Quantification of mRNA expression of Her2/neu, estrogen receptors ERalpha and ERbeta, and progesterone receptor using real-time reverse transcription PCR in small tumor biopsies, in human breast cancer
Date of Award
Masters of Science (Research)
Biological and Pharmaceutical Science
Dr Jerry Clifford
Dr Sean Connolly
Early detection is an effective means of reducing cancer mortality. Early detection greatly improves treatment options and the chances for successful treatment. The prognosis for patients with breast cancer is determined by well-established biological features associated with biological aggressiveness, histological grade, tumour size, and nodal involvement. Breast cancer results from multiple genetic abnormalities, and cellular differentiation, tumour growth, and metastatic potential are the end result of these changes. Identification of molecular changes is of considerable value in enhancing the accuracy of prognostic indices, and more importantly, it may indicate various changes as putative targets for therapy. In recent years a plethora of such changes have been identified, such as estrogen receptor and HER2/neu status. However such indices have largely been examined following tumour excision due to inherent limitations in the methodology used for their assessment. A number of methods are currently used for measuring various targets relating to breast cancer with limitations varying from technical difficulty (very large sample requirement) to subjectivity in interpretation and scoring of results. All such methods are only routinely applicable to excised tissue and are not used for samples such as serum, nipple fluids and fine needle aspirates The purpose of the study was to apply the recent development of real-time quantitative PCR to the identification of molecular indices associated with the detection and diagnosis of breast cancer. The study looked at the establishment of the methodology for HER2/neu, estrogen receptors a and p, and progesterone receptors in solid tumours, and ultimately to develop their effectiveness as a screening tool using other sample types. Quantitative PCR assays for the four molecular indices HER2, ERa, ERp and progesterone receptor were developed for their potential implementation in the clinical laboratory. Assays were developed using sequence specific hybridisation probes to detect the target mRNA and the reference gene GAPDH. Calibration curves were generated using cDNA standards for each of the molecular indices. RNA was purified and extracted from a series of first episode presentation patient samples. reverse transcribed, and the cDNA product amplified on the Lightcyler platform, which utilizes fluorescent resonance energy transfer technology (FRET) for the detection of the amplified product. Gene expression was calculated using Quantification software on the Lightcycler platform. The gene expression results were compared to protein expression measured by Immunohistochemistry (IHC) on the same tumours. To conclude, the Real RT-PCR methodology carried out in this study could be utilized in conjunction with IHC in a clinical setting to elucidate the mRNA copy number for each of the patient samples in creating a receptor profile and aid in the diagnosis and prognosis of individual patients.
Mc Cabe, Evonne Marie., "Quantification of mRNA expression of Her2/neu, estrogen receptors ERalpha and ERbeta, and progesterone receptor using real-time reverse transcription PCR in small tumor biopsies, in human breast cancer" (2006). Theses [online].
Available at: https://sword.cit.ie/allthe/596