All patients were enrolled in the study after taking written informed consent. The study was conducted after the approval of the Ethics committee of Lady Hardinge Medical College with file no-pgthesis2011.
2.1 STUDY DESIGN AND POPULATION:
A case control study was conducted at our institute which is a tertiary care center located in North India. Sample size was 34 cases and controls each. The “case” definition used in our study was female patient of breast carcinoma who presented to our hospital during the study period and satisfying the inclusion and exclusion criteria. Consecutive sampling technique was used to enroll cases in the study. Controls were selected from healthy individuals who visited our hospital for preventive health checkups. Serum levels of fibrinogen and VEGF were compared between cases and controls. The cohort of cases of the case control study was further studied prospectively to study the effect of treatment on the levels of fibrinogen and VEGF.
2.2 INCLUSION CRITERIA:
- Cases: Female patients who were diagnosed with non metastatic breast carcinoma who have consented to participate in our study were enrolled.
- Controls: Healthy adult female volunteers who visited the hospital for preventive health checkups and consented to participate in the study were enrolled.
2.3 EXCLUSION CRITERIA:
- Cases: Pregnant females were excluded from the study. All metastatic breast carcinoma patients were also excluded. Patients with co-morbidities are excluded as they may interfere with fibrinogen and VEGF values.
- Controls: Individuals with any acute or chronic diseases, disorders of coagulation and present or past history of malignancy were excluded as controls.
All cases underwent diagnostic procedures such as ultrasound breast, mammography and core needle biopsy of the lesion. Tissue specimens were examined for the following characteristics:
- Histological type of tumor
- Histological grade: Modified Scarff-Bloom-Richardson grading was used and grade1 to 3 were assigned according to differentiation of tumor.
- Mitotic index: 40x objective was used to count mitotic figures and points were given as follows: 1 point- 0 to5 mitosis per high power field, 2 points- 6 to10 mitosis per high power field,3 points- >11 mitosis per high power field.
- Estrogen, progesterone and HER-2-neu receptor status: These were ascertained by staining the paraffin sections using monoclonal antibodies to estrogen receptor (ER), progesterone receptor (PR) and HER -2-neu receptor utilizing streptovidin biotin peroxidase method with diaminobenzidine as chromogen after antigen retrieval in citrate buffer at pH 6.0.
Patients with enlarged lymph nodes underwent fine needle aspiration cytology to confirm lymph node metastasis. All cases with symptoms suggestive of systemic metastasis and locally advanced breast carcinoma underwent metastatic workup to rule out metastasis. Blood samples were taken from cases and controls for measurement of fibrinogen and VEGF. All cases later underwent modified radical mastectomy. Two weeks post surgery blood samples were taken from the cases to measure the fibrinogen and VEGF levels.
5 ml of blood was drawn from the antecubital vein of the patients after applying tourniquet to arm with 24 gauge needle. Venous blood was collected in a plain vial. Blood was centrifuged within 30 minutes and serum and plasma were kept at -80 degree Celsius. Fibrinogen and VEGF levels were quantified later by Enzyme-linked immunosorbent assay (ELISA).
2.4.1 Fibrinogen measurement:
Fibrinogen value was calculated by commercially available Human Fibrinogen ELISA kit- AssayMax Human Fibrinogen ELISA Kit (Assaypro, USA, Catalog No. EF1040-1, Lot No. 07251330) which employs a quantitative competitive enzyme immunoassay technique. Murine antibody specific to fibrinogen was pre-coated on to a 96-well microplate. Then standards and specimens were added. This was followed by adding biotinylated fibrinogen and incubation. Then the wells were washed with buffer. Streptavidin-peroxidase conjugated antibody was added and the wells were washed to remove unbound antibody. Peroxide enzyme substrate was added for the colour to develop. The colour development was stopped after adding acidic stop solution and the intensity of colour was measured. Normal assay range by this test was 2.0-3.5 mg/ml.
2.4.2 VEGF measurement:
VEGF value was calculated by commercially available Human VEGF ELISA kit- EIA- 4819/96 (DRG Instruments GmbH,Germany) which employs enzyme immunoassay for the quantitative determination of human VEGF. Murine antibody specific to fibrinogen was pre-coated on to a 96-well microplate. Then standards and specimens were added. This assay employs an antibody specific for human VEGF coated onto 96-well microplate. Biotinylated anti-human VEGF was added later. After washing away unbound biotinylated antibody, horse radish peroxidase-conjugated streptavidin was added to the wells. The wells were again washed. Following this, substrate solution was added to the wells. The colour development was stopped after adding acidic stop solution and the intensity of colour was measured. Normal assay range by this test was approximately700 pg/ml.
2.5 STATISTICAL ANALYSIS:
The sample size was 34 patients and 34controls calculated with a margin of error of 5%. The data acquired was coded and recorded in the MS Excel spreadsheet (Microsoft Office, Microsoft, Washington). Data was analyzed using Statistical Package for Social Sciences (SPSS) version 17.0 (IBM SPSS Statistics, International Business Machines Corporation, New York). Data was normally distributed. Normally distributed continuous variables were compared using the unpaired t test for two groups and ANOVA for three or more groups. Categorical variables were analyzed using either the chi square test or Fisher’s exact test as appropriate. For all statistical tests, a p value of less than 0.05 was considered significant.