Prognostic significance of uPA/PAI-1 level, HER2 status, and traditional histologic factors for survival in node-negative breast cancer patients

Nina Fokter Dovnik 1  and Iztok Takac 1 , 2
  • 1 Maribor University Clinical Center, Maribor, Slovenia
  • 2 Faculty of Medicine, University of Maribor, Maribor, Slovenia
Nina Fokter Dovnik and Iztok Takac
  • Maribor University Clinical Center, Maribor, Slovenia
  • Faculty of Medicine, University of Maribor, Maribor, Slovenia
  • Search for other articles:
  • degruyter.comGoogle Scholar

Abstract

Background

The association of HER2 status with urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1) levels raises the question whether uPA/PAI-1 level carries additional clinically relevant prognostic information independently from HER2 status. The aim of our study was to compare the prognostic value of uPA/PAI-1 level, HER2 status, and traditional prognostic factors for survival in node-negative breast cancer patients.

Patients and methods

A retrospective analysis of 858 node-negative breast cancer patients treated in Maribor University Clinical Center, Slovenia, in the years 2000–2009 was performed. Data were obtained from patient medical records. The median follow-up time was 100 months. Univariate and multivariate analyses of disease-free (DFS) and overall survival (OS) were performed using the Cox regression and the Cox proportional hazards model.

Results

In univariate analysis, age, tumor size, grade, lymphovascular invasion, HER2 status and UPA/PAI-1 level were associated with DFS, and age, tumor size, grade, and uPA/PAI-1 level were associated with OS. In the multivariate model, the most important determinants of DFS were age, estrogen receptor status and uPA/PAI-1 level, and the most important factors for OS were patient age and tumor grade. The HR for death from any cause in the multivariate model was 1.98 (95% CI 0.83–4.76) for patients with high uPA and/or PAI-1 compared to patients with both values low.

Conclusions

uPA/PAI-1 level clearly carries an independent prognostic value regardless of HER2 status in node-negative breast cancer and could be used in addition to HER2 and other markers to guide clinical decisions in this setting.

Introduction

One of the greatest challenges in the treatment of node-negative breast cancer is deciding in which patients the benefit from adjuvant cytotoxic chemotherapy would outweigh its adverse effects. Traditionally, this decision has been based on clinical and histomorphologic prognostic factors, such as patient age, tumor size, tumor grade, presence of lymphovascular invasion, and steroid hormone receptor status. Human epidermal growth factor receptor 2 (HER2) status, first used as a prognostic marker, became an important factor for predicting response to anti-HER2 therapy and is now a crucial part of this decision-making.

The serine protease urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor-1 (PAI-1) are markers of tumor invasion and metastasis that have reached the highest level of evidence for clinical utility as prognostic factors in breast cancer.1,2 Node-negative patients with high values of uPA and/or PAI-1 have been shown to benefit from adjuvant chemotherapy in a prospective randomized multicenter therapy trial.3,4 In addition, independent prognostic value of uPA/PAI-1 was confirmed in a pooled analysis of 8377 breast cancer patients that showed high levels of uPA and PAI-1 to be the strongest predictors of relapse-free survival and overall survival apart from lymph node status.5 In spite of this evidence, these biomarkers are still not widely used in the clinic.1

Unfortunately, none of the large trials investigating the prognostic value of uPA and PAI-1 included HER2 status in the survival analysis. Only limited information is available on the relative prognostic impact of these factors when considered along with traditional prognostic markers in the same group of breast cancer patients. Therefore, it is still uncertain whether uPA and PAI-1 can give additional clinically relevant prognostic information after traditional prognostic factors and HER2 status have been taken into account.

To address this issue, we undertook the present study with the aim of comparing the prognostic impact of HER2 status, uPA, PAI-1, and traditional prognostic factors tumor size, grade, histological subtype, lymphovascular invasion, steroid hormone receptor status, and patient age on diseasefree, overall, and breast cancer specific survival in node-negative breast cancer patients.

Patients and methods

Our retrospective analysis included all patients with lymph node-negative invasive breast cancer without distant metastases who underwent primary surgical treatment in Maribor University Clinical Center, Slovenia, in the ten-year period between January 1 2000 and December 31 2009. Exclusion criteria were neoadjuvant chemotherapy and presence of another active malignancy during breast cancer treatment. Considering the Helsinki Declaration principles the Slovenian National Medical Ethics Committee approved this study (Approval No. 55/11/13).

Clinical information on diagnosis, treatment, and follow-up was obtained from patient medical records. Survival data were completed with updated information from Slovenian Cancer Registry. Data on tumor size, histological subtype, grade, lymph node status, steroid hormone receptor status, and HER2 immunohistochemistry were obtained from original histology reports from the primary surgery. HER2 gene amplification, uPA and PAI-1 levels were obtained from our institution’s Medical Genetics Laboratory. Due to economical limitations, uPA and PAI-1 could not be assessed in all patients. Some other histological data were missing in a small fraction of patients due to inconsistent hospital guidelines on histology reports in the past.

All patients underwent either modified radical mastectomy or breast conserving surgery and radiotherapy. Adjuvant systemic treatment was given according to the guidelines followed at our institution at the time and was not influenced by uPA and/or PAI-1 values. Patients who completed primary treatment were followed-up at our institution at regular intervals.

HER2 status was determined immunohistochemically and additional fluorescent in situ hybridization (FISH) with PathVysion HER-2 DNA Probe Kit (Abbott Molecular, Abbott Park, IL, USA) was performed in samples with an immunohistochemical result of 2+. Since the patients were diagnosed before the publication of new ASCO/ CAP guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors6 and human epidermal growth factor receptor 2 testing in breast cancer7 and treated accordingly, steroid hormone receptor status and HER2 status were determined using the old guideline recommendations in order to avoid cases with diagnostic-therapeutic mismatch.

uPA and PAI-1 were analyzed prospectively from representative pieces of tumor tissue that were frozen in liquid nitrogen after histologic evaluation. The frozen samples were pulverised using a micro-dismembrator, suspended in a buffer (pH 8.5) containing 0.02 M Tris-HCl, 0.125 M NaCl and 2% Triton X-100, and shaken for three hours at 4°C. The obtained suspension was centrifuged for 30 minutes at 100,000 x g. Protein content was determined with the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and uPA and PAI-1 content was determined with commercially available ELISA assay kits (American Diagnostica, Greenwich, CT, USA). uPA and PAI-1 content was expressed as nanograms of analyte per milligram of tissue protein.

The correlations between different variables were tested with Spearman’s rank correlation, Kruskal-Wallis test, Mann-Whitney U test and chisquare test, depending on the type of variables. The continuous variables uPA and PAI-1 were coded as binary variables using the previously optimized cutoffs of 3 ng uPA/mg protein and 14 ng PAI-1/mg protein to distinguish between low- and high-risk patients8, and combined into one variable (both low versus one or both high) as previously shown to be of greatest clinical relevance.9 Because adjuvant trastuzumab was not available for the whole cohort, the patients were divided into three groups based on HER2 status and adjuvant trastuzumab treatment for the survival analyses: HER2 negative, HER2 positive who were not treated with adjuvant trastuzumab, and HER2 positive who were treated with adjuvant trastuzumab. Disease-free survival (DFS) was calculated from the date of primary surgery until the date of disease recurrence or death from any cause, or the date of the last follow-up visit in case of no recurrence or death. Overall survival (OS) was calculated from the date of diagnosis until the date of death from any cause, or the date of the last follow-up visit. Breast cancer specific survival (BCSS) was calculated from the date of diagnosis until the date of death from breast cancer, or the date of the last follow-up visit or death from other causes for censored patients. Kaplan-Meier method was used to calculate survival curves and univariate Cox regression was used to assess the differences between the curves in univariate analysis. Multivariate analyses were performed by applying the multivariate Cox proportional hazards model. All variables regardless of univariate analysis results were initially included in the Cox model and the method used for the model was backward stepwise likelihood ratio (LR). Model if term removed was reported separately. All tests were performed at a significance level of p=0.05 and a confidence interval (CI) of 95%. All p values were two-sided. Statistical analysis was performed using the SPSS software package v. 21 (IBM, Armonk, NY, USA).

Results

Patient characteristics

Eight hundred fifty-eight node-negative distant metastasis-free breast cancer patients who underwent primary surgery with curative intent were included in the study. The median age of the patients was 62 years (range, 24-95 years). The distribution of the traditional prognostic factors, HER2 status, uPA and PAI-1 in the study group is presented in Table 1. 787 (91.7%) patients received some kind of adjuvant systemic therapy. Of these, 132 received adjuvant chemotherapy, 522 adjuvant hormone therapy, and 133 a combination of both. Among patients who were given adjuvant chemotherapy, 79.2% received anthracycline-based therapy and the majority of the others received CMF (cyclophosphamide, methotrexate, 5-fluorouracil). Among patients who were given hormone therapy, 28.9% received tamoxifen, 52.7% received an aromatase inhibitor, and the rest received a combination of both. Adjuvant trastuzumab was given in combination with chemotherapy in 6.4% of all patients and in 35.8% of HER2-positive patients. The median follow-up time was 100 months (range, 49–181 months).

Table 1

Distribution of traditional prognostic factors, HER2 status, uPA and PAI-1 in the study group of node-negative breast cancer patients (N = 858)

FactorsNo. of patients%
Age858  
≥ 40 years82195.7
< 40 years374.3
Pathological tumor size846  
< 2 cm47456.0
≥ 2 cm37244.0
Pathological tumor type858  
Ductal invasive72083.9
Other invasive13816.1
Histological grade799  
G1-255769.7
G324230.3
Lymphovascular invasion795  
No72090.6
Yes759.4
Estrogen receptor status854  
Positive67478.9
Negative18021.1
Progesterone receptor status803  
Positive46658.0
Negative33742.0
HER2 status761  
Negative61080.2
Positive, without adjuvant trastuzumab9712.7
Positive, with adjuvant trastuzumab547.1
uPA and PAI-1 level332  
Both low15947.9
One or both high17352.1

Some factors could not be assessed in all tumors.

Associations between traditional prognostic factors, HER2 status, uPA and PAI-1

Positive HER2 status was observed more frequently in younger patients (p = 0.008), in larger tumors (p < 0.001), tumor types other than ductal invasive (p = 0.035), tumors of a higher differentiation grade (p < 0.001), with lymphovascular invasion (p = 0.018), those with a lower expression of estrogen (p < 0.001) and progesterone receptors (p = 0.002), as well as in tumors with higher levels of uPA (p = 0.026) and PAI-1 (p = 0.023). uPA and PAI-1 levels correlated positively with each other (r = 0.553, p < 0.001). Higher uPA and PAI-1 values were seen in larger tumors (p < 0.001 for uPA; p = 0.004 for PAI-1), histologic types other than ductal invasive (p < 0.001 for uPA; p = 0.048 for PAI-1), less differentiated tumors (p < 0.001 for both), and tumors with lower estrogen receptor expression (p < 0.001 for both). In PAI-1 was higher in tumors with lymphovascular invasion (p = 0.047). There were no associations between uPA or PAI-1 values and patient age or progesterone receptor status.

Disease-free survival

A total of 228 events occurred in the DFS analysis. Univariate and multivariate analyses of DFS are presented in Table 2. In univariate analysis, older age, tumors larger than 2 cm, grade 3, with evident lymphovascular invasion, and high uPA and/or PAI-1 (Figure 1A) were associated with worse DFS. HER2 positive patients who were not treated with adjuvant trastuzumab had significantly worse DFS than HER2 negative patients. DFS of HER2 positive patients who received adjuvant trastuzumab was not significantly different than DFS of HER2 negative patients (Figure 1B). In backward stepwise LR multivariate model, the three variables that remained significant for DFS after the final step were patient age at diagnosis, estrogen receptor status and uPA/PAI-1 level. uPA/PAI-1 level was the second most important variable after patient age in the model if term removed (Table 2).

Effect of uPA/PAI-1 level and HER2 status on disease-free survival (DFS) in lymph-node negative breast cancer patients. (A) uPA/PAI-1 low (19 of 159 relapsed or died) versus uPA/PAI-1 high (43 of 173 relapsed or died). (B) HER2 negative (154 of 610 relapsed or died) versus HER2 positive not treated with adjuvant trastuzumab (NT) (37 of 97 relapsed or died) and HER2 positive treated with adjuvant trastuzumab (T) (8 of 54 relapsed or died).

NT = not treated with adjuvant trastuzumab; T = treated with adjuvant trastuzumab

Citation: Radiology and Oncology 51, 1; 10.1515/raon-2016-0024

Table 2

Univariate and multivariate analysis of disease–free survival in lymph node–negative breast cancer patients with a median follow–up time of 100 months. Multivariate analysis was performed in the 273 patients for whom complete data were available

Univariate analysisMultivariate analysis withall variablesMultivariate analysis –backward LR modelModel if term removed
hazard ratio (95 % CI)phazard ratio (95 % CI)phazard ratio (95 % CI)pp
Age

(continuous, unit = 10 years)
1.80

(1.60–2.02)
< 0.0011.53

(1.20–1.96)
0.0011.51

(1.19–1.93)
0.001< 0.001
Tumor size

(≥2 vs. <2 cm)
1.41

(1.08–1.83)
0.0121.01

(0.56–1.80)
0.9820.982
Tumor type

(other invasive vs. ductal invasive)
1.08

(0.76–1.55)
0.6581.28

(0.51–3.20)
0.5980.606
Grade

(G3 vs. G1–2)
1.37

(1.03–1.81)
0.0311.54

(0.75–3.17)
0.2400.240
Lymphovascular invasion

(present vs. absent)
1.54

(1.03–2.29)
0.0351.43

(0.65–3.15)
0.3770.393
Estrogen receptors

(negative vs. positive)
1.19

(0.87–1.61)
0.2711.81

(0.79–4.16)
0.1652.25

(1.24–4.09)
0.0080.158
Progesterone receptors

(negative vs. positive)
1.10

(0.84–1.45)
0.4961.03

(0.48–2.23)
0.9350.935
HER2 status  0.005  0.355    0.379
positive NT vs. negative1.73

(1.21–2.47)
0.0031.66

(0.83–3.31)
0.150  
positive T vs. negative0.70

(0.35–1.43)
0.3321.16

(0.46–2.91)
0.758  
uPA/PAI–1

(one or both high vs. both low)
2.16

(1.23–3.72)
0.0051.76

(0.89–3.49)
0.1061.99

(1.05–3.77)
0.0350.098

NT = not treated with adjuvant trastuzumab; T = treated with adjuvant trastuzumab

In multivariate analysis, the HR for disease recurrence or death from any cause in estrogen receptor positive patients with high uPA and/or PAI-1 was 2.78 (95% CI, 1.28–6.03; p = 0.010) compared to those with both values low. The corresponding HR values for estrogen receptor negative, HER2 negative, HER2 positive patients who did not receive adjuvant trastuzumab, HER2 positive patients who received adjuvant trastuzumab, and triple negative patients, were 1.11 (95% CI, 0.33–3.76; p = 0.863), 1.60 (95% CI, 0.70–3.68; p = 0.268), 9.25 (95% CI, 1.06–80.82; p = 0.044), 3.31 (95% CI, 0.10–109.67; p = 0.503), and 1.08 (95% CI, 0.30–3.86; p = 0.902), respectively.

Overall survival

A total of 172 events occurred in the OS analysis. Univariate and multivariate OS analyses are shown in Table 3. Patient age at diagnosis, tumor size, grade, and uPA/PAI-1 levels (Figure 2a) were found to have a significant impact on OS in univariate analyses. HER2 (Figure 2B) and the other traditional prognostic factors were not found to influence OS in our series of node-negative breast cancer patients. In backward stepwise LR multivariate model, the remaining variables significantly associated with OS after the last step were patient age and tumor grade (Table 3). uPA/PAI-1 level was the third most important factor in the model if term removed and was removed from the model at the last step.

Effect of uPA/PAI-1 level and HER2 status on overall survival (OS) in lymph-node negative breast cancer patients. (A) uPA/PAI-1 low (10 of 159 died) versus uPA/PAI-1 high (31 of 173 died). (B) HER2 negative (118 of 610 died) versus HER2 positive not treated with adjuvant trastuzumab (26 of 97 died) and HER2 positive treated with adjuvant trastuzumab (5 of 54 died).

NT = not treated with adjuvant trastuzumab; T = treated with adjuvant trastuzumab

Citation: Radiology and Oncology 51, 1; 10.1515/raon-2016-0024

Table 3
Univariate analysisMultivariate analysis withall variablesMultivariate analysis –backward LR modelModel if term removed
hazard ratio (95 % CI)phazard ratio (95 % CI)phazard ratio (95 % CI)pp
Age

(continuous, unit = 10 years)
2.18

(1.88–2.52)
< 0.0011.61

(1.19–2.17)
0.0021.68

(1.25–2.27)
0.0010.001
Tumor size

(≥2 vs. <2 cm)
1.38

(1.02–1.88)
0.0360.94

(0.47–1.88)
0.8650.865
Tumor type

(other invasive vs. ductal invasive)
1.24

(0.83–1.85)
0.2982.24

(0.76–6.59)
0.1420.165
Grade

(G3 vs. G1-2)
1.40

(1.01–1.94)
0.0422.39

(0.95–6.02)
0.0662.69

(1.34–5.40)
0.0050.064
Lymphovascular invasion

(present vs. absent)
1.34

(0.83–2.16)
0.2361.36

(0.46–4.01)
0.5820.594
Estrogen receptors

(negative vs. positive)
1.24

(0.88–1.75)
0.2241.80

(0.67–4.81)
0.2420.233
Progesterone receptors

(negative vs. positive)
1.01

(0.74–1.38)
0.9550.62

(0.25–1.54)
0.2990.279
HER2 status  0.190  0.843    0.837
positive NT vs. negative1.37

(0.89–2.09)
0.1520.76

(0.31–1.90)
0.562  
positive T vs. negative0.63

(0.26–1.55)
0.3180.90

(0.26–3.18)
0.871  
uPA/PAI-1

(one or both high vs. both low)
2.73

(1.33–5.58)
0.0061.98

(0.83–4.76)
0.1260.114

NT = not treated with adjuvant trastuzumab; T = treated with adjuvant trastuzumab

Breast cancer specific survival

A total of 65 events occurred in the BCSS analysis. Univariate and multivariate BCSS analyses are shown in Table 4. Tumor size, grade, estrogen and progesterone receptor status, HER2 status and uPA/PAI-1 levels were associated with BCSS in univariate analyses. The only variable retained in the final multivariate model using the backward stepwise LR method was tumor grade. uPA/PAI-1 level was the third most important factor in the model if term removed.

Table 4

Univariate and multivariate analysis of breast cancer specific survival in lymph node-negative breast cancer patients with a median follow-up time of 100 months. Multivariate analysis was performed in the 273 patients for whom complete data were available

Univariate analysisMultivariate analysis withall variablesMultivariate analysis –backward LR modelModel if term removed
hazard ratio (95 % CI)phazard ratio (95 % CI)phazard ratio (95 % CI)pp
Age

(continuous, unit = 10 years)
1.16

(0.95–1.41)
0.1480.76

(0.51–1.12)
0.1620.160
Tumor size

(≥2 vs. <2 cm)
2.44

(1.45–4.13)
0.0011.72

(0.55–5.38)
0.3500.340
Tumor type

(other invasive vs. ductal invasive)
1.20

(0.63–2.29)
0.5873.33

(0.64–17.23)
0.1520.196
Grade

(G3 vs. G1-2)
3.58

(2.12–6.05)
< 0.0017.10

(1.23–40.86)
0.02810.34

(2.33–45.97)
0.0020.014
Lymphovascular invasion

(present vs. absent)
1.48

(0.70–3.12)
0.3051.43

(0.36–5.65)
0.6080.618
Estrogen receptors

(negative vs. positive)
2.21

(1.33–3.66)
0.0021.24

(0.28–5.36)
0.7780.776
Progesterone receptors

(negative vs. positive)
1.74

(1.06–2.86)
0.0280.81

(0.17–3.77)
0.7890.788
HER2 status  0.031  0.663    0.614
positive NT vs. negative2.18

(1.21–3.93)
0.0091.29

(0.39–4.31)
0.683  
positive T vs. negative0.96

(0.30–3.10)
0.9440.46

(0.06–3.68)
0.460  
uPA/PAI-1

(one or both high vs. both low)
6.46

(1.47–28.43)
0.0142.79

(0.57–13.67)
0.2050.166

NT = not treated with adjuvant trastuzumab; T = treated with adjuvant trastuzumab

Discussion

The results of our study indicate an important prognostic value of uPA/PAI-1 level in node-negative breast cancer patients. Even though uPA and PAI-1 values were associated with most of the prognostic factors currently in use for clinical decision-making in the adjuvant setting, multivariate analysis showed that uPA/PAI-1 level carries additional, independent prognostic information. This was particularly true for DFS analysis where uPA/PAI-1 level was the second most important variable in the survival model after age and was retained in the backward LR model after the final step along with age and estrogen receptor status. In OS analysis, the final model included only age and tumor grade, which is probably a reflection of the very strong association between higher uPA and PAI-1 values and less differentiated tumors. However, in the multivariate model that included all variables, the HR for death from any cause was 1.98 (95% CI 0.83–4.76) for patients with high uPA and/or PAI-1 compared to patients with both values low, which shows a substantial possibility of an important effect on OS that would have probably remained statistically significant in a larger sample. The same three variables were the most important in the multivariate model of BCSS as well. Here, the singular prognostic importance of tumor grade was even more evident. As opposed to DFS and OS which both included deaths from other causes as events in the analysis, younger age seemed associated with worse BCSS. It is important to emphasize that BCSS analysis must be interpreted with caution due to the small number of events. Considering the HRs and confidence intervals, it is probable that both age and uPA/PAI-1 level importantly influence BCSS and would have retained statistical significance in a larger sample. On the other hand, HER2 status only showed prognostic significance in univariate DFS and BCSS analyses when comparing HER2 negative to HER2 positive patients who were not treated with adjuvant trastuzumab. It did not remain an important determinant of DFS and BCSS in multivariate analyses and lost all prognostic value in OS analysis.

A subgroup analysis of DFS according to estrogen receptor and HER2 status showed that apart from the no longer relevant group of HER2 positive patients who did not receive adjuvant trastuzumab treatment, uPA/PAI-1 level was prognostically by far the most important in estrogen receptor positive tumors. In contrast, its prognostic value in multivariate analysis was practically null in HER2 positive patients treated with adjuvant trastuzumab and in triple negative patients. Conveniently, estrogen receptor positive patients are the ones with the highest uncertainty regarding the benefit of adjuvant chemotherapy, while the other two subgroups are generally recommended adjuvant chemotherapy ± anti-HER2 therapy regardless of other prognostic factors.

Our findings confirm those of numerous other studies that have reported uPA and PAI-1 to be statistically independent prognostic factors in lymph node-negative breast cancer patients.10-18 However, these studies did not include HER2 status which is now an important part of the decision about adjuvant systemic therapy. Because of the observed associations between overexpression of the HER2/ neu protein and tumor proteolytic factors in breast and in other cancers19-21, HER2/neu has been suggested to up-regulate the proteolytic enzymes, including uPA and PAI-1, and thus play a direct role in tumor invasion and metastasis.19 This possible association might be one of the additional reasons besides methodological difficulties why uPA and PAI-1 testing is still not that frequently used in the clinic in spite of the excellent evidence of its clinical utility.1 Even so, the main reason for this inconsistency are practical issues. uPA and PAI-1 are determined using validated ELISA assay kits as described above. This measurement requires relatively large amounts of fresh or freshly frozen tumor tissue, which is not practical for clinical use, especially in needle or surgical biopsies and in small tumors. Besides, the results may not be available at the time of the histology report due to sample pooling. However, attempts to develop immunohistochemistry assays on formalin-fixed and paraffin-embedded tissues are ongoing.1

Among the few studies that considered the clinical relevance of both HER2 and proteolytic enzymes for survival in breast cancer patients, the results are somewhat conflicting. Harbeck et al.22 showed that PAI-1 was the only independent prognostic factor for DFS and OS when considered along with tumor size, tumor grade, steroid hormone receptor status, uPA, HER2 status, MIB1, SPF, p53, and cathepsin D in 100 node-negative breast cancer patients. Similarly, Bouchet et al.23 found no additional prognostic information of HER2/neu protein levels when evaluated in multivariate analysis together with uPA, PAI-1 and traditional histologic factors in the subgroup of 226 node-negative patients. They reported DFS to be independently influenced by PAI-1 and tumor size and OS by PAI-1 and uPA levels. Recently, Buta et al.24 also reported superior DFS in 73 node-negative, postmenopausal, steroid hormone receptor positive breast cancer patients with smaller tumors and low PAI-1, independent of HER2 status. On the other hand, Konecny et al.19 reported that both uPA/PAI-1 level and HER2 status independently influenced DFS in addition to tumor size and nodal status in a group of 542 patients with a short follow-up not selected by nodal status. The same factors were found to be important in multivariate OS analysis as well, but this time HER2 status did not quite reach statistical significance. Interestingly, among 118 node-negative patients with long-term follow-up, Zemzoum et al.25 found uPA/PAI-1 to be the only variable independently influencing DFS, and HER2 status to be the most important factor in the multivariate analysis of OS. uPA/PAI-1 and tumor grade were of borderline significance for OS. Complicating the matter even further, without reference to uPA and/ or PAI-1, Schmidt et al.26 reported HER2 status to be prognostically significant in node-negative breast cancer patients only when determined by FISH and not when determined immunohistochemically. In our group of 273 node-negative patients with long-term follow-up who were available for multivariate analysis, the combination of uPA and PAI-1 levels clearly carried independent prognostic value for DFS and was important although not formally statistically significant in multivariate analysis of OS as well, while HER2 status determined with the usual combination of immunohistochemistry and FISH did not prove to be an important determinant of DFS or OS in multivariate analyses regardless of adjuvant anti-HER2 treatment.

Most of the studies confirming the prognostic value of uPA and PAI-1 in breast cancer patients have focused on patients who received no adjuvant systemic treatment. In fact, the prognostic information from these biomarkers seems diminished in patients who receive adjuvant treatment, particularly adjuvant hormone therapy27,28, indicating a possible predictive role of uPA and PAI-1 for response to therapy. However, the prognostic importance of uPA/PAI-1 level was evident in our group of patients although the vast majority received adjuvant hormone therapy and a significant fraction were given adjuvant chemotherapy. Contrastingly, based on our results, HER2 status is clearly more important in its predictive than in its prognostic role.

Our study has several limitations, the principal one being its retrospective character and the associated possibility of bias. A major problem is missing data, particularly on uPA and PAI-1 values which could only be determined in about 40% of the patients because of our institution’s economical limitations and the fact that these markers still largely serve only academic purposes. Because of inconsistent histology reports in the past, some other information is missing in a fraction of patients. The total number of patients with available information was used for each univariate analysis and multivariate analyses were performed in the 273 patients in whom complete data were available. Even so, we are aware of only one study comparing the prognostic values of uPA, PAI-1, HER2, and traditional prognostic factors that included a slightly larger subgroup of 283 lymph node-negative patients.19 Another possible limitation is the comparatively high proportion of deaths from causes other than breast cancer, which may have somewhat obscured the results of both DFS and OS analysis and is probably also the cause of such a marked association of older age and worse prognosis, an assumption confirmed by the fact that older age was associated with improved breast cancer specific survival. Furthermore, our results must be considered in the light of the unavoidable multiple analyses and the possibility of a type I statistical error. To facilitate interpretation, 95% confidence intervals and not just the p values have been stated wherever possible. On the other hand, one of the main strengths of our study in addition to the combination of analyzed prognostic factors is the long follow-up which was in the range of 49-181 months, the median of 100 months being more than three times the median follow-up of the larger study by Konecny et al.19 Moreover, we believe that the scientifically sound and straightforward statistical analysis is a strength of our study as well.

Based on the results from our retrospective analysis of node-negative breast cancer patients with long-term follow-up, we conclude that the combined uPA/PAI-1 level carries important additional prognostic information, particularly for DFS, even after all traditional prognostic factors as well as HER2 status have been taken into account. We believe that routine use of uPA/PAI-1 level would further improve risk stratification and adjuvant therapy decisions in this setting, especially in estrogen receptor positive patients.

Acknowledgements

The authors would like to thank prof. Lara Lusa, Ph.D., from the Institute for Biostatistics and Medical Informatics, Faculty of Medicine, University of Ljubljana, for advice regarding statistical methods. We would also like to thank Assoc. Prof. Darja Arko, M.D., Ph.D., and Nina Čas Sikošek, M.D., from Maribor University Clinical Center, for coordinating patient treatment and follow-up.

References

  • 1

    Duffy MJ, McGowan PM, Harbeck N, Thomssen C, Schmitt M. uPA and PAI-1 as biomarkers in breast cancer: validated for clinical use in level-ofevidence-1 studies. Breast Cancer Res 2014; 16: 428.

    • Crossref
    • PubMed
    • Export Citation
  • 2

    Hayes DF, Bast RC, Desch CE, Fritsche H Jr, Kemeny NE, Jessup JM, et al. Tumor marker utility grading system: a framework to evaluate clinical utility of tumor markers. J Natl Cancer Inst 1996; 88: 1456-66.

    • Crossref
    • PubMed
    • Export Citation
  • 3

    Jänicke F, Prechtl A, Thomssen C, Harbeck N, Meisner C, Untch M, et al. Randomized adjuvant chemotherapy trial in high-risk, lymph node-negative breast cancer patients identified by urokinase-type plasminogen activator and plasminogen activator inhibitor type 1. J Natl Cancer Inst 2001; 93: 913-20.

    • Crossref
    • PubMed
    • Export Citation
  • 4

    Harbeck N, Schmitt M, Meisner C, Friedel C, Untch M, Schmidt M, et al. Ten-year analysis of the prospective multicentre Chemo-N0 trial validates American Society of Clinical Oncology (ASCO)-recommended biomarkers uPA and PAI-1 for therapy decision making in node-negative breast cancer patients. Eur J Cancer 2013; 49: 1825-35.

    • Crossref
    • Export Citation
  • 5

    Look MP, van Putten WL, Duffy MJ, Harbeck N, Christensen IJ, Thomssen C, et al. Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients. J Natl Cancer Inst 2002; 94: 116-28.

    • Crossref
    • PubMed
    • Export Citation
  • 6

    Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, et al. American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol 2010; 28: 2784-95.

    • Crossref
    • Export Citation
  • 7

    Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. American Society of Clinical Oncology; College of American Pathologists. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol 2013; 31: 3997-4013.

  • 8

    Harbeck N, Dettmar P, Thomssen C, Berger U, Ulm K, Kates R, et al. Riskgroup discrimination in node-negative breast cancer using invasion and proliferation markers: 6-year median follow-up. Br J Cancer 1999; 80: 419-26.

    • Crossref
    • Export Citation
  • 9

    Harbeck N, Kates RE, Schmitt M. Clinical relevance of invasion factors urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 for individualized therapy decisions in primary breast cancer is greatest when used in combination. J Clin Oncol 2002; 20: 1000-7.

    • Crossref
    • PubMed
    • Export Citation
  • 10

    Foekens JA, Schmitt M, van Putten WL, Peters HA, Bontenbal M, Jänicke F, et al. Prognostic value of urokinase-type plasminogen activator in 671 primary breast cancer patients. Cancer Res 1992; 52: 6101-5.

    • PubMed
    • Export Citation
  • 11

    Jänicke F, Schmitt M, Pache L, Ulm K, Harbeck N, Höfler H, et al. Urokinase (uPA) and its inhibitor PAI-1 are strong and independent prognostic factors in node-negative breast cancer. Breast Cancer Res Treat 1993; 24: 195-208.

    • Crossref
    • PubMed
    • Export Citation
  • 12

    Eppenberger U, Kueng W, Schlaeppi JM, Roesel JL, Benz C, Mueller H, et al. Markers of tumor angiogenesis and proteolysis independently define high- and low-risk subsets of node-negative breast cancer patients. J Clin Oncol 1998; 16: 3129-36.

    • Crossref
    • PubMed
    • Export Citation
  • 13

    Peyrat JP, Vanlemmens L, Fournier J, Huet G, Révillion F, Bonneterre J. Prognostic value of p53 and urokinase-type plasminogen activator in nodenegative human breast cancers. Clin Cancer Res 1998; 4: 189-96.

  • 14

    Kim SJ, Shiba E, Kobayashi T, Yayoi E, Furukawa J, Takatsuka Y, et al. Prognostic impact of urokinase-type plasminogen activator (PA), PA inhibitor type-1, and tissue-type PA antigen levels in node-negative breast cancer: a prospective study on multicenter basis. Clin Cancer Res 1998; 4: 177-82.

    • PubMed
    • Export Citation
  • 15

    Malmström P, Bendahl PO, Boiesen P, Brünner N, Idvall I, Fernö M. S-phase fraction and urokinase plasminogen activator are better markers for dist ant recurrences than Nottingham Prognostic Index and histologic grade in a prospective study of premenopausal lymph node-negative breast cancer. J Clin Oncol 2001; 19: 2010-9.

    • Crossref
    • Export Citation
  • 16

    Meo S, Dittadi R, Peloso L, Gion M. The prognostic value of vascular endothelial growth factor, urokinase plasminogen activator and plasminogen activator inhibitor-1 in node-negative breast cancer. Int J Biol Markers 2004; 19: 282-8.

    • Crossref
    • PubMed
    • Export Citation
  • 17

    De Cremoux P, Grandin L, Diéras V, Savignoni A, Degeorges A, Salmon R, et al. Urokinase-type plasminogen activator and plasminogen-activator-inhibitor type 1 predict metastases in good prognosis breast cancer patients. Anticancer Res 2009; 29: 1475-82.

    • PubMed
    • Export Citation
  • 18

    Rabi ZA, Todorović-Raković N, Vujasinović T, Milovanović J, Nikolić-Vukosavljević D. Markers of progression and invasion in short term follow up of untreated breast cancer patients. Cancer Biomark 2015; 15: 745-54.

    • Crossref
    • PubMed
    • Export Citation
  • 19

    Konecny G, Untch M, Arboleda J, Wilson C, Kahlert S, Boettcher B, et al. Her-2/neu and urokinase-type plasminogen activator and its inhibitor in breast cancer. Clin Cancer Res 2001; 7: 2448-57.

    • PubMed
    • Export Citation
  • 20

    Berney CR, Yang J, Fisher RJ, Russell PJ, Crowe PJ. Correlates of urokinasetype plasminogen activator in colorectal cancer: positive relationship with nm23 and c-erbB-2 protein expression. Oncol Res 1998; 10: 47-54.

  • 21

    Allgayer H, Babic R, Gruetzner KU, Tarabichi A, Schildberg FW, Heiss MM. c-erbB-2 is of independent prognostic relevance in gastric cancer and is associated with the expression of tumor-associated protease systems. J Clin Oncol 2000; 18: 2201-9.

    • Crossref
    • PubMed
    • Export Citation
  • 22

    Harbeck N, Dettmar P, Thomssen C, Henselmann B, Kuhn W, Ulm K, et al. Prognostic impact of tumor biological factors on survival in node-negative breast cancer. Anticancer Res 1998; 18: 2187-97.

    • PubMed
    • Export Citation
  • 23

    Bouchet C, Ferrero-Poüs M, Hacène K, Becette V, Spyratos F. Limited prognostic value of c-erbB-2 compared to uPA and PAI-1 in primary breast carcinoma. Int J Biol Markers 2003; 18: 207-17.

    • Crossref
    • PubMed
    • Export Citation
  • 24

    Buta M, Džodić R, Đurišić I, Marković I, Vujasinović T, Markićević M, et al. Potential clinical relevance of uPA and PAI-1 levels in node-negative, postmenopausal breast cancer patients bearing histological grade II tumors with ER/PR expression, during an early follow-up. Tumour Biol 2015; 36: 8193-200.

    • Crossref
    • PubMed
    • Export Citation
  • 25

    Zemzoum I, Kates RE, Ross JS, Dettmar P, Dutta M, Henrichs C, et al. Invasion factors uPA/PAI-1 and HER2 status provide independent and complementary information on patient outcome in node-negative breast cancer. J Clin Oncol 2003; 21: 1022-8.

    • Crossref
    • PubMed
    • Export Citation
  • 26

    Schmidt M, Lewark B, Kohlschmidt N, Glawatz C, Steiner E, Tanner B, et al. Long-term prognostic significance of HER-2/neu in untreated node-negative breast cancer depends on the method of testing. Breast Cancer Res 2005; 7: R256-66.

    • Crossref
    • PubMed
    • Export Citation
  • 27

    Harbeck N, Alt U, Berger U, Krüger A, Thomssen C, Jänicke F, et al. Prognostic impact of proteolytic factors (urokinase-type plasminogen activator, plasminogen activator inhibitor 1, and cathepsin B, D, and L) in primary breast cancer reflects effects of adjuvant systemic therapy. Clin Cancer Res 2001; 7: 2757-64.

    • PubMed
    • Export Citation
  • 28

    Cufer T, Borstnar S, Vrhovec I. Prognostic and predictive value of the urokinase-type plasminogen activator (uPA) and its inhibitors PAI-1 and PAI-2 in operable breast cancer. Int J Biol Markers 2003; 18: 106-15.

    • Crossref
    • PubMed
    • Export Citation

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • 1

    Duffy MJ, McGowan PM, Harbeck N, Thomssen C, Schmitt M. uPA and PAI-1 as biomarkers in breast cancer: validated for clinical use in level-ofevidence-1 studies. Breast Cancer Res 2014; 16: 428.

    • Crossref
    • PubMed
    • Export Citation
  • 2

    Hayes DF, Bast RC, Desch CE, Fritsche H Jr, Kemeny NE, Jessup JM, et al. Tumor marker utility grading system: a framework to evaluate clinical utility of tumor markers. J Natl Cancer Inst 1996; 88: 1456-66.

    • Crossref
    • PubMed
    • Export Citation
  • 3

    Jänicke F, Prechtl A, Thomssen C, Harbeck N, Meisner C, Untch M, et al. Randomized adjuvant chemotherapy trial in high-risk, lymph node-negative breast cancer patients identified by urokinase-type plasminogen activator and plasminogen activator inhibitor type 1. J Natl Cancer Inst 2001; 93: 913-20.

    • Crossref
    • PubMed
    • Export Citation
  • 4

    Harbeck N, Schmitt M, Meisner C, Friedel C, Untch M, Schmidt M, et al. Ten-year analysis of the prospective multicentre Chemo-N0 trial validates American Society of Clinical Oncology (ASCO)-recommended biomarkers uPA and PAI-1 for therapy decision making in node-negative breast cancer patients. Eur J Cancer 2013; 49: 1825-35.

    • Crossref
    • Export Citation
  • 5

    Look MP, van Putten WL, Duffy MJ, Harbeck N, Christensen IJ, Thomssen C, et al. Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients. J Natl Cancer Inst 2002; 94: 116-28.

    • Crossref
    • PubMed
    • Export Citation
  • 6

    Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, et al. American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol 2010; 28: 2784-95.

    • Crossref
    • Export Citation
  • 7

    Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. American Society of Clinical Oncology; College of American Pathologists. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol 2013; 31: 3997-4013.

  • 8

    Harbeck N, Dettmar P, Thomssen C, Berger U, Ulm K, Kates R, et al. Riskgroup discrimination in node-negative breast cancer using invasion and proliferation markers: 6-year median follow-up. Br J Cancer 1999; 80: 419-26.

    • Crossref
    • Export Citation
  • 9

    Harbeck N, Kates RE, Schmitt M. Clinical relevance of invasion factors urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 for individualized therapy decisions in primary breast cancer is greatest when used in combination. J Clin Oncol 2002; 20: 1000-7.

    • Crossref
    • PubMed
    • Export Citation
  • 10

    Foekens JA, Schmitt M, van Putten WL, Peters HA, Bontenbal M, Jänicke F, et al. Prognostic value of urokinase-type plasminogen activator in 671 primary breast cancer patients. Cancer Res 1992; 52: 6101-5.

    • PubMed
    • Export Citation
  • 11

    Jänicke F, Schmitt M, Pache L, Ulm K, Harbeck N, Höfler H, et al. Urokinase (uPA) and its inhibitor PAI-1 are strong and independent prognostic factors in node-negative breast cancer. Breast Cancer Res Treat 1993; 24: 195-208.

    • Crossref
    • PubMed
    • Export Citation
  • 12

    Eppenberger U, Kueng W, Schlaeppi JM, Roesel JL, Benz C, Mueller H, et al. Markers of tumor angiogenesis and proteolysis independently define high- and low-risk subsets of node-negative breast cancer patients. J Clin Oncol 1998; 16: 3129-36.

    • Crossref
    • PubMed
    • Export Citation
  • 13

    Peyrat JP, Vanlemmens L, Fournier J, Huet G, Révillion F, Bonneterre J. Prognostic value of p53 and urokinase-type plasminogen activator in nodenegative human breast cancers. Clin Cancer Res 1998; 4: 189-96.

  • 14

    Kim SJ, Shiba E, Kobayashi T, Yayoi E, Furukawa J, Takatsuka Y, et al. Prognostic impact of urokinase-type plasminogen activator (PA), PA inhibitor type-1, and tissue-type PA antigen levels in node-negative breast cancer: a prospective study on multicenter basis. Clin Cancer Res 1998; 4: 177-82.

    • PubMed
    • Export Citation
  • 15

    Malmström P, Bendahl PO, Boiesen P, Brünner N, Idvall I, Fernö M. S-phase fraction and urokinase plasminogen activator are better markers for dist ant recurrences than Nottingham Prognostic Index and histologic grade in a prospective study of premenopausal lymph node-negative breast cancer. J Clin Oncol 2001; 19: 2010-9.

    • Crossref
    • Export Citation
  • 16

    Meo S, Dittadi R, Peloso L, Gion M. The prognostic value of vascular endothelial growth factor, urokinase plasminogen activator and plasminogen activator inhibitor-1 in node-negative breast cancer. Int J Biol Markers 2004; 19: 282-8.

    • Crossref
    • PubMed
    • Export Citation
  • 17

    De Cremoux P, Grandin L, Diéras V, Savignoni A, Degeorges A, Salmon R, et al. Urokinase-type plasminogen activator and plasminogen-activator-inhibitor type 1 predict metastases in good prognosis breast cancer patients. Anticancer Res 2009; 29: 1475-82.

    • PubMed
    • Export Citation
  • 18

    Rabi ZA, Todorović-Raković N, Vujasinović T, Milovanović J, Nikolić-Vukosavljević D. Markers of progression and invasion in short term follow up of untreated breast cancer patients. Cancer Biomark 2015; 15: 745-54.

    • Crossref
    • PubMed
    • Export Citation
  • 19

    Konecny G, Untch M, Arboleda J, Wilson C, Kahlert S, Boettcher B, et al. Her-2/neu and urokinase-type plasminogen activator and its inhibitor in breast cancer. Clin Cancer Res 2001; 7: 2448-57.

    • PubMed
    • Export Citation
  • 20

    Berney CR, Yang J, Fisher RJ, Russell PJ, Crowe PJ. Correlates of urokinasetype plasminogen activator in colorectal cancer: positive relationship with nm23 and c-erbB-2 protein expression. Oncol Res 1998; 10: 47-54.

  • 21

    Allgayer H, Babic R, Gruetzner KU, Tarabichi A, Schildberg FW, Heiss MM. c-erbB-2 is of independent prognostic relevance in gastric cancer and is associated with the expression of tumor-associated protease systems. J Clin Oncol 2000; 18: 2201-9.

    • Crossref
    • PubMed
    • Export Citation
  • 22

    Harbeck N, Dettmar P, Thomssen C, Henselmann B, Kuhn W, Ulm K, et al. Prognostic impact of tumor biological factors on survival in node-negative breast cancer. Anticancer Res 1998; 18: 2187-97.

    • PubMed
    • Export Citation
  • 23

    Bouchet C, Ferrero-Poüs M, Hacène K, Becette V, Spyratos F. Limited prognostic value of c-erbB-2 compared to uPA and PAI-1 in primary breast carcinoma. Int J Biol Markers 2003; 18: 207-17.

    • Crossref
    • PubMed
    • Export Citation
  • 24

    Buta M, Džodić R, Đurišić I, Marković I, Vujasinović T, Markićević M, et al. Potential clinical relevance of uPA and PAI-1 levels in node-negative, postmenopausal breast cancer patients bearing histological grade II tumors with ER/PR expression, during an early follow-up. Tumour Biol 2015; 36: 8193-200.

    • Crossref
    • PubMed
    • Export Citation
  • 25

    Zemzoum I, Kates RE, Ross JS, Dettmar P, Dutta M, Henrichs C, et al. Invasion factors uPA/PAI-1 and HER2 status provide independent and complementary information on patient outcome in node-negative breast cancer. J Clin Oncol 2003; 21: 1022-8.

    • Crossref
    • PubMed
    • Export Citation
  • 26

    Schmidt M, Lewark B, Kohlschmidt N, Glawatz C, Steiner E, Tanner B, et al. Long-term prognostic significance of HER-2/neu in untreated node-negative breast cancer depends on the method of testing. Breast Cancer Res 2005; 7: R256-66.

    • Crossref
    • PubMed
    • Export Citation
  • 27

    Harbeck N, Alt U, Berger U, Krüger A, Thomssen C, Jänicke F, et al. Prognostic impact of proteolytic factors (urokinase-type plasminogen activator, plasminogen activator inhibitor 1, and cathepsin B, D, and L) in primary breast cancer reflects effects of adjuvant systemic therapy. Clin Cancer Res 2001; 7: 2757-64.

    • PubMed
    • Export Citation
  • 28

    Cufer T, Borstnar S, Vrhovec I. Prognostic and predictive value of the urokinase-type plasminogen activator (uPA) and its inhibitors PAI-1 and PAI-2 in operable breast cancer. Int J Biol Markers 2003; 18: 106-15.

    • Crossref
    • PubMed
    • Export Citation
OPEN ACCESS

Journal + Issues

Search

  • View in gallery

    Effect of uPA/PAI-1 level and HER2 status on disease-free survival (DFS) in lymph-node negative breast cancer patients. (A) uPA/PAI-1 low (19 of 159 relapsed or died) versus uPA/PAI-1 high (43 of 173 relapsed or died). (B) HER2 negative (154 of 610 relapsed or died) versus HER2 positive not treated with adjuvant trastuzumab (NT) (37 of 97 relapsed or died) and HER2 positive treated with adjuvant trastuzumab (T) (8 of 54 relapsed or died).

    NT = not treated with adjuvant trastuzumab; T = treated with adjuvant trastuzumab

  • View in gallery

    Effect of uPA/PAI-1 level and HER2 status on overall survival (OS) in lymph-node negative breast cancer patients. (A) uPA/PAI-1 low (10 of 159 died) versus uPA/PAI-1 high (31 of 173 died). (B) HER2 negative (118 of 610 died) versus HER2 positive not treated with adjuvant trastuzumab (26 of 97 died) and HER2 positive treated with adjuvant trastuzumab (5 of 54 died).

    NT = not treated with adjuvant trastuzumab; T = treated with adjuvant trastuzumab