Cancer Therapy Vol 3, 167-176, 2005

 

HER-2/neu overexpression in Croatian breast cancer patients: results of one-year multicentric prospective study

Research Article

 

Jasminka Jakic-Razumovic1,*, Jadranka Bozikov2, Bozena Sarcevic3, Viktor Separovic3 Smiljana Kosanovic3, Nives Jonjic4, Elvira Mustac4, Snjezana Tomic5, Josko Bezic5, Bozo Kruslin6, Majda Vucic6, Davor Tomas6, Branko Dmitrovic7, Valerija Blazicevic V7, Tratincica Jakovina8, Drazen Svagelj9, Igor Boric10, Zdenko Njiric10, Vesna Stitic11, Mira Mlinac-Lucijanic M11, Nada Restek-Samarzija12, Hrvoje Predrijevac12, Milan Gosev13

1Department of pathology, Clinical Hospital Center and Medical School Zagreb

2Andrija Stampar School of Public Health-Medical School Zagreb

3Department of pathology Clinic for tumors Zagreb

4Department of pathology Medical faculty Rijeka

5Department of pathology Clinical Hospital Split

6Department of pathology Clinical Hospital Ò Sestre milosrdniceÓ Zagreb

7Department of pathology Clinical Hospital Osijek

8Department of pathology General Hospital Slavonski Brod

9Department of pathology General Hospital Vinkovci

10Department of pathology General Hospital Dubrovnik

11Department of pathology General Hospital Karlovac

12Hoffmann-La Roche Zagreb11, Croatia

__________________________________________________________________________________

*Correspondence: Jasminka Jakic-Razumovic M.D, Ph.D., Department of Pathology, Clinical Hospital Center Zagreb, Kispaticeva 12, 10000 Zagreb, Croatia; phone: + 385 1 2388 523; fax: + 385 1 2421 861; e-mail: drazumov@hotmail.com

Key words: breast carcinoma, HER-2/neu, overexpression, prognostic factors

Abbreviations: cyclophosphamide, metothrexate and fluorouracil, (CMF); disease free survival, (DFS); estrogen, (ER); progesterone, (PR); vascular invasion, (VI);

 

Received: 28 February 2005; Accepted: 8 March 2005; electronically published: March 2005

 

Summary

Treatment approaches and prognosis for breast carcinoma patients depend on different prognostic factors. Uniform reporting of breast carcinoma specimens in sense of traditional prognostic and additional factors such as HER-2/neu is very important for oncologists managing breast carcinoma patients. The aim of this study was to uniform reporting of breast carcinoma specimens, to standardize HER-2/neu testing and reporting as part of routine pathological report in multicentric study for Croatian population, and to correlate HER-2/neu overexpression with other prognostic factors. In this study 1442 breast carcinoma patients during one year from 9 Hospitals in Croatia were prospectively collected and analyzed with data of age, menopausal status, tumor size, type and grade, nuclear grade, axillary node status, intratumoral and extratumoral vascular invasion (VI), estrogen (ER) and progesterone (PR) receptor status, and HER-2/neu expression. The standardization of HER-2/neu staining by HercepTest (Dako, Glostrup, Denmark) was made by repeated collective analysis of doubtful slides, and by repeating of staining in one referral center. All traditional prognostic factors were compared with HER-2/neu status. The results showed that there is statistically significant correlation between HER-2/neu expression and histological grade, nuclear grade, tumor size, lymph node status (p<0.001), extratumoral vascular invasion (p=0.039), and premenopausal status (p=0.040). Negative correlation was found between HER-2/neu overexpression and steroid receptor positivity (p<0.001). When lymph node status was correlated with other prognostic factors lymph node positivity correlates with extratumoral VI (p<0.001). It seems that this prospective study showed correlation between HER-2/neu and some traditional prognostic factors. Oncologists will follow this prospectively collected group of patients carefully, and prognostic influence of investigated factors will be determined in the future.

 

 

I. Introduction

In Croatian female population the incidence and mortality of breast carcinoma is unfortunately very high. The mammography-screening program for female population in Croatia does not exists, except some sporadic actions in certain regions of country. That is probably reason for detection primary breast carcinomas of larger size and advanced clinical stage in comparison with some other European countries. Despite of that, in recent years mortality rate is slightly dropping down, which is probably result of improvement of therapeutic approaches. It is known that the choice of therapy regiments in primary breast carcinoma patients is based on clinical stage and pathological predictive and prognostic factors such as tumor size, grade, lymph node and steroid receptor status 1. For that reason uniform reporting of breast carcinoma specimens is very important for oncologists making predictions for patient therapy respond and survival. Uniform pathological management and reporting, which among others includes standardization of HER-2/neu testing and reporting, is very important not only for metastatic breast carcinoma patients, than probably also for at least some certain group of newly diagnosed patients (Jacobs et al, 2000; Rampaul et al, 2001; Hsu et al, 2002).

The aim of this study was to uniform reporting of breast carcinoma specimens, as well as to standardize HER-2/neu testing and reporting as part of routine pathological report in prospective multicentric study including 9 centers in Croatia. Additionally, verification of association of HER-2/neu expression with other prognostic factors was done. This group of prospectively collected patients will be clinically carefully followed, and effect of combination of prognostic factors in lymph node negative and positive patients on disease free survival (DFS) and overall survival will be investigated in the future. It is known that HER-2/neu positivity is prognostic and predictive factor for therapy respond in patients with metastatic disease, and that selection of patients with strongly HER-2/neu positive tumors is required to obtain clinical benefit from the HER-2/neu targeted therapy in the metastatic breast cancer studies. In some reports HER-2/neu is very powerful prognostic factor indicating poor prognosis in primary diagnosed patients, as well as predictive factor for certain adjuvant therapy respond in particular group of patients. Indeed, several studies suggest that HER-2/neu positive patients experience decreased responses to hormonal therapy. Therefore, HER-2/neu status probably should be part of pathologic report in patients with primary breast cancer, as is recently recommended in St. Gallen conference in January 2005.

 

II. Materials and methods

In this study 1442 consecutive breast carcinoma patients during one year (September 2001 to September 2002) from 9 centers in Croatia were collected. For all patients data of age, menopausal status, hystological type, tumor size, grade, nuclear grade, axillary node status, intratumoral and extratumoral VI, ER and PR status by immunohistochemistry were collected (Table 1). The median age was 60 years (range, 23 to 94). Figure 1 shows age distribution in investigated breast carcinoma patients. The tumor size ranged from 0.1 cm to 20.0 cm (median 2.0 cm). Majority of breast carcinomas were of ductal invasive type (77.2%), lobular invasive carcinoma was found in 145 (10.1%) patients, while other types as mixed, mucinous, papillar, medullar, tubular or other rare types were found in 184 (12.7%) patients. Peritumoral and intratumoral lymphatic/vascular invasion was assessed in hematoxillin and eosin stained slides, and were determined as negative and positive. Peritumoral VI was present in 346 (24%) cases, while intratumoral VI was found in 111 (7.7%) cases. There were 544 cases (37.7%) with and 662 cases (45.9%) without axillary lymph node metastases. For 236 (16.4%) patients axillary node status was unknown. Histological grade 1 and 3 were equally distributed, while grade 2 was found in 767 (55.7%) of patients (Elston et al, 1991; Frierson et al, 1995). Majority of patients were postmenopausal (74.0%), while 374 (26.0%) were premenopausal.

Imunohistochemistry was carried out in formalin fixed, paraffin-embedded tissue sections by streptavidin-biotin peroxidase complex method using an automatized immunostainer (TechMate, Dako). The antibodies employed in the study were anti-ER (clone ER1D5), diluted at 1/100, Dako), PR (clone 1A6, diluted at 1/100, Dako). Positive scoring of immunohistochemistry for ER and PR was assessed on the basis of the visually estimated percentage of neoplastic cells with nuclear staining. All cases without any staining were considered as negative. Positive nuclear staining was semiquantitatively divided in three groups of positivity as mild staining (<10% cells), moderate (10-50% cells) and strong staining (> 50% cells), but for final statistical analysis any nuclear staining was used as positive reaction. For all patients HER-2/neu status was determined using standard Herceptest (Dako). At the beginning of this study there were some differences in intensity of staining interpretation even all participants were using the same scoring system proposed by the manufacturer. Given the potential clinical importance HER-2/neu status in patients management, interlaboratory variability in HER-2/neu IHC results is a matter of legitimate concern. Because of that, slides were first read in each institution, and after that two experts evaluated only positive stained slides (2+, 3+) in blinded fashion. Expert pathologists after adequate training in HercepTest evaluation could reach excellent intraobserver (k=0.921) and interobserver reproducibility (k=0.832). The experts discussed discordant results and complete agreement was achieved. After that the results from the 9 laboratories and experts were compared. Discrepancy between negative (0.1+) and positive (3+) results occurred in few cases (k=0.96, k=0.98 respectively). Distinguishing weakly (2+) from strongly (3+) positive results showed agreement in only 62% of positive cases (k=0.37). After consensus was agreed by analyzing doubtful slides by all


 

Table 1. Distribution of breast carcinoma patients in 9 centers in Croatia during one year (September 2001 to September 2002)

Institution

Number

%

Clinical Hospital Split

196

13.6

Clinical hospital center Zagreb

219

15.2

General Hospital Vinkovci

30

2.1

Clinical Hospital Osijek

163

11.3

General Hospital Slavonski Brod

44

3.1

Medical School Rijeka

293

20.3

Clinic for tumors Zagreb

369

25.6

Clinical Hospital ÒSisters of mercyÓ Zagreb

85

5.9

General Hospital Dubrovnik

43

3.0

Total

 

1442

 

Figure 1. Age distribution in investigated breast carcinoma patients

 

 

pathologists included in this study, the generalized k values indicated substantial agreement (k=0.85).

 

A. Statistical analysis

Analysis was carried out using data analysis software package Statistica ver. 6.0 (StatSoft, 2001). Results were expressed as median and range for quantitative variables and as frequencies and percentages for categorical variables that were grouped following logical classes. Association of categorical variables was tested using the Pearson chi-square test. Results were considered statistically significant at p<0.05.

 

III. Results

Table 2 shows the distribution of all investigated parameters in 1442 breast carcinoma patients. Of 1442 tumors 197 (13.7%) were HER-2/neu positive and 1241 were negative (86.3%). There were 312 (21.8%) patients younger than 50 years, and majority of patients (78.2%) were 50 years or older. In 865 (65.0%) patients ER were positive, while PR were positive in 972 (59.5%) breast carcinoma patients. Almost equal number of patients was treated by mastectomy and spearing surgery procedure (quadrantectomy and tumorectomy with free margins). There was a difference between 9 centers in type of surgical procedure, e.g. in some centers majority of cases were treated with spearing surgery, while in others simple mastectomy was performed in majority of patients (Table 2).

The association of HER-2/neu overexpression with classical prognostic factors is shown in Table 3. Statistically significant association between HER-2/neu expression and lymph node status (p<0.001), tumor size (p<0.001), histological (p<0.001) and nuclear grade (p<0.001), extratumoral VI (p=0.039) and premenopausal status (p=0.040) were found. Statistically significant negative association between HER-2/neu and steroid receptor expression was found (p<0.001).

 

Table 2. Distribution of all investigated parameters in 1442 breast carcinoma patients

 

Factors

Number

%

 

 

 

Age

 

 

< 50

312

21.8

³ 50

1120

78.2

Menopause

 

 

no

374

26.0

yes

1067

74.0

Histologic type

 

 

ductal invasive

1113

77.2

lobular invasive

145

10.1

others

184

12.7

Tumor size

 

 

< 1cm

128

9.1

1 - 2 cm

506

35.9

2 - 5 cm

674

47.8

> 5 cm

101

7.2

Grade

 

 

1

295

21.4

2

767

55.7

3

316

22.9

Nuclear grade

 

 

1

267

19.9

2

750

55.8

3

326

24.3

VI (intratumoral)

 

 

no

1330

92.3

yes

111

7.7

VI (extratumoral)

 

 

no

1094

76.0

yes

346

24.0

ER

 

 

negative

465

35.0

positive

865

65.0

PR

 

 

negative

538

40.5

positive

972

59.5

Lymph nodes

 

 

negative

662

45.9

positive

544

37.7

unknown

236

16.4

HER-2/neu

 

 

negative

1241

86.3

positive

197

13.7

Type of surgery

 

 

quadrantectomy

741

51.4

mastectomy

701

48.6

 

 

 

Table 3. Crosstabulation of HER-2/neu expression with other prognostic factors in 1442 breast carcinoma patients

 

Factors

HER-2/neu (%)

 

 

 

negative

positive

Hi2

p

Lymph nodes

 

 

 

 

negative

595 (90.1)

65 (10.9)

10.37

<0.001

positive

455(84.0)

87(16.0)

 

 

Tumor size

 

 

 

 

< 1cm

121 (94.5)

7 (5.5)

17.67

<0.001

1-2 cm

449(88.9)

56 (11.1)

 

 

2-5 cm

556 (82.7)

116 (17.3)

 

 

> 5 cm

84 (84.0)

16 (16.0)

 

 

Grade

 

 

 

 

1

278 (94.2)

17 (5.8)

48.83

<0.001

2

665 (87.0)

99 (13.0)

 

 

3

236 (74.9)

79 (25.1)

 

 

Nuclear grade

 

 

 

 

1

248 (92.9)

19 (7.1)

43.14

<0.001

2

657 (88.1)

89 (11.9)

 

 

3

246 (75.5)

80 (24.5)

 

 

VI (intratumoral)

 

 

 

 

negative

1148 (86.6)

178 (13.4)

1.18

0.277

positive

92 (82.9)

19 (17.1)

 

 

VI (extratumoral)

 

 

 

 

negative

952 (87.3)

138 (12.7)

4.28

0.039

positive

287 (83.0)

59 (17.0)

 

 

ER

 

 

 

 

negative

365 (78.7)

99 (21.3)

37.86

<0.001

positive

785 (90.8)

80 (9.2)

 

 

PR

 

 

 

 

negative

420 (78.2)

117 (21.8)

53.51

<0.001

positive

730 (92.2)

62 (7.8)

 

 

Menopause

 

 

 

 

no

311 (83.2)

63 (16.8)

4.20

0.040

yes

929 (87.4)

134 (12.6)

 

 

 

 

 

Lymph node status is crosstabulated with other investigated parameters and results are shown in Table 4. It is found statistically significant association between lymph node status and tumor size (p<0.001), histological (p<0.001) and nuclear grade (p<0.001) and intra (p=0.038) and extratumoral VI (p<0.001), while association was negative with both, ER and PR positivity (p<0.001).

Menopausal status and other investigated parameters were crosstabulated and results are shown on the Table 5. No association was observed between menopausal status and lymph node status, tumor size, intratumoral VI, and PR status. Postmenopausal patients more often had ER negative tumors (p<0.001), higher-grade tumors (p=0.007), higher tumor nuclear grade (p=0.037), and presence of extratumoral VI (p<0.001) than premenopausal.

 

IV. Discussion

Treatment and prognosis of breast carcinoma patients depend on histological diagnosis including basic prognostic factors and additional markers. Recently, a number of studies have indicated that overexpression of HER-2/neu in breast carcinoma patients indicate worse prognosis and lower probability of responsiveness to some therapy protocols (Ellis et al, 2001; Love et al, 2003; Schiff et al, 2004). Particularly, it is shown that HER-2/neu positive breast cancer is resistant to endocrine therapy, and that HER-2/neu positivity is associated with shortened survival in ER-positive breast cancer patients treated with hormonal agents, compared with those who are HER-2/neu negative. It is known that testing of HER-2/neu overexpression or amplification is in usage in metastatic breast carcinoma patients because trastuzumab is widely established as an essential and well-tolerated treatment for the management of previously treated HER-2/neu positive metastatic breast caracinoma patients. The testing of newly diagnosed breast cancer specimens for HER-2/neu still not has achieved Òstandard of practiceÓ status for the management of breast carcinoma patients in the United States and Europe. According to the St. Gallen,s conference highlights the Panel was not ready to accept the information suggesting that overexpression of HER-2/neu may indicate a lower probability of responsiveness to tamoxifen and perhaps cyclophosphamide, metothrexate and fluorouracil (CMF) (Paik et al, 2000; Osborne et al, 2003) as currently useful for patient care. Therefore, the predictive utility of HER-2/neu overxpression in newly diagnosed breast carcinoma patients still awaits confirmation, but it seems that after collecting valuable data the Panel finally accepted HER-2/neu testing as a part of routine pathological report at the last St.Gallen conference in January 2005 (data not published yet). Additionally, the discussion as to the best method to determine HER-2/neu status in breast carcinoma samples continuous, with the fluorescenece in situ hybridization method gaining popularity, owing to the recent evidence that in comparison with immunohistochemical analysis this method gives more reliable results.

 

 

Table 4. Crosstabulation of lymph node status with other prognostic factors in 1442 breast carcinoma patients

 

Factors

lymph node status (%)

Hi2

p

 

negative

positive

 

 

Tumor size

 

 

 

 

< 1cm

81 (63.3)

17 (13.3)

94.85

<0.001

1-2 cm

272(53.8)

149 (29.4)

 

 

2-5 cm

282 (41.8)

308 (45.7)

 

 

> 5 cm

19 (18.8)

66 (65.3)

 

 

Grade

 

 

 

 

1

174 (69.3)

77 (30.7)

33.35

<0.001

2

347 (53.1)

306 (46.9)

 

 

3

120 (44.6)

149 (55.14

 

 

Nuclear grade

 

 

 

 

1

156 (65.6)

82 (34.5)

20.79

<0.001

2

351 (54.5)

293 (45.5)

 

 

3

122 (45.4)

147 (54.6)

 

 

VI (intratumoral)

 

 

 

 

negative

619 (55.8)

491 (44.2)

4.29

0.038

positive

43 (44.8)

53 (55.2)

 

 

VI (extratumoral)

 

 

 

 

negative

550 (60.0)

367 (40.0)

39.98

<0.001

positive

112 (38.8)

177 (61.1)

 

 

ER

 

 

 

 

negative

182 (47.6)

200 (52.4)

13.08

<0.001

positive

433 (59.0)

301 (41.0)

 

 

PR

 

 

 

 

negative

215 (48.9)

225 (51.1)

11.45

<0.001

positive

400 (59.2)

276 (40.8)

 

 

Menopause

 

 

 

 

no

162 (52.4)

147 (47.6)

1.02

0.313

yes

500 (55.7)

397 (44.3)

 

 

 

Table 5. Crosstabulation of menopausal status with other prognostic factors in 1442 breast carcinoma patients

 

Factors

Menopause (%)

Hi2

p

 

no

yes

 

 

Lymph nodes

 

 

 

 

negative

162 (24.5)

500 (75.5)

1.02

0.31

positive

147(27.0)

397 (73.0)

 

 

Tumor size

 

 

 

 

< 1cm

33 (25.8)

95 (74.2)

0.94

0.816

1-2 cm

132(26.1)

373 (73.9)

 

 

2-5 cm

170 (25.2)

504 (74.8)

 

 

> 5 cm

30 (29.7)

71 (70.3)

 

 

Grade

 

 

 

 

1

84 (28.5)

211 (71.5)

9.76

0.007

2

176 (23.0)

590 (77.0)

 

 

3

100 (31.7)

216 (68.3)

 

 

Nuclear grade

 

 

 

 

1

66 (24.7)

201 (75.3)

6.62

0.037

2

182 (24.30)

567 (75.7)

 

 

3

103 (31.6)

223 (68.4)

 

 

VI (intratumoral)

 

 

 

 

negative

352 (26.5)

977 (73.5)

2.37

0.124

positive

22 (19.8)

89 (80.2)

 

 

VI (extratumoral)

 

 

 

 

negative

302 (80.7)

791 (19.3)

33.14

<0.001

positive

72 (91.7)

274 (8.4)

 

 

ER

 

 

 

 

negative

153 (32.9)

312 (67.1)

20.13

<0.001

positive

187 (21.6)

677 (78.4)

 

 

PR

 

 

 

 

negative

143 (26.6)

395 (73.4)

0.47

0.492

positive

197 (24.9)

594 (75.1)

 

 

 

 

However, at this point it is accepted that HER-2/neu testing by immunohistochemistry is valid method for screening and should be supplemented by FISH method in some cases of doubtful positive reaction (2+ staining intensity) before the trastuzumab treatment (Falo et al, 2003; Goldhirsch et al, 2003). Standardization of these parameters remains an important objective to optimize interlaboratory agreement, and therefore to compare HER-2/neu immunohistochemical staining results obtained in 9 pathology departments the evaluation of the reproducibility of staining and assessments was done. After some repeating of staining in one referral center, and discussing of doubtful slides, there was good interlaboratory and interobserver agreement, and valuable data were collected that may be used in the development of quality assurance policies.  The present study showed the usefulness of multicentric comparative studies in initiating the development guidelines as has been shown in some other studies (Jacobs et al, 2000; Hsu et al, 2002; Santinelli et al, 2002; Gunhan et al, 2004).

Investigation of adjuvant monoclonal antibody treatment against HER-2/neu for breast carcinoma patients overexpressing HER-2/neu are currently ongoing, but this type of treatment in the adjuvant setting outside of clinical trials is not currently justified.  Ongoing prospective studies will show importance of HER-2/neu testing in making decisions of antibody usage in some subgroups of primary breast carcinoma patients with HER-2/neu overexpression in combination with other poor prognostic markers.

Well-established diagnosis of breast cancer with all relevant traditional prognostic markers is a basis of good patients treatment. Therefore, we conducted this study to make consensus about histological prognostic factors (tumor size, grade, nuclear grade, vascular invasion), immunohistochemical predictive factors (ER, PR) and HER-2/neu overexpression in newly diagnosed breast carcinoma patients during one year in Croatian population. The aim also was to make quality control study for HercepTest in 9 centers in Croatia. Since recently HER-2/neu overexpression has gained therapeutic implications and following these developments demand for pathologists to evaluating properly HER-2/neu in breast cancer specimens has been rapidly increasing. For all that  we succeeded in great amount, and finally an consensus was made for uniform reporting of prognostic factors in breast carcinoma patients. Particularly, cconsensus was made about using Nottingham model for determination of histological grade in breast carcinoma patients, which is more objective, has excellent reproducibility when used by experienced pathologists (Elston et al, 1991; Frierson et al, 1995; Robbins et al, 1995). Using this scheme incidence of G1-G3 was similar as described in many publications. Additionally, according to the St.Gallen,s recommendation there was agreement to use immunohistochemical method and scale of positivity for determination of ER and PR in breast carcinoma patients (Goldhirsch et al, 2003). It is known that most laboratories worldwide have switched to immunohistochemistry to assess steroid receptors, and it is shown in some studies that PR by immunohistochemistry provided significantly better results than by ligand-binding assay in predicting outcome. It is also shown that ER and PR are codependent variables and PR was a weaker predictor of response to endocrine therapy (Mohsin et al, 2004). Using immunohistochemical method and St.Gallen,s criteria we found 65.0% ER positive and 59.5 % PR positive breast carcinomas, which is very similar to other published studies using the same method (Colon et al, 2002).

Controversy surrounds the correlation between HER-2/neu expression and other prognostic markers, as has been discussed in preclinical and clinical studies. The objective of the current study was to investigate association of HER-2/neu overexpression with parameters that are assessed routinely in clinical practice (age, hormonal status, cancer grade, nuclear grade, vascular invasion and axilary lymph node status). The results showed that HER-2/neu overexpression was associated significantly with negative ER and PR status, tumor and nuclear high grade, larger tumors, extratumoral VI, positive lymph nodes and menopausal status, as similarly showed some other authors (Coradini and Daidone, 2004). In subgroup of patients presenting with hormone-responsive Taucher et al, (2003) also showed recently that likelihood of HER-2/neu overexpression in G1/G2 tumors is very small, and therefore, that the assessment of HER-2/neu status in this group of patients with breast carcinoma may be considered unnecessary, unless the role of HER-2/neu status in adjuvant treatment has been proven. 

Despite relevant research efforts and identification of many putative good prognostic factors, few of these factors are clinically useful for identifying patients at minimal risk of relapse and with worse prognosis, or patients likely to benefit from specific treatments. Some of them such as HER-2/neu, EGFr, cyclin E, VEGF, urokinase type plasminogen activator-1 and recently discovered anti-apoptosis protein survavin, are suggested to fit in the category high-level clinico-laboratory effective biomarkers.  However, it is known that there is no single biomarker that is able to identify patients with the best (or worse) prognosis or those that would be responsive to a given therapy. Rapid implementation of laboratory findings to clinical practice is followed by many difficulties, including technical statistical concerns, a lack of assay standardization and comparability, and the modern design of studies. Many studies are performed on too small group of patients to provide reliable results. The studies are often heterogeneous in terms of treatment, patients and tumor characteristics, and data may be evaluated using different analytical approaches and thus no easily comparable. Adequately planned prospective studies are required to assess clinical utility of biomarker determinations. The present study showed the usefulness of multicentric comparative studies in initiating the development guidelines, and we hope that collected data will serve as a reference point for future studies of the epidemiological aspect breast cancer among women living in Croatia.

 

Acknowledgements

The authors greatly acknowledge the contribution and support of Ankica Ajdukovic, Smilja Bumber-Bolanca, and Hoffmann-La Roche Zagreb for providing HercepTests for all study participants, and their generous guidance, suggestions and human support during this period. We also appreciate the assistance of the pathology laboratory staff from all pathology departments included in this study.

 

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Jasminka Jakic-Razumovic