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
1
Department for Medical Statistics, Epidemiology and Medical Informatics, Andrija Stampar
School of Public Health, Medical School, University of Zagreb, Zagreb, Croatia
2
Clinical Hospital Center „Zagreb“, Zagreb, Croatia
3
Department 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
1
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.
2
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
3
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
4
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; 5fluorouracil, 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
5
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
6
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
7
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
8
molecule belongs to a family of four homologous receptors involved in the tyrosine kinasemediated 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 nonX 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 (3133). 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
9
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
10
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).
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13
Table 1. Patients characteristics.
Characteristics
p; 2 x / Fxx
Five years relapse
free (n=23)
Breast cancer
Locoregional
relaps (n=30)
patients
Bone metastases
(n=28)
Total (n=81)
Age at diagnosis (years)
30-39
40-49
50-59
60-69
70-79
80-89
0
6
7
6
3
1
3
7
7
10
2
1
0
7
7
12
1
1
3
20
21
28
6
3
Year of diagnosis
1995x23
1992x1, 1993x2
1994x8, 1995x15
1996x2, 1997x2
1995x9, 1997x10
1998x7, 1999x2
20
30
26
76
3
0
2
5
Type of operation
Mastectomy with axillary
dissection
Segmentectomy with
axillary dissection
0,62 2=8,06
0,14 2=3,89
Year 2000. - median time
of disease relapse
(months, range in
parenthesis)
/
18 (3 – 49)
0,00000
13 (2 – 44)
F=30,348
Tumor size
<2 cm (T1)
2-5 cm (T2)
>5 cm (T3)
7
16
0
7
20
3
13
13
2
27
49
5
Histologic grade
I
II
III
4
13
6
3
18
9
1
16
11
8
47
26
Axillary nodal status
0
1-3
≥4
0
14
9
4
10
16
2
12
14
6
36
39
0,19 2=5,99
0,52 2=3,22
0,20 2=5,94
Nottingham prognostic
index (NPI)
NPI I
NPI II
NPI III
1
14
8
3
11
16
1
16
11
5
41
35
0,38 2=6,37
Estrogen receptor
negative
positive (≥5 fmol/mg
protein)
10
13
22
8
21
7
53
28
0,03 2=6,86
Progesterone receptor
negative
positive (≥10 fmol/mg
protein)
Adjuvant chemoterapy
8
15
15
15
8
20
31
50
0,22 2=2,98
7
CMFx6, FACx1
23
CMFx20, FACx3
25
CMFx16, FACx9
55
0,00002
2=21,74
Adjuvant tamoxifen
12
10
12
34
Five-years survival
Alive
Dead
Uknown
23
-
7
18
5
2
22
4
32
40
9
0,38 2=1,91
0,00000
F=43,122
Ten-years survival
14
Alive
Dead
Uknown
14
7
2
1
24
5
1
23
4
16
54
11
0,00000
2=34,45
x
2= Pearson's Chi-Square Test
xx
F= F-test within Analysis of variance (ANOVA) test
15
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.
p; 2 x
Immunohistochemistry Breast cancer
patients
staining
Five years
relapse free
(n=23)
Locoregional
relapse
(n=30)
Bone
metastases
(n=28)
All
19 (83%)
4 (17%)
23 (77%)
7 (23%)
22 (79%)
6 (21%)
64 (79%)
17 (21%)
HER-2
negative (0,1+,2+)
positive (3+)
0,87
2=0,28
MAGE-A4
negative (0)
positive (1+,2+,3+)
2 (9%)
21 (93%)
13 (43%)
17 (57%)
6 (21%)
22 (79%)
21 (26%)
60 (74%)
0,013
2=8,58
NY-ESO-1
negative (0)
positive (1+,2+,3+)
13 (57%)
10 (43%)
22 (73%)
8 (27%)
14 (50%)
14 (50%)
49 (60%)
32 (40%)
0,17
2=3,51
x
2= Pearson's Chi-Square Test
16
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
MAGE-A4
negative (0)
Tumor size
T1
T2
T3
8
11
2
p; 2 x
patients
positive
(1+,2+,3+)
all
19
38
3
27
49
5
0,60 2=1,01
Histologic grade
I
II
III
2
13
6
6
34
20
8
47
26
0,91 2=0,18
Axillary lymph nodes
negative
positive
3
18
3
57
6
75
0,36 2=1,99
Estrogen receptors
ERER+
17
4
36
24
53
28
0,08 2=3,02
Progesteron receptors
PRPR+
10
11
21
39
31
50
0,30 2=1,04
HER-2
negative
positive
19
2
44
16
63
18
0,13 2=2,25
NY-ESO-1
negative
positive
18
3
31
29
49
32
0,006 2=7,54
x
2= Pearson's Chi-Square Test
17
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).
18
Figure 2. Patient’ survival (abscise in days) in correlation with MAGE-A4 expression.
Cumulative Proportion Surviving (Kaplan-Meier)
Complete
Censored
1,0
0,9
0,8
0,7
0,6
0,5
0,4
0,3
Cumulative Proportion Surviving
0,2
0,1
0,0
0
500
1000
1500
2000
2500
3000
3500
4000
pos (1+,2+3+)
neg (0+)
Time
19