Heterogeneity of HER2 amplification in breast cancer – the use of liquid biopsy to identify patients with discrepancies between primary tumor and metastatic lesions

In general, when a patient is diagnosed with invasive breast cancer the amplification status of the HER2 gene is evaluated by immunohistochemistry (IHC) and/or fluorescent in situ hybridization (FISH) on the primary tumor. International literature demonstrates that in 7-20% of cases HER2 heterogeneity takes place, especially between the primary tumor and bone metastasis. Furthermore, heterogeneity in time can occur due to treatment selection. Although reports have demonstrated that heterogeneity of the HER2 status at different sites occurs, only seldom new biopsies are analyzed to re-evaluate the HER2 status. By liquid biopsy, we can overcome this issue as liquid biopsies can (more) easily be repeated over time and contains cell-free tumor DNA (ctDNA) from the whole tumor mass. Hence this research can lead to a more detailed analysis of the presence of heterogeneity of HER2 amplification in this patient group. With this project, we plan to develop an assay that can accurately detect HER2 amplification in a liquid biopsy of breast cancer patients. Furthermore, we will investigate HER2 heterogeneity in breast cancer patients who progress upon first-line treatment (such as hormone therapy). By analysis of a liquid biopsy upon progression to first-line therapy in patients without HER2 amplification (at diagnosis on primary tissue biopsy), we expect to identify the patient group in which the HER2 amplification status has changed (significantly) and might benefit from anti-HER2 therapy. The innovative aspect of this project is that we will evaluate HER2 amplification (by ddPCR) in both the blood and urine simultaneously. Whereas blood sampling is minimally invasive, urine sampling is non-invasive and can easily be repeated over time. We will combine the HER2 amplification data with the results of a methylation assay (ddPCR) or the results of other frequent occurring gene aberrations (AVENIO ctDNA) to know whether enough tumor DNA is present in the circulation/urine for reliable analysis. Currently knowledge on the presence of ctDNA in the cfDNA fraction is lacking in many liquid biopsy studies. As long as we have no idea on the presence of ctDNA in our sample, we cannot distinguish a negative result from a non-informative result due to low ctDNA content by liquid biopsy. The overall aim of this project is to evaluate the discrepancies of HER2 amplification status between primary tumor and metastatic lesions that develop during therapy.To meet this objective we will pursue following aims:- Develop and evaluate an assay to measure HER2 amplification in the blood and urine by ddPCR.- Develop and evaluate a methylation assay to estimate the amount of cfDNA derived from tumor cells present in the blood and urine by ddPCR.- Evaluate an NGS assay detecting multiple breast-specific gene aberrations in the cfDNA from blood (AVENIO ctDNA assay).- Evaluate HER2 amplification in patients without a FISH positive HER2 status of the primary tumor who develop multiple organ metastasis during therapy.

Keywords:EXPERIMENTAL STUDY

Disciplines:Cancer biology

Project type:Service project