Breast cancer is the most common malignancy in women and ...



Immunohistochemical analysis of MAGE-A4, NY-ESO-1 and HER-2 antigen expression in patients with relapsing invasive ductal breast cancer

Daniela Bandić, Antonio Juretić*, Božena Šarčević, Viktor Šeparović, Mirjana Kujundžić Tiljak1, Tvrtko Hudolin2, Giulio C Spagnoli3, Dinko Čović, Mirko Šamija

University Hospital for Tumors, Zagreb, Croatia

1Department for Medical Statistics, Epidemiology and Medical Informatics, Andrija Stampar

School of Public Health, Medical School, University of Zagreb, Zagreb, Croatia

2Clinical Hospital Center „Zagreb“, Zagreb, Croatia

3Department of Surgery, Research Laboratory, University Hospital Basel, Switzerland

*Present address: Clinical Hospital Center „Zagreb“, Zagreb, Croatia

Short title: Expression of antigens MAGE-A4, NY-ESO-1 and HER-2 in breast cancer

Correspondence to:

Professor Antonio Juretić, MD, PhD

radiation oncologist

Clinics for Oncology,

Clinical Hospital Center „Zagreb“

Kispatićeva 12

HR-10000 Zagreb, Croatia.

e-mail: antonio.juretic(zg.htnet.hr

Summary

Objective: The aim of the study was to evaluate the possible prognostic role of the expression of cancer/testis antigens MAGE-A4 and NY-ESO-1 in patients with invasive ductal breast cancer.

Design: Expression of these antigens together with the HER-2 antigen was evaluated immunohistochemicaly (IHC) on archival paraffin embedded breast cancer tissues from 81 patients. The patients were initially selected on the basis of timing of tumor relapse. Initially all patients (T1 to T3, N0 to N1, M0 tumors) and as adjuvant therapy they all received postoperative irradiation and, if indicated, systemic therapy (chemotherapy and hormonal therapy).The patients who were disease free after a five years of follow up (n=23) were compared with the ones who either had locoregional relapse (n=30) or bone metastases (n=28). After a ten years of follow up patient’s survival was also evaluated.

Results: The three groups of patients were comparable in terms of age, type of operation, tumor size, tumor grade, number of metastatically involved axillary lymph nodes, Nottingham prognostic index (NPI), progesterone receptor (PR) status, and adjuvant hormonal therapy. The “five-year” relapse-free group of patients had a significantly higher number of patients with positive estrogen receptors (ER; p=0.03) and it received significantly less adjuvant chemotherapy (p=0,00002). Expectably, this group also had a a significantly better ten year survival (p(0.00000). The three groups of patients did not differ in the NY-ESO-1 or HER-2 expression but in the group of patients with locoregional relapse there was a a statistically significant lower number of patients expressing MAGE-A4 antigen (p=0.006). Furthermore, in all groups MAGE-A4 antigen expression was significantly associated with the NY-ESO-1 antigen expression (p=0,005) but not with tumor size and grade, number of metastatically involved axillary lymph nodes, and the ER and PR status. MAGE-A4 positive patients showed significantly improved survival had the than the MAGE-A4 negative patients (p=0.04). This was not observed regarding the NY-ESO-1 and HER-2 antigen.

Conclusions: The obtained results suggest that the MAGE-A4 antigen might represent a tumor marker of potential prognostic relevance.

Key words: Breast cancer, cancer / testis (C/T) antigens, HER-2 immunoreactivity, immunohistochemistry, MAGE-A4 immunoreactivity, NY-ESO-1 immunoreactivity, tumor antigens.

Introduction

Breast cancer is the most common malignancy in women and its clinical course may vary from indolent and slowly progressive to rapidly metastatic disease. Identification of prognostic and predictive factors that reflect the biology of breast cancer is important for refining our assessment of prognosis and the selection of patients who may benefit from adjuvant and/or systemic therapy. When choosing among prognostic factors suitable for clinical use, it is also important to consider aspects such as their availability, reproducibility, and cost. In routine clinical practice, the standard prognostic factors such as age, menopausal status, tumor size, tumor grade, steroid-hormone receptor status and nodal metastases form the basis for treatment decisions and selection of treatment modalities for individual patients (1-5).

Variability in breast cancer clinical course is undoubtedly related at least in part to tumor cell growth rate and other features such as invasiveness or metastatic potential. Research in molecular biology has identified genes and their products involved in or associated with the malignant cell transformation and behavior. Moreover, expression of some of these molecules, such as p53, Ki-67, nm23, catepsin D, Ep-CAM, HER-2, urokinase-type plasminogen activator and its inhibitor, is usually found to be also associated with the patient’s prognosis. Since it seems that many genes and molecules might be involved in, respectively, malignant transformation and malignant cell behavior, additional other molecules can also be tested as potential prognostic factors (1,2,6-11).

The cancer/ testis (C/T) genes encode tumor associated antigens (TAA) found in various tumors of different histological origins, but not in normal tissues other than testis. Their physiological function is unknown. Peptides derived from these antigens could be used as targets for active immunotherapy. Expression analysis of these genes or of their products in malignancies could also be of potential diagnostic and/or prognostic relevance (12,13). We present here therefore data on the immunohistochemical expression of antigens MAGE-A4 and of NY-ESO 1. Since the HER-2 antigen has a prognostic and predictive role it was also included into our analysis.

Patients and methods

This is a retrospective study which included 81 patients who were diagnosed with the invasive breast cancer without distant metastasis (pT1-3pN0-1M0). Patients were identified retrospectively on the year 2000 from the medical records at the Department of Radiotherapy, University Hospital for Tumors, Zagreb, Croatia. Before the inclusion into the study they all already had primary surgical treatment and adjuvant treatment: radiotherapy in all patients and if indicated systemic treatment (chemotherapy and hormonal therapy).

Patients were selected based on a five year disease (relapse) free period or relaps for which irradiation was used (medical records at the Department of Radiotherapy). Therefore, the year 1995 was chosen as the year for the retrospective identification of patients without breast cancer recurrence (five-years of follow up). For the disease relapse patients having either bone metastases or locoregional relapses were selected. To have approximately a similar number of patients in these two groups (between 20 to 30), additional patients diagnosed in other calendar years were then also included. Locoregional recurrence was defined as the first recognized recurrence in the chest wall or breast or axilla and supraclavicular region. Before reirradiation some of these patients were also treated surgically (reoperation). Patients with bone metastases might also have metastases at other sites. Moreover, they might also have received systemic anticancer treatment. Patients’ survival analysis was performed in the year 2005. Their survival data were also checked in the Croatian national cancer registry and rechecked by making, whenever possible, personal telephone calls to the presumed alive patients. The study protocol was approved by the Ethics Committee of the Hospital.

The following clinical, pathological, and laboratory data were presented and analyzed (Table 1): age, year of diagnosis, type of surgical operation, median time of disease relapse, tumor size, histological grade, axillary node status, Nottingham prognostic index (NPI), estrogen and progesteron receptor positivity, administration of adjuvant chemotherapy or of adjuvant tamoxifen and the patients’ survival.

Pathological examination of primary tumors and axillary lymph nodes was performed in the same hospital at the Department of Pathology. For routine histological analysis resected material was fixed in 10% buffered formalin, embedded in paraffin and stained with haematoxylin and eosin. The histological grade of tumors was determined according to the method by Elston (14). Tumors were divided into three groups on two criteria, regarding their tumor size (0,1-2 cm (pT1), 2,1-5 cm (pT2), more than 5 cm (pT3)) and regarding the ipsilaterally axillary lymph node status (patients without positive lymph node (N0), and positive lymph nodes (N1)) (15). NPI scores were calculated according to Rampault et al. (16): NPI = 0.2 x tumor size (cm) + lymph-node stage (1, 2 or 3) + histological grade (1, 2 or 3), where size is measured in centimeters; lymph node stage 1 is lymph node–negative, stage 2 is one to three positive lymph nodes, stage 3 is ≥ four positive lymph nodes; and the scoring of histologic grade is 1 to 3 (see below). For prognostic considerations, NPI was categorized into three groups: low (good prognosis), NPI ≤ 3.4; intermediate (moderate prognosis), NPI 3.41 to ≤ 5.4; and high (poor prognosis), NPI > 5.4. Concentrations of estrogen and progesteron receptors in tumor cytosol were evaluated by the dextran-coated charcoal assay as described (17). For estrogen receptors (ER) and progesterone receptors (PR), levels of 5 fmol/mg of protein or more and of 10 fmol/mg of protein or more, respectively, were considered to be positive (17).

Adjuvant therapies were based and prescribed according to the University Hospital Treatment Protocol for Breast Cancer (unpublished document for internal usage). Adjuvant radiotherapy consisted of external megavoltage irradiation delivered from the linear accelerator (18). Adjuvant chemotherapy included either the “CMF protocol” (cyclophosphamide, 600 mg/m2 i.v. on day 1; methotrexate, 40 mg/m2 i.v. on day 1; 5-fluorouracil, 600 mg/m2 i.v. on day 1) or the “FAC protocol” (5-fluorouracil, 500 mg/m2 i.v. on day 1; doxorubicine, 50 mg/m2 i.v. on day 1; cyclophosphamide, 500 mg/m2 i.v. on day 1). Cycles were repeated every 3 weeks, 6 cycles in total. Tamoxifen was administered as systemic hormonal therapy (2x10 mg over 5 years) (19).

Immunohistochemistry (IHC)

Expression of MAGE-A4 or of NY-ESO-1 tumor associated cancer/testis antigens in primary breast cancer tissue was studied by using “57B” monoclonal antibody (mab) (20) or “B9.8.1.1” mab (21), respectively. Briefly, tissue sections from paraffin-embedded breast tumor samples (0,5 mm thick) were placed on Silane (3-aminopropyltriethoxysilane, A 3648, Sigma, St Louis, MO, USA) treated microscope glass slides. After deparaffinization the sections were heated in a 800 W household microwave oven at maximum power for 8,5 and 5 minutes in 10 mmol/L citric buffer (pH 6,0) and washed with phosphate buffered saline (PBS; pH 7,2). The sections were H2O2 treated in order to suppress endogenous peroxidase activity. Following an additional PBS wash the sections were also incubated for 20 minutes with 1:10 diluted normal rabbit sera (DAKO X0902, DAKO A/S, Glostrup, Denmark) at room temperature in a humidified chamber to prevent nonspecific immunoglobulin binding. Mab “57B” or mab “B9.8.1.1.”, in the form of undiluted hybridoma supernatant, were applied to them for 90 minutes at room temperature. Specific binding was revealed by using a streptoavidin-biotinylated horseradish peroxidase based detection system (DAKO K 0355, DAKO A/S, Glostrup, Denmark) (22,23).

Immunoreactivity was scored in the following way: 0, no positive tumor cells; +, up to 20% positive cells („mild reaction“); ++, 21 to 50% positive cells („middle strong reaction“); +++, over 50% positive tumor cells (strong reaction). In all specimens non neoplastic cells, such as normal ductal epithelial cell, fibroblasts, etc., were indeed present but were not stained, and thus served as internal negative controls (22,23). In tables, for the sake of simplicity, we presented the immunoreactivity scores only as either “negative” or “positive” which encompasses mild, middle and strong reactions.

DAKO Hercept TestTM kit (FDA-approved reagent) was used for the HER-2 immunohistochemical staining in accordance with producer instructions. Samples with staining intensity score 3+ (standard control slides were included in the Hercept TestTM kit) were considered to be HER-2 positive. When using the HERCEP test scoring system a strong positive reaction implied a complete (diffuse) membrane staining in more than 10% of tumor cells (24).

Statistical Analysis

The complete statistical analysis was performed by the use of the Statistics 6.1 software package (StatSoft, Inc.; Tulsa, USA). The following statistical tests were used: frequency tables, crosstabulation tables, ANOVA (analysis of variance) and survival analysis. The p value of less than 0,05 was considered as statistically significant.

Results

Patients characteristics

The characteristics of the 81 patients studied are listed in Table 1. The patients’ age ranged from 36 to 82 years. The majority of patients were diagnosed with breast cancer in the year 1995 and modified radical mastectomy was the predominant operational procedure. In the group of patients with locoregional relapse the median time of relapse was 18 months (3-49 months), while in the group with bone metastases it was 13 months (2-44 months) (p=0.766). When these three groups of patients were compared regarding the standard clinical parameters no statistically significant difference was found in any parameter except in ER status (p=0.03) and in adjuvant chemotherapy (p=0.00002). In the five relapse-free group of patients there was a significantly higher number of patients with the positive ER. Moreover, this group also received significantly less adjuvant chemotherapy. This group of patients (as expected) had also significantly better survival outcome (p(0.00000). After the ten years of follow up (the year 2005.) 14 out of 23 patiens were alive (61%; 2 patients with uknown survival status). In the other two groups of patiens only one patien per group was alive.

IHC results

Within our panel of monoclonal antibodies, the mab “57B” (20) recognizes a number of related MAGE-A gene products, including MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, and MAGE-A12 (25). In paraffin-embedded specimens however it has been shown to predominantly recognize MAGE-A4 TAA (26). The mab “B9.8.1.1” is specific to NY-ESO-1 TAA (21) and the monoclonal antibody in the DAKO Hercept TestTM kit is FDA approved as the specific reagent for the HER-2 detection. Representative examples of immunohistochemical staining obtained with these mab are presented in Figure 1. Immunohistochemical staining of cancer-testis protein, MAGE-A4 and NY-ESO-1 is predominatly visible as cytoplasmatic staining limited to tumor cells. HER-2 staining is visible as HER-2 staining is visible as membrane staining in normal and tumor cells. HER-2 positivity is based in relation to the percentage of tumor cells and intensity of membrane staining. The scoring system was the following: negative reaction (-) - up to 10% of tumor cells with negative or weak membrane staining, weak reation (+) - more than 10% tumor positive cells but partial membrane staining, moderate reaction (++) - more than 10% tumor positive cells with moderate membrane staining and strong reaction (+++) - strong positive completly membrane staining in more than 10% of tumor cells. The results from the immunohistochemical expression of MAGE-A4, NY-ESO-1 C/T and HER-2 protein in the three analyzed groups of patients are presented in table 2. The overall positivity of mab “57” and of mab “B9.8.1.1” was 60/81 (74,0%) and 32/81 (40%), respectively. A statistically significantly lower expression of MAGE-A4 antigen was detected in the group of patients with locoregional relapse (p=0.013). No significant difference was observed when these three groups of patients were compared for NY-ESO-1 antigen expression. Positive HER-2 reaction was found in 18/81 patients (22.2% ) with equal distribution in all three groups.

Survival of patients was also analyzed in the relation to the expression of MAGE-A4, NY-ESO-1 and of HER-2 antigen. It was found that MAGE-A4 positive patients had significantly better survival than MAGE-A4 negative patients (p =0.04569; figure 2). In contrast NY-ESO-1 and HER-2 antigen expression was not correlated with survival (data / figures not shown).

Relation of MAGE-A4 antigen to the standard prognostic and predicitive factors

Since the three groups of breast cancer patients analyzed differed significantly in MAGE-A4 antigen expression, the relationship between MAGE-A4 antigen and standard prognostic and predictive factors was analyzed (Table 3). MAGE-A4 antigen expression was found to be associated to a significant degree only with the NY-ESO-1 antigen expression (p=0,006), but not with tumor size and grade, number of metastatically involved axillary lymph nodes, or with ER and PR status. Such results suggest that in our analyzed sample the MAGE-A4 antigen might behave as a prognostic factor unrelated to the above standard prognostic and predictive factors.

Discussion

Breast cancer biology is complex, with multiple factors contributing to breast cancer development, tumor growth, and metastatic progression. The clinical data of the follow-up and studies of the biology of breast carcinoma can be used, for example, in order to identify parameters which could serve as prognostic or predictive factors. Decision making is usually based on a combination of clinical and tumor characteristics, such as age, tumor size and histology (type, grade), lymph-node and ER and PR status (1-5). However, since the prognostic value of these criteria is imperfect, it is apparent that additional and still unidentified molecular factors influence and determine the clinical course of the breast cancer disease. By identifying these additional factors therapeutic approaches to patients with breast cancer could be further individualized thus increasing both their survival rate and quality of life. Novel high-performance screening methods, such as the DNA microarray, analyzing simultaneously in a single experiment the expressions of thousands of genes in a tissue may allow the identification of disease subsets that correlate with clinical outcomes. Clearly such gene-expression profiling (holistic approach) will provide highly useful prognostic information but at the moment there is still no routine clinical use of this new technology as of yet (6-11).

Since detection of MAGE-A4 and NY-ESO-1 antigen expression is based on IHC expression, as a control IHC detection of HER-2 antigen expression was employed. HER-2 molecule belongs to a family of four homologous receptors involved in the tyrosine kinase-mediated regulation of normal breast tissue growth and development. Overexpression of HER-2 molecule in breast cancer cells was associated with poor prognosis (7,8, 24). C/T TAA antigens were discovered in the 1990s, initially as targets in CD8 T cell recognition of autologous human melanoma cells (27). To date, 44 C/T gene have been identified and their expression has been studied in numerous cancer types. Briefly, they code for products with the following characteristics: (i) mRNA expression in normal tissues appears to be restricted to testis, fetal ovary, and placenta. (ii) mRNA expression in cancers of diverse origin is common - up to 30 - 40% of a number of different cancer types, e.g., melanoma, bladder cancer, sarcoma express one or more C/T antigens. (iii) The X chromosome codes for the majority of C/T antigens, but a number of more recently defined C/T coding genes have non-X chromosomal loci. (iv) The function of most C/T antigens is unknown, although some role in regulating gene expression appears likely. One possibility to account for the aberrant C/T expression in cancer relates to the global demethylation associated with certain cancers. Another important issue is whether expression of these genes in the cancer cell contributes to its malignant behavior. (v) There is increasing evidence that C/T expression is correlated with tumor progression and takes place in tumors of higher malignant potential (28-30)

Regarding the expression of C/T genes, they have mostly been studied in clinical materials at the gene expression level by polymerase chain reaction (PCR). This technology does not distinguish if the analyzed genes are expressed in low percentages of tumor cells or in majority (preferably all) tumor cells. Considering possible clinical immunotherapy studies and trials they should be performed and/or directed against antigens expressed in majority (preferably all) tumor cells. Therefore, studies where it is possible to quantify tumor cells expressing tumor antigens are of therapeutic relevance. Owing to the development of serological reagents (mabs) against C/T TAA this become also possible and this can be performed, for example, by immunohistochemical studies (29,30).

In breast cancers expression of MAGE genes has been reported by several groups (31-33). In particular MAGE-A1, -A2, -A3, -A4, -A6 and -A12 specific transcripts have been identified (34). Regarding immunodetection much less is published (29). Results from Kavalar et al. (22) indicate a correlation between the mab “57B” staining and the tumor grade, lymphatic vessel invasion and intratumoral necrosis and an inverse correlation with ER staining.

The present retrospective study shows the results obtained by analyzing the expression of C/T antigens MAGE and NY-ESO-1 and HER-2 in the three groups of breast cancer patients. Initially they all underwent radical surgical treatment and adjuvant radiotherapy. According to the clinical course of their disease (as evident from their medical records from the Department of Radiation Oncology) it was possible, by taking into account tumor relapse and the site of tumor relapse, to identify three groups of patients. One group of patients was still disease-free at the time of study initiation (five years disease free group) while the other two groups consisted of patients who had in the meantime either developed locoregional breast disease relapse or bone metastatic disease. The three groups of patients did not show a statistically significant difference between themselves in the following analyzed parameters: age, tumor size, tumor grade, number of metastatically involved axillary lymph nodes, NPI, PR status, in NY-ESO-1 or HER-2 IHC expression. Differences appear, however, in the ER status, adjuvant systemic therapy and MAGE-A4 expression. In the group of patients with locoregional disease relapse there was a significantly lower number of patients with positive ER (p=0.03). Also, fewer patients from this group showed MAGE-A4 antigen IHC staining (p=0.013). No association was found between the MAGE-A4 IHC expression and standard prognostic and predictive markers: tumor size, lymph node status, tumor grade, ER and PR status and HER-2 expression. A link with the NY-ESO-1 IHC expression was found. When the data regarding the survival were compared with antigen expression a significant difference in the survival was observed between MAGE-A4 positive and MAGE-A4 negative patients. Knowing the prognostic and predictive role of the HER-2 antigen one would expect to observe a similar difference for the HER-2 antigen (7,8,24), but this was not detected. Such data suggest the possibility of MAGE-A4 having a prognostically independent relevance in breast cancer along with traditional prognostic factors. However, our study presented a retrospective cohort design and the number of patients analyzed was relatively small. Therefore, potential differences in unknown factors in groups under investigation may lead to erroneous results or conclusions. Also, there was a difference in the adjuvant chemotherapy. Such findings indicate that the physicians in charge considered that some patients had a relatively higher risk of a breast relapse not obviously resulting from the patients’ medical records. Accordingly, the possibility that the MAGE-A4 antigen might have a prognostic role should be examined by tests covering a much greater number of breast cancer patients.

Acknowledgements

This work was partially supported by the Ministry of Science and Technology of the Republic of Croatia (grants no. 074001 and 074004 to AJ) and the Swiss National Fund for Scientific Research (grant no. 31-57473.99 to GCS).

References

1. Chang JC, Hilsenbeck SG. Prognostic and predictive markers. In: Harris JR, Lippman ME, Morrow M, Osborne CK, editors. Diseases of the breast. 3rd edit. Philadelphia (PA, USA): Lipponcott Williams and Wilkins; 2004. p. 675-96.

2. Goldhirsch A, Wood WC, Gelber RD, Coates AS, Thurlimann B, Senn H-J. Meeting highlights: updated international expert consensus on the primary therapy of early breast cancer. J Clin Oncol 2003; 21:3357-65.

3. Colozza M, Cardoso F, Sotiriou C, Larsimont D, Piccart MJ. Bringing molecular prognosis and prediction to the clinic. Clin Breast Cancer 2005;6:61-76.

4. Keen JC, Davidson NE. The biology of breast carcinoma. Cancer. 2003;97(3 Suppl):825-33.

5. Cianfrocca M, Goldstein LJ. Prognostic and predictive factors in early-stage breast cancer. Oncologist 2004;9:606-16.

6. Roukos DH, Pavlidis N, Agnantis NJ. Gene-expression profile: the future in the outcome prediction and treatment of breast cancer. Gastric Breast Cancer 2003;2:5-8.

7. Ross JS, Linette GP, Stec J, Clark E, Ayers M, Leschly N, Symmans WF, Hortobagyi GN, Pusztai L. Breast cancer biomarkers and molecular medicine. Expert Rev Mol Diagn 2003;3: 89-101.

8. Esteva FJ, Hortobagyi GN. Prognostic molecular markers in early breast cancer. Breast Cancer Res 2004, 6:109-18.

9. Van de Vijver MJ. Microarray analysis of human breast cancer. U: Harris JR, Lippman ME, Morrow M, Osborne CK, editors. Diseases of the breast. 3rd edit. Philadelphia (PA, USA): Lipponcott Williams and Wilkins; 2004. p. 433-9.

10. van't Veer LJ, Paik S, Hayes DF. Gene expression profiling of breast cancer: a new tumor marker. J Clin Oncol 2005;23:1631-1635.

11. Sorlie T. Molecular portraits of breast cancer: tumour subtypes as distinct disease entities. Eur J Cancer 2004;40:2667-75.

12. Bolli M, Kocher T, Adamina M, Guller U, Dalquen P, Haas P, Mirlacher M, Gambazzi F, Harder F, Heberer M, Sauter S, Spagnoli GC. Tissue microarray evaluation of melanoma antigen E (MAGE) tumor-associated antigen expression: potential indications for specific immunotherapy and prognostic relevance in squamous cell lung carcinoma. Ann Surg 2002;236:785–93.

13. Kocher T, Zheng M, Bolli M, Simon R, Forster T, Schultz-Thater E, Remmel E, Noppen C, Schmid U, Ackermann D, Mihatsch MJ, Gasser T, Heberer M, Sauter S, Spagnoli GC. Prognostic relevance of Mage-A4 tumor antigen expression in transitional cell carcinoma of the urinary bladder: a tissue microarray study. Int J Cancer 2002;100:702–5.

14. Elston CW. Grading of invasive carcinoma of the breast. In: Page DL, Anderson TJ, ed. Diagnostic hystopathology of the breast. Edinburgh: Churchill Livingston, 1987:300-11.

15. National Cancer Institute. Breast cancer. CancerNet, PDQ - treatment -health professionals, date last modified 02/01/2005. Available: http: // nci. / cancer info / pdq / treatment / breast / health professional/. Date of access May 23rd, 2005.

16. Rampaul RS, Pinder SE, Elston CW, Ellis IO. Prognostic and predictive factors in primary breast cancer and their role in patient management: The Nottingham Breast Team. Eur J Surg Oncol. 2001;27:229-38.

17. Brdar B, Graf D, Padovan R, Nola P, Rudan N, Petrinec Z, Sarcevic B, Belicza M. Estrogen and progesterone receptors as prognostic factors in breast cancer. Tumori 1988;74:45-52.

18. Juretic A, Zivkovic M, Samija M, Bagovic D, Purisic A, Viculin T, Bistrovic M, Stanec M, Juzbasic S, Lesar M, Tomek R. Radiotherapy of stage IEA primary breast lymphoma: case report. Croat Med J 2002; 43: 569-72.

19. Jardines L, Haffy BG, Doroshow JH. Stage II breast cancer. In: Pazdur R et al, eds. Cancer management: a multidisciplinary approach, 8th ed. Melville, NY: PRR, 2004:205-14.

20. Kocher T, Schultz-Thater E, Gudat F, Schaefer C, Casorati G, Juretic A, Williman T, Harder F, Heberer M, Spagnoli G C. Identification and intracellular location of MAGE-3 gene product. Cancer Res 1995;55:2236-9.

21. Schultz-Thater E, Noppen C, Gudat F, Dürmüller U, Zajac P, Kocher T, Heberer M, Spagnoli GC. NY-ESO-1 tumour associated antigen is a cytoplasmic protein detectable by specific monoclonal antibodies in cell lines and clinical specimens. Br J Cancer 2000;83:204–8.

22. Kavalar R, Sarcevic B, Spagnoli G, Separovic V, Samija M, Terraciano L, Heberer M, Juretic A. Expression of MAGE tumor associated antigens is inversely correlated with tumor differentiation in invasive ductal breast cancers: an immunohistochemical study. Virchows Archiv 2001;439:127-31.

23. Sarcevic B, Spagnoli GC, Terraciano L, Schultz-Thater E, Heberer M, Gamulin M, Krajina Z, Oresic T, Separovic R, Juretic A. Expression of cancer/testis tumor associated antigens in invasive squamous cell cervical carcinoma. Oncology 2003;64:443-9.

24. Ross JS, Fletcher JA, Bloom KJ, Linette GP, Stec J, Symmans WF, Pusztai L, Hortobagyi GN. Targeted therapy in breast cancer: the HER-2/neu gene and protein. Mol Cell Proteomics 2004;3:379-98.

25. Van Baren N, Brasseur F, Godelaine D, Hames G, Ferrant A, Lehmann F, André M, Ravoet C, Doyen C, Spagnoli GC, Bakkus M, Thielemans K, Boon T. Genes encoding tumor-specific antigens are expressed in human myeloma cells. Blood 1999;94:1156–64.

26. Landry C, Brasseur F, Spagnoli GC, Marbaix E, Boon T, Coulie P, Godelaine D. Monoclonal antibody 57B stains tumor tissues that express gene MAGE-A4. Int J Cancer 2000;86:835–41.

27. van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B, Knuth A, Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991;254:1643-7.

28. Old LJ. Cancer/Testis (CT) antigens - a new link between gametogenesis and cancer. Cancer Immunity 2001;1:1-7.

29. Juretic A, Spagnoli GC, Schultz-Thater E, Sarcevic B. Cancer/Testis tumour associated antigens: immunohistochemical detection with monoclonal antibodies. Lancet Oncol 2003; 3:104-9.

30. Scanlan MJ, Simpson AJ, Old LJ. The cancer/testis genes: review, standardization, and commentary. Cancer Immun. 2004;4:1-15.

31. Brasseur F, Marchand M, Vanwijck R, Herin M, Lethe B, Chomez P, Boon T. Human gene MAGE-1, which codes for a tumor-rejection antigen, is expressed by some breast tumors. Int J Cancer 1992;52:839-41.

32. Wascher RA, Bostick PJ, Huynh KT, Turner R, Qi K, Giuliano AE, Hoon DS. Detection of MAGE-A3 in breast cancer patients' sentinel lymph nodes. Br J Cancer 2001;85:1340-6.

33. Mashino K, Sadanaga N, Tanaka F, Yamaguchi H, Nagashima H, Inoue H, Sugimachi K, Mori M. Expression of multiple cancer-testis antigen genes in gastrointestinal and breast carcinomas. Br J Cancer 2001;85:713-20.

34. Otte M, Zafrakas M, Riethdorf L, Pichlmeier U, Loning T, Janicke F, Pantel K. MAGE-A gene expression pattern in primary breast cancer. Cancer Res 2001;61:6682-7.

Table 1. Patients characteristics.

| | | | | | |

|Characteristics | |Breast cancer |patients | |p; (2 x / Fxx |

| |Five years relapse |Locoregional relaps |Bone metastases |Total (n=81) | |

| |free (n=23) |(n=30) |(n=28) | | |

| | | | | | |

|Age at diagnosis (years) | | | | | |

|30-39 |0 |3 |0 |3 | |

|40-49 |6 |7 |7 |20 | |

|50-59 |7 |7 |7 |21 | |

|60-69 |6 |10 |12 |28 | |

|70-79 |3 |2 |1 |6 | |

|80-89 |1 |1 |1 |3 | |

| | | | | |0,62 (2=8,06 |

|Year of diagnosis |1995x23 |1992x1, 1993x2 |1995x9, 1997x10 | | |

| | |1994x8, 1995x15 |1998x7, 1999x2 | | |

| | |1996x2, 1997x2 | | | |

| | | | | | |

|Type of operation | | | | | |

|Mastectomy with axillary |20 |30 |26 |76 | |

|dissection | | | | | |

|Segmentectomy with axillary|3 |0 |2 |5 | |

|dissection | | | | | |

| | | | | |0,14 (2=3,89 |

|Year 2000. - median time of| | | | | |

|disease relapse | | | | | |

|(months, range in |/ |18 (3 – 49) |13 (2 – 44) | |(0,00000 F=30,348 |

|parenthesis) | | | | | |

| | | | | | |

|Tumor size | | | | | |

|5 cm (T3) |0 |3 |2 |5 | |

| | | | | |0,19 (2=5,99 |

| | | | | | |

|Histologic grade | | | | | |

|I |4 |3 |1 |8 | |

|II |13 |18 |16 |47 | |

|III |6 |9 |11 |26 | |

| | | | | |0,52 (2=3,22 |

|Axillary nodal status | | | | | |

|0 |0 |4 |2 |6 | |

|1-3 |14 |10 |12 |36 | |

|≥ 4 |9 |16 |14 |39 | |

| | | | | |0,20 (2=5,94 |

|Nottingham prognostic index| | | | | |

|(NPI) | | | | | |

|NPI I |1 |3 |1 |5 | |

|NPI II |14 |11 |16 |41 | |

|NPI III | 8 |16 |11 |35 | |

| | | | | |0,38 (2=6,37 |

|Estrogen receptor | | | | | |

|negative |10 |22 |21 |53 | |

|positive (≥5 fmol/mg |13 |8 |7 |28 | |

|protein) | | | | | |

| | | | | |0,03 (2=6,86 |

|Progesterone receptor | | | | | |

|negative |8 |15 |8 |31 | |

|positive (≥10 fmol/mg |15 |15 |20 |50 | |

|protein) | | | | | |

| | | | | |0,22 (2=2,98 |

|Adjuvant chemoterapy |7 |23 |25 |55 | |

| |CMFx6, FACx1 |CMFx20, FACx3 |CMFx16, FACx9 | | |

| | | | | |0,00002 (2=21,74 |

|Adjuvant tamoxifen |12 |10 |12 |34 | |

| | | | | |0,38 (2=1,91 |

|Five-years survival | | | | | |

|Alive |23 |7 |2 |32 | |

|Dead |- |18 |22 |40 | |

|Uknown |- |5 |4 | 9 | |

| | | | | |(0,00000 F=43,122 |

|Ten-years survival | | | | | |

|Alive |14 |1 |1 |16 | |

|Dead |7 |24 |23 |54 | |

|Uknown |2 |5 |4 |11 | |

| | | | | |(0,00000 (2=34,45 |

| | | | | | |

x (2= Pearson's Chi-Square Test

xx F= F-test within Analysis of variance (ANOVA) test

Table 2. HER-2, MAGE-A-4 and NY-ESO-1 expression, as detected by immunohistochemistry in patients without breast cancer relapse, with locoregional relapse and with bone metastases.

| | | | | | |

|Immunohistochemistry |Breast cancer |patients | | |p; (2 x |

|staining |Five years relapse |Locoregional relapse |Bone metastases |All | |

| |free | | | | |

| |(n=23) |(n=30) |(n=28) | | |

| | | | | | |

|HER-2 | | | | | |

|negative (0,1+,2+) |19 (83%) |23 (77%) |22 (79%) |64 (79%) | |

|positive (3+) | 4 (17%) | 7 (23%) | 6 (21%) |17 (21%) | |

| | | | | |0,87 (2=0,28 |

|MAGE-A4 | | | | | |

|negative (0) | 2 (9%) |13 (43%) | 6 (21%) |21 (26%) | |

|positive (1+,2+,3+) |21 (93%) |17 (57%) |22 (79%) |60 (74%) | |

| | | | | |0,013 (2=8,58 |

|NY-ESO-1 | | | | | |

|negative (0) |13 (57%) |22 (73%) |14 (50%) |49 (60%) | |

|positive (1+,2+,3+) |10 (43%) | 8 (27%) |14 (50%) |32 (40%) | |

| | | | | |0,17 (2=3,51 |

| | | | | | |

x (2= Pearson's Chi-Square Test

Table 3. Relationship of MAGE-A4 expression, as detected by immunohistochemistry, with tumor size, histologic grade, axillary lymph node status, estrogen and progesteron receptor positivity and with HER-2 and NY-ESO-1 expression.

| | | | | |

| |Breast cancer |patients | |p; (2 x |

| |MAGE-A4 | | | |

| |negative (0) |positive (1+,2+,3+) |all | |

| | | | | |

|Tumor size | | | | |

|T1 |8 |19 |27 | |

|T2 |11 |38 |49 | |

|T3 |2 |3 |5 | |

| | | | |0,60 (2=1,01 |

|Histologic grade | | | | |

|I |2 |6 |8 | |

|II |13 |34 |47 | |

|III |6 |20 |26 | |

| | | | |0,91 (2=0,18 |

|Axillary lymph nodes | | | | |

|negative |3 |3 |6 | |

|positive |18 |57 |75 | |

| | | | |0,36 (2=1,99 |

|Estrogen receptors | | | | |

|ER- |17 |36 |53 | |

|ER+ |4 |24 |28 | |

| | | | |0,08 (2=3,02 |

|Progesteron receptors | | | | |

|PR- |10 |21 |31 | |

|PR+ |11 |39 |50 | |

| | | | |0,30 (2=1,04 |

|HER-2 | | | | |

|negative |19 |44 |63 | |

|positive |2 |16 |18 | |

| | | | |0,13 (2=2,25 |

|NY-ESO-1 | | | | |

|negative |18 |31 |49 | |

|positive |3 |29 |32 | |

| | | | |0,006 (2=7,54 |

| | | | | |

x (2= Pearson's Chi-Square Test

Figure 1. Immunohistochemical staining in ductal invasive breast cancer tissue, non otherwise specified

A Intense, cytoplasmic MAGE-A4 staining with 57mab observed in the absence of staining of normal ducts (PAP 400x).

B NY-ESO-1 positivity with specific cytoplasmic tumor distribution detected by B9.8.1.1 mab (PAP 400x).

C Overexpression of HER-2 detected by HERCEP test with strong positive, completly membrane staining in more than 10% of tumor cells noticed as strong immunohistochemical reaction (PAP 400x).

Figure 2. Patient’ survival (abscise in days) in correlation with MAGE-A4 expression.

[pic]

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