Copy-Number Gain and Overexpression of TNF-α in Breast Cancer: Clinicopathological Correlates in Patients from North Bengal, Bangladesh
Authors: Chandan Barai1, Nelofar Yasmin2, Md Abdul Auwal1, Rabeya Khatun2, Tasnima Kamal1, Md Rashel Mollah1, Al-Amin Hossain1, Azadur Rahman Bhuiyan1, Md Ashraful Islam1, Zinnat Ara Moni1, Asma Ul Husna Biswas1, Md Asadur Rahman3, Md Farhadul Islam1,4*
Institution: 1Rajshahi University, Rajshahi, 2Institute of Biological Science, Rajshahi University, Rajshahi, 3Rajshahi Medical College, Rajshahi,4School of Medicine and Dentistry, Griffith University, Australia
Introduction
Gene expression profiling can reveal molecular differences between normal and malignant breast tissue. Tumour necrosis factor-alpha (TNF-α) is a key pro-inflammatory cytokine in the breast tumour microenvironment. It is secreted by tumour-associated macrophages, stromal cells, and cancer cells, and it promotes tumour proliferation, progression, metastasis, and survival, which makes it an attractive therapeutic target. However, data on TNF-α copy number and expression in Bangladeshi breast cancer patients are limited. This study aimed to analyse TNF-α copy number variation (CNV) and mRNA expression in breast cancer tissue from patients in North Bengal, Bangladesh, and also to examine the correlation between CNV and mRNA expression, and to assess their association with clinicopathological parameters.
Methods
Formalin-fixed tissue samples were collected from 100 patients with histopathologically confirmed breast cancer, and 10 normal tissue samples served as controls. Twelve cancer samples were excluded for poor tissue quality, leaving 88 tumours for analysis. Nucleic acids were extracted by the TRIzol method. TNF-α CNV and mRNA expressions were measured by RT-qPCR with GAPDH as the reference gene. Tumours were classified into low, normal, and high expression groups and into low and high copy-number groups. Both measures were compared between tumour and normal tissue, correlated with each other and tested against clinicopathological parameters. A p value below 0.05 was considered significant. The study was approved by the Ethics Committee of the University of Rajshahi, and written informed consent was obtained from all participants.
Results
Patients were aged 33-70 years. Most tumours were histological grade 2 (70.5%) and stage IIIA (38.6%) or IIA (37.5%); 70.5% of patients had regional lymph-node involvement, 68.2% had tumour necrosis, and 37.5% had lymphovascular invasion. A high TNF-α copy number was found in 86 of 88 tumours (97.7%), and copy number was significantly higher in tumour than in normal tissue (p = 0.005). TNF-α mRNA was overexpressed in 46 tumours (52.3%), normal in 17 (19.3%) and low in 25 (28.4%), and expression was significantly higher in tumour than in normal tissue (p = 0.006). Expression correlated positively with copy number (r = 0.28, p = 0.01). TNF-α expression was significantly associated with regional lymph-node involvement (p = 0.04): involvement rose from 52.0% in low expressers to 70.6% in normal and 80.4% in high expressers. mRNA expression was also associated with histological grade (p = 0.02) and stage (p = 0.04). Grade 1 tumours were concentrated in the low-expression group (20.0% vs 2.2% of high expressers), and stage IIB disease was more frequent among high expressers (28.3% vs 4.0%). Copy-number status was associated with lymphovascular invasion and regional lymph-node involvement (both p = 0.04). Neither expression nor copy number was associated with age, tumour size, tumour focality, or necrosis.
Conclusion
TNF-α copy-number gain is very common in breast tumours from the North Bengal region of Bangladesh, and about half of these tumours overexpress TNF-α mRNA. The modest positive correlation between copy number and expression suggests that copy-number gain contributes to TNF-α overexpression. The association of TNF-α expression with lymph-node involvement, histological grade, and stage links TNF-α to tumour progression. TNF-α may therefore serve as a prognostic marker and potential therapeutic target in this population. However, larger studies are needed to validate these findings.