Immunohistochemical evaluation of endothelial nitric oxide synthase expression in primary breast cancer

ARTICLE IN PRESS The Breast (2005) 14, 230–235

THE

BREAST www.elsevier.com/locate/breast

ORIGINAL ARTICLE

Immunohistochemical evaluation of endothelial nitric oxide synthase expression in primary breast cancer S. Loibla,, C. Stranka, G. von Minckwitza, H.-P. Sinnb, A. Bucka, C. Solbacha, K. Strebhardta, M. Kaufmanna a

Department of Gynaecology and Obstetrics, Johann-Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany b Department of Gynaecological Pathology, University Women’s Hospital Heidelberg, Vossstr. 9, 69112 Heidelberg, Germany Received 18 May 2004; received in revised form 21 September 2004; accepted 3 November 2004

KEYWORDS Breast cancer; Endothelial nitric oxide synthase; NO; Nitric oxide; Prognosis

Summary To evaluate the clinical potential of endothelial nitric oxide synthase (eNOS) in human breast cancer, we performed immunohistochemical (IHC) staining of paraffin-embedded primary breast cancer tissue of 163 patients for e-NOS using a monoclonal antibody. A correlation was found between e-NOS expression and both the classic prognostic factors and survival rates. Under half the patients were premenopausal (38.5%), 61.5% being postmenopausal. The median tumour size was 2 cm; in 41.7% of the patients there was involvement of the axillary lymph nodes. Most (84.1%) of the tumours were hormone receptor positive. e-NOS staining was positive in 62%, most of the positive tumours having weak (32.5%) or medium (21.5%) staining for e-NOS. The median follow-up time was 42 months, during which 46 (28%) patients had a local recurrence or metastatic disease. A positive correlation of e-NOS with the hormone receptor status was found (P ¼ 0:031). However, no impact on survival rates was observed. & 2004 Elsevier Ltd. All rights reserved.

Introduction The role of nitric oxide synthases (NOS) in breast cancer was first investigated in 1995.1 To date three Corresponding

author. Tel.: +49 69 6301 7024; +49 69 6301 7938. E-mail address: [email protected] (S. Loibl).

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isoforms of NOS are known: (1) inducible i-NOS, which is not calcium dependent, and two constitutively expressed isoforms, (2) an endothelial form (e-NOS) and (3) a neuronal form (n-NOS), both of which are calcium dependent. Nitric oxide (NO) production by endothelial cells might be of interest, because of its effects of increasing blood flow, inducing angiogenesis, killing tumour cells and

0960-9776/$ - see front matter & 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.breast.2004.11.005

ARTICLE IN PRESS Immunohistochemical evaluation of endothelial nitric oxide synthase expression reducing tumour cell adhesion.2–5 NO protects endothelial cells from TNFa-induced apoptosis. On the other hand, cytokine-activated endothelial NO production can kill adherent tumour cells.6 In view of these conflicting results, the role of NO in tumour biology remains to be elucidated. We have already reported that i-NOS and e-NOS are expressed in tumour cells of in situ and invasive breast cancer tissue but not in benign lesions.7 In addition, our observations demonstrate that i-NOS and e-NOS in cells of primary breast cancer are active enzymes that produce NO, as determined by electro-spin trapping (ESR) analysis. Since NOS expression differs between benign and malignant breast lesions, we wished to define the prognostic impact of e-NOS expression in breast cancer more closely. Therefore, we conducted the present study in tissue taken from 163 primary breast cancer patients and sought correlations between e-NOS expression and the classic prognostic factors, such as tumour size, lymph node status, age, grading, and steroid hormone receptor status. Special emphasis was placed on correlations with the endocrine-related factors.

Patients and methods Patients Two hundred patients who had undergone surgery at the Women’s University Hospital in Frankfurt between 1995 and 1998 were enrolled in the study, and 163 patients were evaluable after complete follow-up with records of any tumour recurrence and of survival status. Patients with primary metastatic disease, or with surgery after primary chemotherapy, were excluded (see Table 1 for more details).

Methods The immunochemical method used has been described in detail elsewhere, but is summarised briefly here.2 Tissue blocks were fixed for 18–24 h in 4% buffered formalin and embedded in paraffin. Immunohistochemical (IHC) reactions were performed using antibodies against e-NOS (anti-ECNOS; Transduction Laboratories, Lexington, KY), which detected a 133-Da protein. There was no cross-reactivity with n-NOS or i-NOS. All other reagents for immunohistochemistry were obtained from Biogenex (San Ramon, USA), unless otherwise indicated. Rabbit anti-human e-NOS monoclonal antibody (diluted 1:000, with an incubation time of

231

Table 1 Patient characteristics and e-NOS expression (N ¼ 163). Characteristics

%

(n)

Tumour size T1 T2 T3 T4

49.8 38.0 6.1 6.1

(81) (62) (10) (10)

Menopausal status Premenopausal Postmenopausal

38.5 61.5

(63) (100)

Hormone receptor status Positive Premenopausal Postmenopausal Negative Missing

84.1 37.5 62.5 14.1 1.8

(137)

(23) (3)

Oestrogen receptor status Positive Negative Missing

78.5 19.6 1.8

(128) (32) (3)

Progesterone receptor status Positive Negative Missing

71.2 24.5 4.3

(116) (40) (7)

e-NOS Positive 1+ 2+ 3+ 4+ Negative

62.0 32.5 21.5

7.4 0.6

38.0

60 min at room temperature) was used after the tissue sections had been pretreated in a microwave oven (5  3 min in citrate buffer, 700 W). After this, the APAAP method was applied.8 An automated cell-staining system (Optimax plus, Biogenex, San Ramon, USA) was used for the staining procedures. In a semi-quantitative analysis the staining was independently evaluated by two observers (H.P.S. and S.L.), who used a ranking score of 0–4 (0 ¼ negative, 4 ¼ strongly positive). In any case of different ranking, a third observer was asked to score and the cases were discussed. The cut-off median was 1. To control for the specificity of the IHC staining, several control experiments were performed. Controls included replacement of the primary antibody in the initial incubation with nonimmune mouse immunoglobulin, with normal rabbit serum, and with the secondary antibody only. As a positive

ARTICLE IN PRESS 232 control, sections derived from human umbilical cord were stained for e-NOS, and as a negative control, sections from a fibroadenoma were stained in the same way. Oestrogen and progesterone receptor analysis was performed with sections cut from formalinfixed and paraffin-embedded tissue. After dewaxing through xylol and graded alcohols the sections were pretreated in a pressure cooker for 45 s using EDTA buffer (pH 8.0). Oestrogen and progesterone receptor expression is examined using mouse-monoclonal antibody oestrogen receptor (NCL-ER-6F11, 1:60) and progesterone receptor (NCL-PgR, 1:40) (Novocastra Laboratories Ltd. Newcastle-upon-Tyne, UK); the incubation time for both was 30 min, and Biogenex Supersensitive MultiLink (detection system, 30 min) (Biogenex, San Ramon, USA) was used with alkaline phosphatase and fast red as chromogen (2  8 min). All incubations were performed at room temperature, and all staining steps were performed by a TECAN robot (Tecan GmbH, Crailsheim, Germany). Between single incubation steps, sections were washed with TBS (pH 7.6). Sections were counterstained with Gill’s haematoxylin. The Remmele score was used, and a score of p3 out of 12 was classed as hormone receptor negative.9 Grading was performed according to the Elston and Ellis10 modification of Bloom and Richardson’s grading system.

S. Loibl et al. of the patients (38.5%) were premenopausal and the remaining 61.5% were postmenopausal. Most (65.8%) of the patients underwent breast-conserving surgery, while 31.9% underwent mastectomy with axillary lymph node dissection. A median number of 15 lymph nodes were excised (range 5–31). Most patients (58.3%) had no involvement of the axillary lymph nodes. Node-positive patients were divided into different groups according to the TNM 2002 staging system.13 In 24.1% of patients 1–3 lymph nodes were found to be involved (pN1), while 9.4% had 4–9 lymph nodes involved (pN2) and 8.2% had 10 or more lymph nodes involved (pN3). For more patient characteristics the reader is referred to Table 1. All women who underwent breast-conserving surgery also received irradiation to the breast. Risk-adapted systemic therapy was given according to menopausal status, lymph node involvement and hormone receptor status. During the median observation period of 42 months (range 12–84 months) in all 44 events (local recurrences and distant metastases) were detected among these 163 patients, and 27 of the patients died during the observation period. Expression of e-NOS was found in 62% of the tumours, but most tumours had weak (score 1) staining (32.5%); in 21.5% a staining score of 2 was recorded; and in only 8% was strong expression of eNOS (scores: 3+ and 4+) found. A median of 1 was taken as the cut off point (Fig. 1).

Statistical analysis Correlations were analysed using the Spearman-r test. For univariate analysis of metastasis-free and overall survival (OAS) Kaplan–Meier estimation was performed11 and then compared using the log-rank test. Multivariate Cox proportional hazard analysis was performed in a stepwise backward fashion for lymph node status, grading, tumour size, age, hormone receptor status and e-NOS by using the SPSS software package (SPSS, Inc. Chicago, IL).12 All test decisions were made at a significance level of a ¼ 0:05: Estimated values were given with the 95% confidence interval.

Correlations and associations between e-NOS and established prognostic factors The Spearman-r-test revealed a significant correlation between e-NOS and hormone receptor status. Hormone receptor-positive patients were significantly more likely to be e-NOS positive than were hormone receptor-negative patients (P ¼ 0:016) (Table 2). We were unable to detect any correlation between e-NOS expression and lymph node status, tumour size, grading or age. There were demonstrable correlations of hormone receptor status with age and with grading; lymph node status with tumour size; and grading with tumour size.

Results Univariate and multivariate survival analysis The patient population was made up of 163 women with invasive breast cancer who had had primary surgery at the University Hospital Frankfurt, Department of Obstetrics and Gynaecology. The median age of these women at the time of diagnosis was 55 (31–88) years. More than one-third

In univariate analysis grading, lymph node status and hormone receptor status were both linked with overall and disease-free survival in a statistically significant manner. e-NOS failed to emerge as a prognostic factor for disease-free survival (DFS)

ARTICLE IN PRESS Immunohistochemical evaluation of endothelial nitric oxide synthase expression Disease free survival rate

1.0

Proportion alive

233

0.8 e-NOS neg. events 17/62

0.6 e-NOS pos. events 33 /101

P = 0.76

0.4 0

10

20

30

40

50

60

Time since surgery (months)

Figure 2 Disease-free survival: Kaplan–Meier estimates of e-NOS expression in breast cancer patients (log-rank test P ¼ 0:76). (e-NOS negative: 17/62, e-NOS positive: 33/101).

Overall survival rate

1.0

Figure 1 IHC detection of e-NOS in paraffin-embedded tissue of breast cancer. (a) Invasive ductal carcinoma, showing strong staining of tumour cells for e-NOS. Original magnification  400. (b) Umbilical cord (positive control for e-NOS). Original magnification  100.

Proportion alive

0.9 e-NOS neg.

0.8

events 8 /62

e-NOS pos.

0.7

events 19 /101 P = 0.38

0.6

Table 2 Correlation of e-NOS expression with the menopausal- and hormonereceptor status (Spearman-r). Number of patients e-NOS ve Menopausal status Pre 21 Post 40

P

r

e-NOS +ve 42 60

Hormone receptor status Positive 56 81 Neg 4 19

10

0.17

(Fig. 2) or for OAS (Fig. 3). Of the established prognostic factors are concerned, age and tumour size failed to demonstrate significance. In the multivariate analysis (Cox model) for DFS and OAS only the lymph node status and the tumour grade proved to be statistically significant prognostic factors (Table 3).

20

30

40

50

60

Time since surgery (months)

Figure 3 OAS: Kaplan–Meier estimation of e-NOS expression in breast cancer patients (log-rank test P ¼ 0:38). (e-NOS negative: 8/62, e-NOS positive: 19/ 102). Patients at riskH e-NOS pos.: e-NOS neg.:

0.29

0.031

0

62 101

55 94

51 87

44 67

35 54

18 27

6 14

Discussion It remains debatable whether e-NOS has a role in human breast cancer. Shear stress and pregnancy, possibly due to an increased oestradiol concentration, are known to increase e-NOS activity.14,15 However, e-NOS has a constitutive effect, and only a small increase of NO results from stimulation of eNOS. NO itself activates i-NOS, and the rise of NO by way of e-NOS could be a trigger for i-NOS stimulation, which leads to a 100- to 1000-fold increase in NO.16

ARTICLE IN PRESS 234 Table 3

S. Loibl et al. Univariate and multivariate analysis of known prognostic factors (Cox’s regression) for overall survival. Univariate analysis

Grading LN status Receptor status Tumour size Age e-NOS

Multivariate analysis

HR

Confidence interval

P-value

HR

Confidence interval

P-value

3.67 5.35 2.32 2.39 0.78 1.36

1.66–8.15 2.01–14.24 0.97–5.56 1.00–5.71 0.35–1.75 0.59–3.14

0.0014 0.0008 0.05 0.049 0.54 0.47

3.6 5.19 — — — —

1.61–8.04 1.94–13.88 — — — —

0.0018 0.001 — — — —

It has been known for some time that e-NOS activity in endothelial cells of the cardiovascular system is regulated by oestrogen.17 However, e-NOS is present in breast cancer cells as well as in intratumoural endothelial cells.7,18–20 Breast cancer itself is a hormone-dependent disease, and the expression of e-NOS might be dependent on the endocrine environment of the tumour. We started a study on 163 patients to investigate whether there was an association between e-NOS expression and the classic prognostic factors, especially hormone receptor status, and whether there was an impact on survival rate. We expected a correlation with patient age at the time of diagnosis, and also with menopausal status or receptor status, because oestrogen is known to increase the constitutive e-NOS expression.21 In our subset of 163 patients we found a significant correlation between e-NOS expression and hormone receptor status, which is consistent with results from other groups.20,22 However, unlike Martin et al.,22 who demonstrated a correlation between e-NOS and oestrogen receptor status, tumour differentiation and lymph node status, we found a correlation only between e-NOS and hormone receptor status. This confirms previous data recorded in 80 patients by Vakkala et al.,20 which indicate an association between e-NOS expression and oestrogen and progesterone receptor status but not between e-NOS expression and tumour differentiation or TNM status. In our investigation either the oestrogen or the progesterone receptor had to be expressed for a tumour to be defined as hormone receptor positive, which is appropriate and reflects the clinical use of the receptor status. There was no correlation between oestrogen receptor expression and e-NOS expression, or between expression of progesterone receptor and of e-NOS. We found no difference in recurrence-free survival or OAS linked with e-NOS expression in tumour cells, whereas hormone

receptor-positive patients had significantly better recurrence-free survival and OAS. Mortensen et al.23 did find an impact of e-NOS expression in peritumoural vessels on the recurrence-free and OAS rates in 56 patients younger than 55 years who were categorised as premenopausal; however, they also failed to demonstrate the same for e-NOS expression in tumour cells and intratumoural vessels. e-NOS expression had no impact on survival in the postmenopausal patients. Oestrogen replacement therapy in postmenopausal women temporarily elevates the plasma NO level.24 Exogenous oestrogen might cause increased e-NOS expression, but this was not investigated separately. In premenopausal women there is an increased NO level in the follicular phase, which is a strong indication that oestradiol is involved in NO release.25 Recent data support results observed by our group showing that premenopausal breast cancer patients who had undergone surgery during the follicular phase had a significantly worse OAS rate.26,27 We were not able to demonstrate a correlation between e-NOS expression and menstrual phase in the premenopausal patients. To the best of our knowledge this is the largest sample of breast cancer patients that has been investigated for e-NOS expression in tumour cells of primary breast cancer. As opposed to i-NOS expression in breast cancer, e-NOS has not been shown to have any impact on survival rates. In a corresponding investigation of i-NOS in breast cancer patients, there was a correlation between i-NOS expression and both tumour size and tumour grade, and i-NOS was an independent prognostic factor for survival.28 There is clear evidence that e-NOS expression is regulated by hormonal stimulation in endothelial cells of the cardiovascular system and breast cancer tumour cells. However, if e-NOS does have a role in breast cancer this has not yet been defined.

ARTICLE IN PRESS Immunohistochemical evaluation of endothelial nitric oxide synthase expression

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