Pooled analysis of prognostic impact of uPA and PAI-1 in 8, 377 breast cancer patients. J Natl Cancer Inst 94:116–128, 2002
6. Harbeck N, Kates R, Schmitt M: Clinical relevance of invasion factors uPA and PAI-1 for individualized therapy decisions in primary breast cancer is greatest when used in combination. J Clin Oncol 20:1000–1009, 2002 7. Andreasen PA, Kjöller L, Christensen L, et al: The urokinase-type plasminogen activator system in cancer metastasis: A review. Int J Cancer 72:1–22, 1997[CrossRef][Medline] 8. Schmitt M, Wilhelm OG, Reuning U, et al: The plasminogen activation system as a novel target for therapeutic strategies. Fibrinolysis 14:114–132, 2000[CrossRef]
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10. Yamauchi H, Stearns V, Hayes DF: When is a tumor marker ready for prime time? A case study of c-erbB-2 as a predictive factor in breast cancer. J Clin Oncol 19:2334–2356, 2001 11. Harbeck N, Ross J, Yurdseven S, et al: HER-2/neu gene amplification determined by in-situ hybridization (FISH) allows risk group assessment in node-negative breast cancer. Int J Oncology 14:663–671, 1999[Medline]
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14. Harbeck N, Kates RE, Look MP, et al: Enhanced benefit from adjuvant systemic chemotherapy in breast cancer patients classified high-risk according to urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) (n=3,424). Cancer Res 62:4617–4622, 2002 15. Harbeck N, Thomssen C, Berger U, et al: Invasion marker PAI-1 remains a strong prognostic factor after long-term follow-up both for primary breast cancer and following first relapse. Breast Cancer Res Treat 54:147–157, 1999[CrossRef][Medline] 16. Press MF, Bernstein L, Thomas PA, et al: HER-2/neu gene amplification characterized by fluorescence in situ hybridization: Poor prognosis in node-negative breast carcinomas. J Clin Oncol 15:2894–2904, 1997[Abstract] 17. Harbeck N, Dettmar P, Thomssen C, et al: Prognostic impact of tumor biological factors on survival in node-negative breast cancer. Anticancer Res 18:2187–2198, 1998[Medline]
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19. Lebeau A, Deimling D, Kaltz C, et al: HER-2/neu analysis in archival tissue samples of human breast cancer: Comparison of immunohistochemistry and fluorescence in situ hybridization. J Clin Oncol 19:354–363, 2001 20. Ellis MJ, Hayes DF, Lippman ME: Treatment of metastatic breast cancer, in Harris JR, Lippmann ME, Morrow M, et al (eds): Diseases of the Breast. Philadelphia, PA, Lippincott-Raven Publishers, 2000, pp 749–797 21. McGuire W, Clark GM: Prognostic factors and treatment decisions in axillary node-negative breast cancer. N Engl J Med 326:1756–1761, 1992[Medline]
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25. Muss HB, Thor AD, Berry DA, et al: C-erbB-2 expression and response to adjuvant therapy in women with node-positive early breast cancer. N Engl J Med 330:1260–1266, 1994 26. Revillon F, Bonneterre J, Peyrat JP: Review: ERBB2 oncogene in human breast cancer and its clinical significance. Eur J Cancer 34:791–808, 1998[CrossRef][Medline] 27. Clark G: Prognostic and predictive factors, in Harris JR, Lippmann ME, Morrow M, et al (eds): Diseases of the Breast. Philadelphia, PA, Lippincott-Raven Publishers, 2000, pp 489–514
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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
2.5% of tumor cells positive) versus positive (> 2.5% of tumor cells positive).
