correlation of her-2 neu over-expression with clinico pathological features of carcinoma breast
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Correlation of Her-2 neu over-expression with clinico pathological features of carcinoma breast
Research Article
Correlation of Her-2 neu over-expression withclinico pathological features of carcinoma breast
Uma Krishnaswamy*, Premkumar Balachandran, I. Rajakumar,K. Balachandar, Mohamed Mansoor
Department of General Surgery, Apollo (Main) Hospitals, Chennai, Tamil Nadu, India
a r t i c l e i n f o
Article history:
Received 17 August 2012
Accepted 29 October 2013
Available online 27 November 2013
Keywords:
Her-2 neu amplification
Oestrogen receptor
Progesterone receptor
a b s t r a c t
Purpose: To determine the relationship between Her-2 neu over-expression and clinico
pathological features of carcinoma breast.
Methods: 150 patients with carcinoma breast, were assessed for Her-2 neu over-expression
as a prospective study. Oestrogen (ER) and progesterone receptor (PR) status along with
other clinico pathological parameters of tumour size, node status, type and grade of
tumour were assessed.
Results: Her-2 neu over-expression was seen in 32.30% of patients who were ER and PR
negative and in 11.94% of those who were ER and PR positive. It was unrelated to the other
clinico pathological parameters.
Conclusions: Significant correlation between Her-2 neu amplification and ER/PR negative
tumours was found but not with other clinico pathological parameters.
Copyright ª 2013, Indraprastha Medical Corporation Ltd. All rights reserved.
1. Introduction
The classical clinico pathological features such as tumour
size, axillary node status, histological type and grade have a
well-established prognostic role in breast cancer. However,
they do not allow patients to be stratified for appropriate
therapy on an individual basis. Biological markers such as
oestrogen (ER) and progesterone receptors (PR) are of value in
individualizing therapy and have become standard of care.
Similarly, assessment of Her-2 neu over-expression has
become the norm because it provides both prognostic and
therapeutic information.
There is no consensus in literature on the relationship
between Her-2 neu over-expression and the classical clinico
pathological features.1,2 But, nearly all investigators report an
inverse relationship between Her-2 neu status and hormone
receptors in diverse populations.3e5 The purpose of this study
was to verify whether such correlations existed in our pa-
tients. To this end, the association between Her-2 neu over-
expression and oestrogen and progesterone receptor status
(both independently and jointly) and other clinico patholog-
ical features, that is, tumour size, node status, type of tumour
and grade of tumour were studied.
2. Patients and methods
This was a prospective study accumulating 150 breast cancer
patients over a 5 year period from 2004. All specimens of
definitive surgery were fixed in 10% formaldehyde, embedded
* Corresponding author. Flat 1, No 3 (Old 9), Tiruveedi Amman Street, Ramakrishna Nagar, Chennai 600028, India. Tel.: þ91 44 24937926,þ91 98410 33426 (mobile).
E-mail address: umaks@vsnl.com (U. Krishnaswamy).
Available online at www.sciencedirect.com
ScienceDirect
journal homepage: www.elsevier .com/locate/apme
a p o l l o m e d i c i n e 1 0 ( 2 0 1 3 ) 3 1 3e3 1 7
0976-0016/$ e see front matter Copyright ª 2013, Indraprastha Medical Corporation Ltd. All rights reserved.http://dx.doi.org/10.1016/j.apme.2013.10.012
in paraffin, sectioned and stained with Haematoxylin/Eosin.
Pathological staging (pTNM),6 determination of tumour type
as per WHO classification,7 and grading by the Nottingham
system8,9 was done.
ER and PR status were assessed by immuno histochemistry
either on the pre operative core biopsy sample or the surgical
specimen on 4 mm sections of formalin-fixed, paraffin-
embedded tissues. Monoclonal antibody 6F11, (mouse anti
human oestrogen receptor alpha specific product, MCA1799T,
AbD Serotec, UK) was used to detect ER. For detection of PR,
the monoclonal antibody 1A6 (mouse anti human progester-
one receptor specific product MCA1800T, AbD Serotec, UK)
was used. With the use of monoclonal antibody and antigen
retrieval, immuno histochemical reaction for ER and PR is
usually an all-or-none phenomenon. Therefore, quantitation
of results was not done.
HER-2 neu expression was assessed on either the pre
operative core biopsy samples or the surgical specimen on
4 mm sections of formalin-fixed, paraffin-embedded tissues
after an antigen retrieval procedure, (monoclonal antibody to
c-erbB-2 protein, Mouse IgG1, Biogenex). Her-2 neuwas scored
on a 0e3 scale according to the criteria set by Dako.10 No
staining or membrane staining in less than 10% of tumour
cells is score 0 (Her-2 neu negative), faint incomplete mem-
brane staining in more than 10% of the tumour cells is score
1þ (trace negative), a weak to moderate complete membrane
staining in more than 10% of the tumour cells is score 2þ(weak positive) and a strong complete membrane staining
in more than 10% of the tumour cells is score 3þ (strong
positive). A score of 0 or 1þ is interpreted as negative, 3þ is
considered positive for Her-2 neu over-expression.
A score of 2þwas considered borderline and was confirmed
by fluorescence in situ hybridization (FISH) test, which assesses
whether Her-2 neu gene amplification has occurred in breast
cancer cells11 (Religare SRL Diagnostics. Probe: PathVysion�Her-2 DNA Probe Kit, Vysis (Abbott); FDA approved. Image
Analysis method: Metasystems Isis, Carl Zeiss; Germany). The
cut-off point for Her-2 neu amplification is a Her-2/CEP17
(centromere 17) ratio of more than or equal to 2.0 with appro-
priate positive and negative controls run with the test sample.
3. Statistical analysis
All statistical analyses were performed with SPSS software
version 15.1 forWindows (SPSS Inc, Chicago, Illinois, USA) and
Epi Info� (Version 6). The c2 test was used to examine the
association between Her-2/neu status and the various cate-
gorical variables in univariate analysis. p values less than 0.05
were accepted as significant.
4. Results
Table 1 summarises the clinico pathological features of all 150
patients.
Her-2 neu over-expressionwas seen in 35 (23.3%) out of the
150 patients. Themeanage of thepatientswas 51.29 years. The
youngestpatientwas26and theoldest85years.Only7outof 25
patients (28%) who were �age 40 were Her-2/neu positive. In
the 125whowere�41 years, 28 (22.4%)wereHer-2neupositive.
For younger vs. older patients: Odds ratio ¼ 1.35. 95% CI
0.46e3.87. Relative risk ¼ 1.25 (p ¼ 0.7298443 Yates corrected).
Her-2 neu over-expression was present in 2 out of 6
(33.33%) Tis tumours, 5 out of 32 (15.62%) T1 lesions, 15 out of
63 (23.80%) T2 lesions, 11 out of 39 (28.20%) T3 lesions and 2 out
of 10 (20%) T4 lesions. Out of 38 lesions <2 cm in size, 7
(18.42%) were Her-2/neu positive. In 112 lesions >2 cm size, 28
(25%) were Her-2 neu positive. For larger tumours vs. smaller
tumours: Odds ratio ¼ 0.68. 95% CI 0.24e1.84. Relative
risk ¼ 0.74 (p ¼ 0.5441040 Yates corrected).
Her-2 neu over-expressionwas seen in 21 out of 82 (25.60%)
N0 cases, 10 out of 59 (16.94%) N1 cases, 3 out of 8 (37.5%) N2
cases and 1 out of 1 N3 case. A total of 14 patients out of 68
(20.58%) node positive disease (N1, N2 and N3) were Her-2 neu
positive. For node positive vs. node negative disease: Odds
ratio ¼ 1.33. 95% CI 0.58e3.08. Relative risk ¼ 1.24
(p ¼ 0.5961294 Yates corrected).
There were 2 instances of Her-2 neu over-expression out of
6 (33.33%) patients with ductal carcinoma in situ and in 33
instances out of 144 (22.60%) infiltrating carcinomas. For
infiltrating carcinoma vs. in situ lesions: Odds ratio¼ 1.68. 95%
CI 0.20e11.39. Relative risk¼ 1.16 (p¼ 0.6242803 Fisher exact 2-
tailed). The infiltrating duct carcinomas were mainly not
otherwise specified variety (NOS) (78.66%). There were 2 in-
stances of infiltrating lobular carcinoma which did not show
Her-2 neu over-expression.
There was 1 instance of Her-2 neu over-expression out of
10 (10%) grade 1 tumours, 13 out of 67 (19.40%) grade 2 tumours
Table 1 e Clinico pathological features (n [ 150).
Categorical variables Number Percentage
Age
�40 years 25 16.66
�41 years 125 83.33
T stage
Tis 6 4.0
T1 32 21.3
T2 63 42.0
T3 39 26.0
T4 10 6.7
N stage
N0 82 54.7
N1 59 39.3
N2 8 5.3
N3 1 0.7
Type
Infiltrating 144 96.0
In situ 6 4.0
Grade (infiltrating duct cancer)
1 10 7.51
2 67 50.37
3 56 42.10
ER
Positive 82 54.7
Negative 68 45.3
PgR
Positive 70 46.7
Negative 80 53.3
Her-2/neu status
Positive 35 23.3
Negative 115 76.7
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and 18 out of 56 (32.14%) grade 3 tumours. For high grade vs.
lower grade tumours (infiltrating duct carcinomas only): Odds
ratio ¼ 0.47. 95% CI 0.19e1.13. Relative risk ¼ 0.57
(p ¼ 0.0980697 Yates corrected).
Her-2 neuwas amplified in 13 out of a total of 82 (15.85%) ER
positive cases and in 22 out of 68 (32.35%) ER negative cases.
For ER negative vs. ER positive disease: Odds ratio ¼ 0.39. 95%
CI 0.17e0.92. Relative risk ¼ 0.80 (p ¼ 0.0289233 Yates cor-
rected). It was amplified in 9 out of 70 (1.28%) PR positive cases
and in 26 out of 80 (32.5%) PR negative cases. For PR negative
vs. PR positive disease: Odds ratio ¼ 0.31. 95% CI 0.12e0.76.
Relative risk ¼ 0.77 (p ¼ 0.0081887 Yates corrected). Table 2
summarises the frequency of Her-2 neu expression accord-
ing to all of the above independent variables.
When ER and PR status was looked at jointly, it was noted
that out of 67 patients who were both ER and PR positive, 8
(11.94%) were Her-2 neu positive. Out of 65 patients who
were ER and PR negative, 21 (32.30%) were Her-2 neu positive.
Out of 15 patients who were ER positive but PR negative, 5
(33.33%) were Her-2 neu positive. For ER and PR negative vs. ER
and PR positive lesions: Odds ratio ¼ 0.28. 95% CI 0.10e0.76.
Relative risk ¼ 0.37 (p ¼ 0.0089154 Yates corrected). For ER
positive PgR negative lesions vs. ER positive PR positive le-
sions: Odds ratio¼ 3.69. 95% CI 0.84e16.25. Relative risk ¼ 2.79
(p ¼ 0.0552503 Fisher Exact). Table 3 summarises the fre-
quency of Her-2 neu expression according to joint ER/PR
status.
5. Discussion
Classical clinical and morphological prognostic factors, such
as tumour size, axillary node status, histological grade and
type, vascular invasion etc. have a well-established role in the
management of breast cancer.
Advances in the understanding of the molecular and ge-
netic alterations underlying breast cancer development and
progression have resulted in the identification of a great
number of cell biological markers which are of potential value
in individualizing therapy.12,13
The HER-2 neu gene encodes a 185 kDa transmembrane
phosphoglycoprotein with tyrosine kinase activity and is a
member of the human epidermal growth factor receptor gene
family. The HER-2 neu proto-oncogene (also known as c-erbB-
2) is homologous with, but distinct from, the epidermal
growth factor receptor.
Her-2 neu (c-erbB-2) gene amplification, which usually re-
sults in over-expression of the encoded transmembrane pro-
tein, occurs in approximately 15%e30% of invasive breast
cancers.14 In this study, 35 (23.3%) out of 150 patients were
Her-2 neu positive. This figure appears to be within the
commonly accepted rate of occurrence.
It has been suggested that Her-2 neu gene amplification is
more common in younger patients,15 but this study failed to
reveal such a relationship in a statistically significant manner
as this has been the case with other authors as well.16
Tumour size is a valuable predictor of behaviour in breast
cancer. Higher rates of Her-2 neu over-expression in larger
tumours have been documented by some.17 But, this study
shows no such tendency. This is in keeping with other
studies.18
Axillary lymph node status too is a powerful prognosti-
cator. In this study, as well as that of others, there was no
association between Her-2 neu over-expression and axillary
lymph node status.19
Literature suggests that there is generally no correlation
between Her-2 neu status and histological type of cancer.20 A
later study indicated that Her-2 neu over-expression was
more likely in infiltrating duct carcinomas than in infiltrating
lobular carcinomas.21 In this study too, there was no correla-
tion with histological type, but as the number of infiltrating
lobular carcinomas were very small (only 2), when compared
to infiltrating duct carcinomas, no definite conclusions could
be reached.
Her-2 amplification/over-expression in different histologi-
cal grades of breast cancer has been a subject of interest. Most
studies have correlated Her-2 neu over-expression with poor
histological or nuclear grade of the primary tumour22 whereas
others have not.23,24 This study did show a numerically
increasing incidence of Her-2 neu positivity with increasing
tumour grade, but this was not statistically significant.
Table 2 e Frequency of Her-2 neu status and clinicopathological variables.
Categoricalvariables
Her-2 neunegative
Her-2 neupositive
p Value
Number (%) Number (%)
Age
�40 years 18 (72) 7 (28) Not significant
�41 years 97 (77.6) 28 (22.4)
T stage
<2 cm 31 (81.57) 7 (18.43) Not significant
>2 cm 84 (75) 28 (25)
N stage
Node negative 61 (74.39) 21 (25.61) Not significant
Node positive 54 (79.41) 14 (20.59)
Type
In situ 4 (66.66) 2 (33.34) Not significant
Infiltrating 111 (77.08) 33 (22.92)
Grade
Low (1e2) 63 (81.81) 14 (18.19) Not significant
Higher (3) 38 (67.85) 18 (32.15)
ER
Positive 69 (84.15) 13 (15.85) 0.0289233
Negative 46 (67.65) 22 (32.35)
PgR
Positive 61 (98.72) 9 (1.28) 0.0081887
Negative 54 (67.5) 26 (32.5)
Table 3 e Frequency of Her-2 neu status by joint ER/PRexpression.
Categoricalvariables
Her-2 neunegative
Her-2 neupositive
Total
Number (%) Number (%)
ERþ PgRþ 59 (88.05) 8 (11.94) 67
ERþ PgR� 10 (66.66) 5 (33.33) 15
ER� PgRþ 2 (66.66) 1 (33.33) 3
ER� PgR� 44 (6.15) 21 (32.30) 65
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Oestrogen and progesterone receptor studies are estab-
lished procedures in the routine management of breast can-
cer, primarily as predictive factors for response to hormonal
therapy. Despite the great variation in levels of Her-2 neu
positivity, nearly all investigators report a negative relation-
ship between Her-2 neu status and steroid receptors lev-
els.25e27 In this study too, Her-2 neu protein over-expression
was associated with a statistically significant higher rate of ER
and PR negative status.
Interestingly some authors highlight the negative rela-
tionship between PR and Her-2 neu status and that this is seen
after age 45. They found increased Her-2 neu positivity (2.75-
fold increase) in ER positive PR negative tumours compared
with ER positive PR positive tumours.28,29 In this study too a
2.79-fold increase in Her-2 neu positivity was seen in ER pos-
itive, PR negative tumours when compared to ER positive and
PR positive lesions with the mean age of these patients being
48 years. However, no further conclusions could be reached on
an age-related association between Her-2 neu and PR in ER
positive breast cancers because of the limited number of pa-
tients in this study.
In a country such as India, with severe financial con-
straints, while ER testing is offered for establishing hormone
receptor status, Her-2 neu testing is not, because of the crip-
pling cost of Trastuzumab. The trend of a negative relation-
ship between steroid receptors and Her-2 neu amplification/
over-expressionmay be helpful to the clinician in determining
the diagnostic pathway in selected instances where financial
considerations are paramount.
6. Conclusion
The estimation of ER, PR and Her-2 neu in breast cancer is
now standard of care, but the relationship between these and
other traditional prognosticators is still not clear. In this
study of 150 breast cancers, significant correlation between
Her-2 neu amplification and ER/PR negative tumours was
found but such was not so with other clinico pathological
parameters.
Presentation details
Organisation: Association of Surgeons of India, Tamil Nadu &
Pondicherry State Chapter Conference (NAGASICON 2009).
Place: Kanya Kumari, Tamil Nadu.
Date: 2nd August, 2012.
Conflicts of interest
All authors have none to declare.
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