Claudia Vollbrecht’s research while affiliated with Charité Universitätsmedizin Berlin and other places

Background: ESR1 mutations are biomarkers in breast cancer patients who develop metastatic disease after endocrine therapy (ET). Recently, the Food and Drug Administration (FDA) and European Medicines Agency (EMA) have approved Elacestrant, a selective estrogen receptor degrader for patients harboring ESR1 mutations. This has necessitated the establishment of reliable and sensitive NGS- or PCR-based assays to detect these ESR1 resistance mutations in liquid biopsy samples. Methods: We evaluated NGS results of a pan-cancer cohort of almost 6000 patients from two major German institutes of pathology, to show that the occurrence of ESR1 mutations is extremely rare (<1%) in ET-naïve patients. This suggests that ESR1 mutations arise almost exclusively under the pressure of ET. Therefore, we designed a breast cancer-specific hybrid capture-based NGS liquid biopsy assay covering 12 breast cancer-related genes, including ESR1, PIK3CA, AKT1, ERBB2, BRCA1/2, and TP53. We validated the HS2-Mamma-LIQ assay extensively using reference material to detect mutations to 0.1% variant allele frequency (VAF) and compared the performance to a commercially available ESR1 ddPCR assay. Results: We show the results of routine diagnostic analysis of the first consecutive 354 patients with activating ESR1 mutations rate of 43%, with 20% of patients harboring co-mutations in PIK3CA and other genes underlining the relevance of tumor heterogeneity. Our study highlights liquid biopsy as a preferred approach for monitoring ESR1 mutations in breast cancer patients by showing cases where NGS analysis suggests complex tumor heterogeneity with multiple ESR1 as well as PIK3CA mutations at different VAFs. Conclusions: Our findings not only corroborate prior research concerning the rarity of these mutations in unselected patients but also emphasize the importance of robust and broad molecular assays rather than single gene assays in their detection and characterization in the diagnostic setting. Advantages of different approaches are discussed to address the current clinical need.

Upfront KRAS and NRAS gene testing (‘RAS’) is the standard of care for metastatic colorectal cancer (mCRC), to guide first-line treatment. The presence of RAS mutation (MT) is a negative predictor for the efficacy of anti-EGFR antibodies and the use of cetuximab and panitumumab is restricted to RAS wild-type (WT) mCRC. Conversion from RAS WT to RAS MT mCRC after treatment with anti-EGFR antibodies is a known and well-described acquired resistance mechanism. The by far less frequent ‘NeoRAS wild-type’ phenomenon (reversion from RAS MT to RAS WT) has recently drawn attention. The proposed effect of chemotherapy on RAS status in mCRC patients is not fully understood. Because of the intriguing biological consequence of a RAS MT to RAS WT reversion, subsequent treatment of NeoRAS WT patients with anti-EGFR antibodies is increasingly being discussed. Here, we report three clinical cases of NeoRAS WT mCRC patients, which received standard-of-care regimens for RAS MT mCRC. Anti-EGFR antibodies were used in two out of three patients after progression of the disease. One of the patients had a long-term response. In line with our observations, NeoRAS WT phenomenon occurs in clinical practice. Retesting of RAS status during treatment should be discussed in patients with unusual long-term clinical courses of RAS MT mCRC to optimise treatment strategy and to evaluate the use of anti-EGFR antibodies.

The impact of liquid biopsies on the analysis of molecular alterations of circulating tumor DNA (ctDNA) has recently increased. PIK3CA is one of the most frequently mutated genes in breast cancer and the expected approval of targeted PIK3CA therapy based on the results of the SOLAR1 trial is likely to lead to the use of liquid biopsies as another promising testing strategy in breast cancer patients who can benefit from a targeted therapy.Choosing an appropriate method for the detection of activating PIK3CA mutations should include factors like sensitivity, specificity, and limit of detection. The test should at least meet the parameters of the assay used in the drug approval study.If carefully used, PIK3CA mutation detection with liquid biopsies can then be a useful addition to standard tissue diagnostics.