Establishment of a RESEARCH USE ONLY Condensed-Efficient-Fast (CEF) PITX2 workflow for analysis of PITX2 DNA methylation in small tumor tissue samples 

Abstract The CE-marked therascreen® PITX2 RGQ PCR Kit (QIAGEN Cat No./ID 873211), a PITX2 DNA methylation assay was introduced to the market in 2018, (hereinafter referred to as the “standard therascreen PITX2 workflow) and represents an in vitro diagnostic to predict outcome to anthracycline-based chemotherapy considered standard-of-care systemic treatment in early breast cancer patients. The Intended Use (IUS) of this kit covers lymph node-positive, estrogen receptor-positive and HER2-negative high-risk breast cancer patients treated in the adjuvant setting with anthracycline-based chemotherapy and requires 400 ng genomic DNA as recommended DNA input extracted from 1-2 x 5 µm Formalin-Fixed-Paraffin-Embedded (FFPE) sections with a minimal total tumor tissue surface area of ≥ 100 mm2. In today’s clinical workflow of early high-risk breast cancer patient therapy, increasing number of patients are treated in the neoadjuvant setting after tumor biopsy with anthracycline-based chemotherapy before tumor resection by surgery. Biopsy material size restriction in this clinical area requires for a predictive marker test to be applicable biopsy specimens with tumor tissue surface area’s in the range of 20-50 mm2. Therefore, a modified PITX2 workflow protocol - the so-called “Condensed Efficient Fast (CEF)” PITX2 workflow – was developed to address this need. The required modifications of the standard therascreen PITX2 workflow is described in the “bisDNA Preparation” section. The “PMR Detection & Analysis” section of the standard therascreen PITX2 workflow remains unchanged. The new CEF PITX2 workflow is for Research Use Only (RUO) and intended to be used by qualified users such as technicians, molecular biologists/clinical chemists, or physicians. The complete CEF PITX2 workflow is optimized for medium sample through-put with highly reliable and robust read-out and can be performed in two working days.