European Journal of Cardiovascular Medicine

Breast Cancer is the most common female cancer worldwide (25%) of all cancers. It is the most frequently diagnosed cancer in the vast majority of the countries (154 of 185) and is also the leading cause of cancer death in over 100 countries. In 2018 there were 2,088,849(11.6%) new cases and 626,679 (6.6%) number of deaths due to breast cancer. (1)

Incidence rate of breast cancer has been rising for most countries in transition over last decades with rapid increase occurring in countries like South America, Africa and Asia. It reflects a combination of demographic factors allied to social and economic development including postponement of child bearing, greater obesity levels and increase in breast cancer screening and evidence. (2)

Breast Cancer has been ranked number one cancer among Indian females with age adjusted rate as high as 25.8 per100,000 women and mortality 12.7 per 100,000 women. The age adjusted incidence rate of carcinoma of the breast was as high as 41 per 100,000 women for Delhi, followed by Chennai (37.9), Bangalore (34.4) and Thiruvananthapuram District (33.7). A statistically significant increase in age adjusted rate over time (1982–2014) in all the PBCRs namely Bangalore (annual percentage change: 2.84%), Barshi (1.87%), Bhopal (2.00%), Chennai (2.44%), Delhi (1.44%) and Mumbai (1.42%) was observed. Mortality-to-incidence ratio was found to be 66 in rural registries whereas 8 in urban registries. Breast cancer projection for India during time periods 2020 suggests the number to go as high as 1797900. (3)

Patients have better survival compared with more fatal cancers possibly because the breast tissue is physically not a necessary organ for human survival. Yet the mental and emotional disturbances from major surgeries as well as deaths by relapse or metastasis seriously endanger women’s health.

Mastectomy and chemotherapy have greatly improved the survival of breast cancer patients and more elegant forms of conservation surgeries are now being applied to minimize the post-treatment psychological impact. However, without fully understanding the underlying mechanism and pathogenesis, the efficiency of prevention and treatment will be always limited.

Due to the rapid progress made in molecular biology, systemic biology and genome sciences in the past decades, our understanding about this disease has been dramatically expanded at cellular, molecular and genomic levels.

Triple negative breast cancer (TNBC) refers to the breast cancer phenotype where the estrogen and progesterone receptor are negative, as assessed by immunohistochemistry (IHC) and there is a lack of overexpression of HER2 as assessed by IHC or the absence of its gene amplification as assessed by fluorescence in situ hybridization technique.

Patients with TNBC do not benefit from hormonal or trastuzumab-based therapy because of the loss of target receptors such as ER, PGR, and HER-2. Hence, surgery and chemotherapy, individually or in combination, appear to be the only available modalities. However, some studies have identified certain receptors as targets for new therapeutic drugs.

In the backdrop of above, an observational study was undertaken to study the response and outcome of different modalities of management in TNBC patients.

This is an Observational study was conducted in the Department of General Surgery at Tertiary Care Teaching Hospital from January 2019 to August 2020. Approximately small breast cancer tissues pieces of not less than 50 patients will be required for successful establishment of primary breast cancer cell line and perform our further studies

Inclusion Criteria: all the female patients with histologically proven triple negative breast malignancies admitted in general surgery wards, except those meeting the exclusion criteria.

Exclusion Criteria:

Fungating breast tumor

Breast carcinoma with distant metastasis

Patients having other malignancies associated with breast carcinoma

Patient not fit for general anesthesia.

STUDY TOOLS AND TECHNIQUES

Isolation and in vitro expansion of progenitor cells from breast tumor specimens:

Tumor specimens obtained from consenting patients will be received in the Laboratory within 30 minutes of surgery and immediately mechanically disaggregated. Occasionally, enzymatic digestion may also require and tissue fragments will be incubated at 37 oC for 2 hours in a 1:1 solution of collagenase/hyaluronidase (Roche Diagnostics GmbH, Mannheim, Germany and Sigma-Aldrich Corp., St. Louis, MO, respectively). After filtration through a 30 μm pore filter, single cells will be plated at 1,000 cells/mL in serum-free DMEM-F12 (CambrexBioScience, Venviers, Belgium), supplemented with 10 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor (EGF), 5 Ag/mL insulin, and 0.4% bovine serum albumin (all from Sigma).Cells grown in these conditions as nonadherent spherical clusters of cells (usually named ‘‘spheres’’ or ‘‘mammospheres’’) will be enzymatically dissociated every 3 days by incubation in a trypsin-EDTA solution (Cambrex) for 2 minutes at 37oC. Conversely, differentiation will be induced by culturing mammosphere derived cells for 8 days on collagen coated dishes in DMEM-F12 supplemented with 5% fetal bovine serum without growth factors

Flow-cytometric analysis:

By using a Flow-cytometer, the expression of a panel of differentiation markers will be distinctly evaluated on cells obtained from mammospheres or from cells cultured in differentiating conditions. The antibodies to be used are anti-CK14, anti-CK18, anti–epithelial-specific antigen (ESA), anti–a-smooth muscle actin (a-SMA), anti-CD44, anti-MUC1 (directly or indirectly conjugated with fluorescein, FITC), anti-CD10, and anti-CD24 (directly or indirectly conjugated with phycoerythrin). For staining of cytoplasmic antigens (CK14, CK18, and aSMA), single cells will be fixed in 70% ethanol at 20oC for 10 minutes. Conversely, before staining of membrane antigens (CD10, ESA, MUC1,CD44, and CD24), unfixed cells will be allowed to recover in fresh medium for 1 hour at 37jC in gentle agitation after dissociation.

3. Immunoblotting for the NFκβ and other markers: Protein lysates will be prepared with a radioimmunoprecipitation assay (RIPA) buffer (20 mMTris-HCl pH 7.5, 2 mM EDTA, 150 mMNaCl, 1 mM sodium vanadate, 10 mMNaF, 2.5 mM sodium pyrophosphate, 1% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations will be determined using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Protein samples (30 μg) will be resolved by SDS-PAGE and transferred onto PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA). Blocked membranes with skim milk will be incubated with primary antibodies. Washed membranes will then be incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody and developed with an ECL plus western blot detection system reagent (GE Healthcare Biosciences, Piscataway, NJ, USA). Protein levels will be quantified using ImageJ software (NIH, Bethesda, MD, USA).

4.Patients will be subjected to different surgical procedures according to the clinical status of the disease and the response and outcome will be studied with every 3 monthly follow-up to look for recurrence, metastasis and overall survival.

Data Collection Forms:

A well designed proforma containing various parameters under study were used for data collection. The data will be maintained computer.

Statistical Analysis

Sample size was calculated with help of Epi Info (TM) EPI INFO which is a trademark of the Centres for Disease Control and Prevention (CDC). The same software will used for statistical analysis of data of this study. 2 test will be used to test the association of different study variables. t-test will used to compare the means.

Parameters To Be Studied:

• Name
• Age
• Comorbidities
• Karnofsky
• Nutritional status
• ER/PR/Her2nu status
• FNAC
• Tru cut biopsy
• of CT/RT
• CT regimen-

Study Tools:

• Clinical observational tool – History of disease with Complete physical examination of patient
• Routine Investigation:
• Hematological – Hb%, TC, DC, ESR.
• Biochemical - RBS, Blood urea, Serum creatinine & Serum electrolyte, LFT
• ECG
• Radiological – Chest X- ray, ultrasound of both breasts, CT scan wherever applicable
• FNAC
• True cut biopsy
• HPE

Study Techniques:

After approval of institute’s ethical committee and obtaining written informed consent patients will undergo thorough evaluation and will be checked against the exclusion criteria of the study. All the assessment mentioned above will be done on included patients and analysis will be expressed in the form of percentage of patient and Input of data in the master table and statistical analysis to find out the significant relation of the various parameters.

In table 1, Basic Demography

50 patients with TNBC were identified. Tumors were categorized based on ER, PR, and HER2 status. Tumors that have ≤1% expression of ER and PR as determined by IHC and that are HER2 negative, either 0–1+ by IHC or 2+ and fluorescence in situ hybridization negative, were identified as triple negative. IHC was done on formalin-fixed paraffin-embedded sections by polymer horseradish peroxidase technique on fully automated immune stainer. TNBCs were classified histologically according to the WHO classification. Patients with Stage I, IIA, or a subset of Stage IIB disease (T2N1) were categorized as having early breast cancer (EBC) and a subset of patients with Stage IIB disease (T3N0) and patients with Stage IIIA to IIIC disease were categorized as having locally advanced breast cancer (LABC). The patients were subjected to Modified Radical Mastectomy (MRM) or Breast Conservation Surgery (BCS) according to TNM staging.

The tumor was right sided in 51%, left sided in 47%, and 2% had bilateral cancer at presentation. Clinically, T2 was the most common (58%) followed by T3 (18.2%), T4 (13.2%), and T1 (10.6%). Nodal involvement was seen in 58% patients. N1, N2, and N3 disease was seen in 26.7%, 16.1%, and 15.1% patients, respectively.

A modified radical mastectomy was done in 76.7%, and 18.2% underwent breast conservation surgery. Adjuvant chemotherapy was given to 79.2%, while 15.6% patients received both neoadjuvant and adjuvant chemotherapy (NACT). Of the patients who received NACT, pathological complete response (pCR) was seen in 25.8% patients. Postmastectomy radiation therapy, as part of adjuvant treatment, was given to 52.6% patients.

50 Breast tissue samples were taken and subjected to western blot study to study protein expression levels and through Q-RT PCR NFkB expression was seen.

Western Blots of NFkB Expression showing protein expression levels

Average Protein Expression Level

(two-tailed Student’s t test, *p < 0.05).

NFKB Expression through Q-RT PCR

(two-tailed Student’s t test, *p < 0.05).

The study has been conducted in a population of 50 patients with TNBC (ER^(-)/PR^(-)/Her2^(-)) who underwent surgery in SSKM hospital with or without NACT. In the present study, TNBCs accounted for 19.3% of all the breast cancers. It is similar to the incidence reported by other studies from the West and other authors from India ranging from 12.5% to 26%. The median age of 50 years in the present study was almost similar to that described in the Western literature (median age of 53 years). Majority of the patients in this study had a tumor size of >2 cm at presentation (88.8%), and more than half of the patients had lymph nodal involvement (58%). Other studies have also reported that TNBCs are relatively large tumors with a high incidence of involvement of the lymph nodes. Majority of the patients in this study underwent modified radical mastectomy (MRM) (76.7%). Various reasons for this are the extent of disease at presentation, concern about recurrence, patient and/or surgeon's choice.

The fact that majority of patients in this study presented with a history of breast lump of <3 months, duration shows that TNBCs have a rapid growth pattern, and they are more likely to be diagnosed clinically than mammographically.

Then the samples have been evenly divided into groups of five with or without NACT. The resected specimens were subjected to analyze NF-kB expression through RNA and protein analysis. RNA was isolated from all the patients with Trizol phenol chloroform method of RNA isolation. The RNA isolated was then purified and using SYBR green, Quantitative Real Time Polymerase Chain Reaction (Q-RT PCR) was performed to analyze the expression of NF-kB gene. It was observed that in samples isolated from patients treated with NACT regime showed higher expression of NF-kB as compared to samples collected from patients undergoing surgery without NACT. 

Similarly, protein was isolated from the samples and subjected to western blot with monoclonal antibody against NF-kB (rel A). The blots were all performed in groups of two (one NACT sample vs. a sample without NACT).  In all the five blots, it was found that the NF-kB showed higher protein level expression in case of the NACT treated samples as compared to the non-NACT treated samples. Even when the blots were quantified, the NACT samples clearly indicated more profound expression indicating a sharp increase in chemoresistance and therefore an increase in the number of Tumor initiating cells (TICs) or what are conventionally referred to as Cancer Stem Cells (CSCs).

Our results combined together indicate that NACT in this case (Tac regime) though effective raises chemo-resistance as our early trends show. In order to substantiate our claim, we need to extend our studies to more samples and reach effective conclusions.

TNBCs are a distinct subtype of breast cancers with unique pathological and clinical behavior. They have a rapid clinical course and early recurrences inspite of timely medical intervention, which reflects the aggressive tumor biology. This warrants further studies on the intensification of chemotherapy and identification and development of targeted therapy aimed at decreasing recurrences and improving survival in this patient population.

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