Apr 27, 2006 - patients. Subsequently, Berger et al7 reported that. HER-2/neu protein ..... Lipton A, Ali SM, Leitzel K, Demers L, Harvey HA,. Chaudri-Ross HA.
Indian J Med Res 125, February 2007, pp 137-142
Clinical utility of serum human epidermal receptor-2/neu detection in breast cancer patients Thriveni K., Vijayalakshmi Deshmane*, P.P. Bapsy**, Lakshmi Krishnamoorthy & Girija Ramaswamy
Departments of Biochemistry, *Surgical Oncology, **Medical Oncology, Kidwai Memorial Institute of Oncology, Bangalore, India
Received April 27, 2006
Background & objectives: In breast cancer, the HER-2/neu gene is amplified in 20-30 per cent of cases. The mechanism by which the amplification/overexpression occurs is not known. Elevated serum HER-2/neu levels have been shown to be associated with a poor clinical prognosis and decreased survival in early stage breast cancer patients, and thus might help in management of the disease. The present study was therefore to estimate the serum HER-2/neu levels in breast cancer patients and associate with other prognostic factors. Methods: Serum HER-2/neu levels were studied in 207 patients with cancer breast, 15 benign breast diseases (BBD) and 175 age-matched healthy controls. Patients’ age, menopausal status, node and hormone receptor status were compared with serum HER-2/neu levels. Results: Serum HER-2/neu overexpression was associated with age, disease stage and positive nodal status but not with menopausal status. Serum HER-2/neu levels were negatively related with hormone receptor positivity. Interpretation & conclusion: HER-2/neu serum test could be done more frequently in women with breast cancer irrespective of the hormone receptor status, to suggest modifications in systemic adjuvant therapy, including possibly the use of Herceptin.
Key words Benign breast disease - breast cancer - HER-2/neu - hormone receptor
called p185HER-2) is derived from human epidermal growth factor receptor, as it shows substantial homology with the epidermal growth factor receptor (EGFR)2,3. HER-2/neu gene amplification has been found to be associated with the development of breast
The human epidermal growth factor receptor-2 (Her-2/neu or c-erbB2) gene is a proto-oncogene mapped on chromosome 17q and encodes a transmembrane tyrosine kinase growth factor receptor 1. The name for the HER-2 protein (also 137
138
INDIAN J MED RES, FEBRUARY 2007
cancer in animal models2. The HER-2/neu protein is a component of a four-member family of closely related growth factor receptors, including EGFR or HER-1 (erb-B1); HER-2 (erb-B2); HER-3 (erb-B3) and HER-4 (erb-B4)4. The full-length glycoprotein has a molecular mass of 185, 000 Daltons (p185) and is composed of the internal tyrosine kinase domain, a short transmembrane portion, and an extracellular domain (ECD) that is similar to the three other members of the HER family5. Slamon et al 6 found that HER-2/neu gene amplification determined by Southern blotting analysis independently predicted time to disease relapse and overall survival in breast cancer patients. Subsequently, Berger et al 7 reported that HER-2/neu protein overexpression determined by immuno-histochemistry correlated with lymph node status and breast cancer tumour grade. However, immunohistochemical study by van de Vijver et al 8 failed to show a significant association of HER-2/neu protein immunostaining with disease outcome. In 1990, Heintz et al 9 reported that the HER-2/neu gene copy number by Southern blotting analysis correlated with mitotic activity and negative estrogen and progesterone receptor status but could not link gene expression with disease outcome. HER-2/neu protein overexpression was first reported in in situ breast cancer by van de Vijver et al 8 . HER-2 gene amplification and/or protein overexpression of c-erbB2 may contribute to transformation and tumourigenesis of cancer. HER2 has been extensively studied in breast cancer, and approximately 20-30 per cent of patients have tumours that overexpress this receptor often as a result of gene amplification10. The amplification of the HER-2/neu gene is regarded as an established predictive and prognostic marker for breast cancer, particularly for the management of advanced breast cancer11. We undertook this study to evaluate the serum HER-2/neu levels in normal healthy individuals,
individuals with benign breast disease and untreated breast cancer patients, and try to associate these levels with other prognostic factors like age, menopausal status, stage of disease, node and hormone receptor status. Material & Methods Selection of subjects: Patients registered in the Breast service unit of Kidwai Memorial Institute of Oncology, Bangalore, during November 2004-July 2005, were included in the study. Among 256 selected patients, 207 female patients were histologically confirmed breast cancer cases, 15 patients had benign breast disease. The remaining 34 patients were excluded from the study, as they were treated cases. Histologically confirmed invasive carcinoma of the breast in different stages of the disease (207 patients), in the age group of 26-75 yr were included. Age matched (± 2 yr) healthy female controls (175 cases) were arbitrarily selected from patients’ relatives. Information on patients’ age, menopausal status, disease stage, estrogen receptor (ER), progesterone receptor (PR) status, clinical nodes was noted from the case files. Blood samples were collected in plain tubes, centrifuged to separate the serum. The serum samples (0.3 ml) were stored at -20°C until analysis (within a month). The study protocol was approved by the Ethics committee of Kidwai Memorial Institute of Oncology, Bangalore. A written informed consent was obtained from each patient. HER-2/neu levels: The levels of serum HER-2/neu were measured by modified sandwich enzyme immunoassay 12 . An anti - sp185 HER-2 human monoclonal coating antibody (Bender Med System, USA) was adsorbed on to micro wells. sp185 HER-2 present in the sample or standards bound to antibodies adsorbed to the micro wells; a horse radish
THRIVENI et al: SERUM HER-2/NEU LEVELS IN BREAST CANCER PATIENTS
peroxidase (HRP) conjugated monoclonal antisp185 HER-2 antibody was then added to the wells. Following incubation, unbound enzyme conjugated anti - sp185 HER-2 was removed by washing and substrate tetramethylbenzidine (TMB) solution reactive with HRP was added to the wells. A coloured product was formed in proportion to the amount of soluble p185HER-2 present in the sample. The reaction was terminated by addition of sulphuric acid and absorbance was measured at 450 nm. A standard curve was prepared from 10 ng/ml stock standard sp185HER-2 by serially diluting as 5, 2.5, 1.25, 0.625, 0.312 and 0.156 ng/ml with assay buffer and sample concentration determined. Statistical analysis: Data were transformed to natural logarithmic scale; analysis of variance with post-hoc Tukey test were used to compare serum HER-2/neu levels, menopausal status and stage of disease. Student ‘t’ test was used to compare serum HER-2/ neu levels between patients with different age groups, node status and hormone receptor status. Multivariate logistic regression was used to find association of risk factors with the elevated serum HER-2/neu levels. Results & Discussion Among 207 histologically confirmed breast cancer patients, 53 were HER-2/neu positive (25.6%). The cut-off value was 40 yr,
139
node positivity, stage 2 and 3 were positively related to the elevated HER-2/neu levels, and ER and PR positivity were negatively related to the elevated serum HER-2/neu levels (Table II). HER-2/neu was not reported to be elevated in benign breast tissue13, whereas the level of HER-2/ neu oncoprotein elevation in malignant specimens was apparent at all stages, from intraductal to invasive phases of primary breast cancer and to subsequent metastases 14 . Elevated HER-2 ECD concentration was related to pT (pathological tumour size), histological grade, presence of comedo-type carcinoma and tissue HER-2/neu expression15. Interestingly, elevated serum HER-2/neu levels have been shown to be correlated with decreased survival and absence of clinical response to Table I. Levels of serum HER-2/neu in breast cancer patients and relationship with other parameters Study parameters
Subjects (n)
HER-2/neu levels
Age (yr)
40 (155)
16.85 ± 1.82 21.32 ± 3.25*
Menopausal status
Pre (84) Peri (7) Post (116)
16.17 ± 1.77 24.96 ± 13.92 15.34 ± 1.16
Node
Negative (105) Positive (102)
13.32 ± 0.91 21.76 ± 2.36**
Stage +
Control (175) BBD (15) I (14) II (64)# III (105)## IV (24)##
8.07 ± 1.22 8.25 ± 0.52 9.40 ± 0.63 14.05 ± 1.09 18.43 ± 1.79 22.94 ± 5.64
ER
Negative (90) Positive (117)
23.42 ± 2.34 12.04 ± 0.80**
PR
Negative (93) Positive (114)
23.19 ± 2.29 12.02 ± 0.84**
Values are mean ± SE BBD, benign breast disease; ER, estrogen receptor; PR, progesterone receptor P *
