Evaluating Microbiota Signatures, Intrinsic Subtypes, PIK3CA Gene Mutations and Alternative Diagnostic Modalities Among Ethiopian Breast Cancer Patients
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Date
2024-06-13
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Addis Ababa University
Abstract
Background: Female breast cancer incidence has risen to unprecedented levels. It is a complex disease presented with distinct morphology, biological behaviors, clinical outcome and prognosis. Consequently, classification of breast cancer, understanding the molecular events including PIK3CA mutations and exploration of environmental factors including microbiota have a central role in understanding tumor biology. However, there is limited evidences in subSaharan Africa (SSA) context including Ethiopia; therefore, this study was conducted to generate evidence-based data to improving breast cancer care in Ethiopia. Additionally, considering the limited infrastructure for breast cancer diagnosis, we did method evaluation between PCR methods and immunohistochemistry to suggest alternative methods. Objectives: The study aims to evaluate microbiota signatures, determine intrinsic subtypes of breast cancer, determine the rate of PIK3CA gene mutations and investigate the diagnostic performance of PCR to determine ER, PR and HER2 markers against immunohistochemistry. Methods: A pathologically confirmed breast cancer patients recruited to the study. Clinical and histopathological data were collected using a questionnaire coupled with a follow up data for survival analysis. Formalin-fixed paraffin-embedded (FFPE) tumor tissue and paired (normal adjacent and breast tumor tissues) samples were collected. IHC analysis, PAM50- based gene expression profiling, and 16S rRNA gene-based sequencing were carried out. PAM50 based assay was employed to classify breast cancer into distinct intrinsic subtypes. Hot spot mutations across exon 9 and 20 in PIK3CA gene was determined using TaqMan® Mutation detection assay applying a competitive Allele-Specific TaqMan® PCR platform. 16S rRNA gene amplicons were PCR-amplified using dual-barcoded primers targeting the V4 region followed by high through put sequencing in Illumina MiSeq. Concordance between IHC and PCR method was evaluated using Cohen kappa coefficient. SPSS, Python, and R-packages were employed for data analysis. Survival analysis was performed using Kaplan-Meier.
Conclusion: The distribution of intrinsic subtypes confirms previous immunohistochemistrybased studies from Ethiopia showing potentially endocrine-sensitive tumors in more than half of the patients. The findings indicated that PAM50-based classification offers a robust method for the molecular classification of tumor. While assessing mutations across PIK3CA gene, the proportion of mutation was high in favorable clinical and histopathological features including HR+ tumors. Considering endocrine therapy resistance challenge, these group of patients with PIK3CA mutation can benefit from combining endocrine therapy, chemotherapy, and targeted PI3K activity inhibitors. Though not yet explored in our setting and statistically significant, a specific microbial abundance noted between NAT and breast tumor. Again, a marked difference in the relative distribution of distinct microbiota across clinical and histopathological features was observed. Furthermore, a strong correlation of certain microbial communities with paired breast tissues and further prognostic features. The study highlighted that mammary microbiota composition differs significantly across tumor and cohort clinical and histopathological features which encourage to conduct further wide-scale study strengthen the current data and further utilize microbiota as a potential marker. Besides understanding the molecular biology and environmental factors including microbiota, working towards alternative diagnostic modalities is essential. Accordingly, applying PCR method, biomarker expression was higher while utilizing FFT than FFPE. Improvement of agreement noted between qPCR and IHC in FFT samples. Finally, the finding confirmed that mRNA-based breast cancer marker detection could be a complementary and/ or an alternative method in Ethiopia taking into account the limited IHC service availability.
Keywords: Breast cancer, Ethiopia, IHC, Intrinsic subtypes, Microbiota, Mutation, Overall survival, PCR, PIK3CA, Prognostic features,
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Keywords
Breast cancer Ethiopia IHC Intrinsic subtypes Microbiota Mutation Overall survival PCR PIK3CA Prognostic features