Evaluation of Breast Cancer Biomarkers using Genexpert® on Core Biopsy-A Retrospective Laboratory Based Validation Study.
Date of Award
Master of Medicine (MMed)
Dr. Zahir Moloo
Dr. Shahin Sayed
Dr. Samuel Gakinya
Pathology (East Africa)
Introduction: Breast cancer is the most common malignancy in women worldwide. It is a significant cause of cancer related morbidity and mortality among Kenyan women. Evaluation of Estrogen receptor (ER), Progesterone receptor (PR), and Human epidermal growth factor receptor 2 (HER2) in breast cancer tissue is standard of care as these biomarkers have been shown to have both prognostic and predictive utility. Immunohistochemistry (IHC) is the gold standard for evaluation of ER/PR/HER2, but its availability in Kenya is limited due to the high operational cost. There have been recent developments in alternative cheaper and readily accessible molecular based testing platforms for ER/PR/HER2. The GeneXpert®’s Breast Cancer Stratifier (BCS), is a quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) simple cartridge based assay developed by Cepheid Inc that quantifies mRNA expression of ER, PR, HER2, and Ki-67,(a marker of proliferation). The study aimed to evaluate sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and accuracy of the BCS assay for ER/PR/HER2 in breast cancer tissue core biopsies compared to IHC. The association of Ki-67 expression as tested by the BCS assay and IHC with tumor grade and mitotic counts was also evaluated.
Methods: Core biopsy slides and Formalin fixed paraffin embedded (FFPE) tissue blocks of pathologically confirmed breast cancer diagnosed between April 2016 and January 2018, were retrieved from the archives at Aga Khan University’s Department of Pathology. Haematoylin and Eosin (H&E) and IHC slides for ER/PR/HER2 were reviewed and the appropriate block selected for nucleic acid extraction. Tissue scrolls were cut at 10um and a single step nucleic acid extraction performed according to the BCS assay protocol. The BCS cartridges were loaded with the extracted RNA and run on the GeneXpert® instrument.
Data Management and Analysis: Data from both the BCS assay and IHC was entered on a Microsoft Excel spreadsheet for each marker and transferred to SPSS version 20 (IBM corporation). Sensitivity, specificity, PPV and NPV with 95% CI was analyzed from a 2X2 table. Kappa statistics and percentage agreement between the two tests was calculated. Diagnostic odd’s ratio was calculated as a measure of association of Ki-67 to the Nottingham tumor grade and mitotic counts.
Results: A total of 162 breast cancer core biopsies were analysed. The predominant histologic type and grade was grade 2 Ductal carcinoma. Sensitivity, specificity, PPV, NPV and accuracy for each marker on the BCS was as follows: 86.96%, 94.74%, 98.04%, 70.59% and 88.89% for ER; 90.00%, 76.47%, 87.10%, 81.25% and 85.11% for PR; 96.88%, 88.57%, 72.09%, 98.94% and 90.51% for HER2. There was moderate agreement for the 3 markers with kappa values of 0.733, 0.673 and 0.763 for ER, PR and HER2 respectively. There was no association between Ki67 expression with both tumor grade and mitotic count.
Conclusion: The BCS assay demonstrates relatively good sensitivities and specificities for ER/PR/HER2 when compared with IHC. However, the study highlights the limitations of the BCS assay in detection of low positive ER and PR samples. There was no demonstrable utility of Ki67 in predicting tumor grade and mitotic activity. Future prospective studies using various sample types are recommended to assess utility of the assay in low resource settings.
Chesori, E. (2018). Evaluation of Breast Cancer Biomarkers using Genexpert® on Core Biopsy-A Retrospective Laboratory Based Validation Study. (Unpublished master's dissertation). Aga Khan University, East Africa.