This prospective study included 220 primarily diagnosed BC patients and 100 controls who were apparently healthy volunteers. All cases were recruited and pathologically diagnosed in our institute, from December 2014 to December 2017. The eligibility criteria included patients who were 20–80 years old, not pregnant, devoid of any other type of cancer, never had any breast surgery including implants or breast reduction, and had been treated surgically with either a mastectomy or breast-conserving surgery.
The control volunteers who were enlisted in this study were females with a similar age as the patients. All volunteers for the control group were female nurses, workers, or doctors who were not first- or second-degree relatives of the patients. All volunteers underwent a mammography accompanied by a breast high-resolution ultrasound (US) prior to obtaining a taking out the blood sample to ensure that they were free from BC.
Whole blood samples were taken from all BC patients and apparently healthy volunteers for the cytogenetic study. The tumors from the BC patients were pathologically staged (pTNM) following the WHO classification of tumors of the breast [17]. The breast specimens were used to evaluate DAXX and ATRX protein expression. Poor prognostic pathologic parameters evaluated included vascular tumor invasion (VI), density of the tumor-infiltrating lymphocytes (TILs), regional lymph node status, and the presence of necrosis. The BC cases ranged from stage I to stage IIIC invasive ductal carcinoma. The cases were molecularly classified according to their HER-2 neu, estrogen receptor (ER), and progesterone receptor (PR) statuses [18].
TILs density can be intratumoral TILs and stromal TILs. The density of TILs in this study was evaluated as the percentage of the tumor stromal area that contains a mononuclear inflammatory infiltrate without direct contact with tumor nests. Density grading varies among studies. In the current study, a 50% cutoff point was used to divide the TILs into focal infiltrate and lymphocyte-predominant breast cancer (LPBC) [19].
A total of 180 patients received adjuvant treatment in the form of four cycles AC (adriamycin cyclophosphamide), and four cycles of paclitaxel, except for four patients who received four cycles of AC only and 30 patients who received neoadjuvant treatment addition to four cycles of AC and four cycles of paclitaxel. We used postoperative tumor tissue samples for the pathological examinations in the patients who received neoadjuvant therapy. All patients who were HER2/neu positive also received trastuzumab at the start of paclitaxel therapy. Ten patients did not receive chemotherapy because they were elderly and presented with early stages of luminal A type BC.
The patient follow-up ended in January 2020. During follow-up, all patients received a physical evaluation every 3 months, an abdominal US and chest X-ray every 6 months, and a routine computerized topography (CT) scan every year or at any time during follow-up if indicated for anything suspicious in the X-ray or US.
Cytogenetic FISH study
In this study, we used the PNA FISH kit, which contains a telomere PNA probe with a TTAGGG sequence (code K5326, DAKO, Denmark). The heparinized blood samples were cultured in RPMI mixed with glutamine, phytohemagglutinin, 20% fetal bovine serum, and penicillin/ streptomycin. An Axioscope Imager M1 microscope was used for the capture of 20 metaphases of each patient, accompanied by DAPI and Cy3 individual excitation filter, at × 63 magnification, by an attached CCD camera. The ISIS software was used to analyze TL.
Active separation and removing the overlapping of chromosomes was performed, followed by transference to karyotype window. A DAPI automatic banding classifier was used to classify the chromosomes. Telomere measurement area was represented as two horizontal lines overlaid to each chromosome (for p- and q-arms) in the karyogram. The reference signal was measured by applying two horizontal lines on the respective chromosome (chromosome 2). TL was calculated as the ratio between the telomere fluorescence (T) and the centromere fluorescence (C) of chromosome 2 ratio (T/C ratio). C of chromosome 2 has a stable length, so it was used as the internal reference in each metaphase image analyzed [20].
Immunohistochemistry
Formalin-fixed paraffin-embedded (FFPE) samples were prepared from tumor tissues of the BC patients and underwent an IHC analysis. The stained hematoxylin and eosin slides produced from these blocks were reviewed histologically for confirmation of infiltrating duct carcinoma before IHC marker staining. The FFPE blocks were cut to a thickness of 3 μm and mounted on positively charged slides. The sections were de-paraffinzed and rehydrated. Tris EDTA was used for antigen retrieval in a heated water bath at 90 °C for 45 min. A hydrogen peroxide block was applied. The sections were incubated at room temperature for 10 min. Then, Ultra V Block was applied to the slides for 5 min. Two primary antibodies were applied into two separate sections from tumor tissues: primary goat polyclonal anti-human DAXX antibody (Catalog # DAXX (S-20) sc 7001, Santa Cruz Biotechnology, Inc., USA) and a primary rabbit polyclonal anti-human ATRX antibody (Catalog # ATRX (H-300) sc-15408, Santa Cruz Biotechnology, Inc., USA). Both were used at a dilution of 1/100 (optimum dilution according to the data sheet). The tissue sections were then incubated in a humid chamber for 1 h at room temperature. Immunostaining was performed a universal staining kit “Ultra Vision Detection System Anti-Polyvalent, HRP/DAB (Ready-To-Use)” (Lab Vision Corporation, catalog # TP-015-HD, Fremont, CA 94539-6406, USA) following the manufacturer’s instructions. Biotinylated Goat anti-Polyvalent was applied to the slides at room temperature for 10 min. Streptavidin was applied for 10 min, and diaminobenzidine (DAB) solution was then applied to the slides for 5–10 min. A counterstain for the tissue sections was performed using Mayer’s hematoxylin. Normal gastric and normal prostatic tissues were used as a positive control for DAXX and ATRX, respectively.
Evaluation of DAXX and ATRX expression
An unequivocal moderate and strong nuclear expression with or without cytoplasmic staining of both DAXX and ATRX positivity identified in > 5% of the nuclei of tumor cells is considered positive (high expression) as previously described (Fig. 1) [21]. Positive nuclear staining of DAXX in the gastric gland and positive nuclear staining of ATRX in prostatic ductal cells were used as a positive control. Sections of the tissue-specific positive controls were stained using the same protocol but with omitting the primary antibody, which was used as a negative control.
Statistical analysis
The statistical analyses were performed by using the statistical package for Social Sciences (SPSS), version 21. The chi-square test was used to detect the associations between each of TL, DAXX protein expression, ATRX protein expression, and various clinicopathological data. An independent t test was used to evaluate the associations between TL, DAXX protein expression, ATRX protein expression, and tumor size. A one-way ANOVA test was used to detect the association between TL in the control and early and late tumor stages. Spearman’s correlation coefficient test used to examine the correlations between TL, DAXX protein expression, and ATRX protein expression.
Kaplan-Meier curves and the log-rank survival test were used to analyze overall survival (OS) and the disease-free interval (DFI). P < 0.05 was defined as statistically significant.