In this cross-sectional study, female patients diagnosed with TNBC who underwent mastectomy at our medical center from 2012 to 2017 comprised the study population.‌ Inclusion criteria comprised living patients 4 weeks after surgery who have completed information of the required clinicopathological factors and given an efficient sample for IHC (immunohistochemistry) and real time PCR (polymerase chain reaction). A data collection checklist was designed and the required variable including age, histopathologic diagnosis, tumor grade, tumor stage and months patient survived after being diagnosed were documented. Eighty-four patients were included. The interval between diagnosis and death or phone call (if they were alive) was considered the survival time. Subsequently, paraffin blocks with sufficient tumoral tissue were taken and two pathologists checked the tumors’ grades. For IHC, 3×4 micron slides were provided and stained by rabbit polyclonal antibody [anti FGFR1 (phospho Y654) antibody cat (No: ab59194, ABCAM, Cambridge, UK). Like similar studies, we have used lung adenocarcinoma tissue as a negative control and lung SCC as a positive control. In terms of the percentage of colorfulness, cells were divided into 5 subsets: under 1%, 1-25%, 25-50%, 50-75%, 75-100% which received scores ranging from 0 to 4, respectively, and the degree of colorfulness was categorized as negative, weak, moderate, severe with scores from 0 to 3. For each sample, the degree of colorfulness was multiplied by the percentage of colorfulness, and the received number was called the FGFR1 expression score for that sample. Therefore, samples were scored from 0 to 12 in terms of FGFR1 expression. Like other studies, scores from 2-12 were considered as positive samples (Figures 1 and 2).

Fig. 1. Severe colorfulness of FGFR1 expression in IHC

Fig. 2. Weak colorfulness of FGFR1 expression in IHC

Below is a brief explanation of IHC steps, similar to other studies and based on a standard process (12): we readied and activated the specimens by placing them in a poly-lysine solution for one hour at 60°C in order to cause the absolute sticking of the tissue on the plate. The tissue was then cut into 3*4 micron slices and put it in an autoclave at 80°C for 45 minutes for fixation. For deparaffination, samples were put in Xylenol for 5 minutes, followed by immersing in ethanol with different percentage (absolute, 90%, 80%, 70%) (5 minutes in each) and then washing in distilled water for hydration. Subsequently, we put the plates in an antigen retrieval buffer in a bain-marie (94-98°C) for 30 minutes, washing them afterwards with distilled water, and then with Tris-Buffered Saline (TBS) in order to delete cross connections, protein was denatured and improving epitopes appearance.
We blocked endogenous peroxidase action by using hydrogen peroxide 5% for 5 minutes at room temperature, then washing for 5 minutes in TBS. In the proprietary antibody attachment step, we put FGFR1 antibody 1/1000 for 90 minutes in the moist dish, and then washed with TBS for 5 minutes. The following step is secondary antibody attachment, in which samples were put for 30 minutes in anti-rat envision horseradish peroxidase antibody, and subsequently washes for 5 minutes with TBS. To see antigen expression, we put samples in Diaminobenzidine which acts as a substrate for peroxidase enzyme, in room temperature for 10 minutes and then we washed samples in TBS for 5 minutes. Finally, after putting samples in non-alcoholic hematoxylin for a few seconds followed by washing, we observed coloring under the microscope. Alcohol (70% up to absolute) and xylenol were respectively used to dehydrate and clarify the samples. At the end, we dried the plate and stuck lamel on it to preserve it.

The section below provides a brief explanation on real time PCR steps as conducted in another similar study (13):
1. DNA extraction from paraffin block of tissue fixed in formalin.
2. Designing primers (direct and reverse) for FGFR1 gene and the control gene, GAPDH.
3. Providing master mix (Tag DNA Polymerase) after primer attachment to single strand DNA proliferates DNA.
4. Providing SYBR Green I, which exclusively bonds double-stranded DNA, fluorescent light is then generated and identified by device.
5. Real-time PCR proliferation of FGFR1 gene by ABI thermal cycler; 1. Denaturation 2. Annealing 3. Polymerization 4. Repetition of these steps up to 40 cycles so that a significant amount of DNA is synthesized.
Real-time PCR steps include:
1. Linear ground phase: until 16^th cycle, the amount of double-stranded DNA is low. Thus, there is no fluorescent light.
2. Early exponential phase: from the 17^th cycle, the number of DNA copies is increased so that the device can identify the fluorescent light from the attached SYBR Green I (Threshold cycle).
3. Log-linear phase: by continuing Real-time PCR cycles, the number of double-stranded DNA is progressively increased, as well as the intensity of fluorescent light.
4. Plateau phase: there is no production of DNA. The emitted fluorescent light will stabilize.
5. Check positive amplification, we use Relative Quantitative Real-time PCR in this study. Therefore, like similar studies, positive samples were considered to be those with fold change >2. (The ratio of double-stranded DNA copies of FGFR1 gene to GAPDH gene copies).
For evaluation of fold changes in each sample deltaCt was calculated for FGFR1 and GAPDH and subsequently;
1. Patients group     
FGFR1 gene Ct = FGFR1 gene Ct – GAPDH gene Ct
2. Control group     
FGFR1 gene Ct = FGFR1 gene Ct – GAPHD gene Ct
Ct  = 1-2
In the Ct  formula, the number 2 is equal to the efficiency of PCR being at the best and the most ideal situation of the reaction (100%). In order to determine PCR efficiency, a series of different dilutions of DNA copies of FGFR1 and GAPDH genes was provided. After the reactions were conducted and the standard curve drawn, the line slope is seen. Efficiency was = [10(-1/slope)]-1; thus, the efficiency for FGFR1 gene and GAPDH gene was 97% and 96% respectively (Table 1).

Table 1. The sequence of direct and reverse primers of FGFR1 and GAPDH genes

Fusion curve:  concerning the usage of SYBR Green I in our study, a fusion curve was drawn for both FGFR1 and GAPDH genes. As seen, this curve has only two peaks, one for FGFR1 and the other for GAPHD. So there is no nonspecific product in our test.

Statistics
For analysis of the correlation among prognostic factors, the Chi-Square test, Student T-test, Fisher's exact test, ANOVA and Cohen's kappa coefficient were used. All analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL., USA). The final sample size was estimated to be 84 with a 30% prevalence of amplification in similar studies, a 95% safety factor and 30% relative precision.
 
Ethics
The study protocol was verified by the Research Council Ethics Committee of Mashhad University of Medical Sciences. No informed consent was required and mastectomy was indicated for the patients. The study was in conformity with the Declaration of Helsinki.