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

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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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