Abstract P5-04-19: Gene variants in circulating tumor cells as markers of resistance in patients with metastatic breast cancer receiving CDK4/6 inhibitors

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Abstract Background: Predictive biomarkers for the stratification of patients with metastatic (M), hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) to receive CDK4/6 inhibitor (i) treatment before therapy start are still missing to protect patients with primary resistant disease from starting ineffective therapies and minimize unnecessary toxicities. Here, we aim to identify markers of primary and acquired resistance to CDK4/6i therapy by sequencing genomic (g) DNA of circulating tumor cells (CTCs) at baseline and at the time of progression. Patients and Methods: CTCs were isolated from 10 ml EDTA blood of 33 HR+/HER2- MBC patients at baseline of CDK4/6i plus endocrine therapy (TX) and at the time of progression using positive immunomagnetic selection (AdnaTest EMT2/StemCell Select). mRNA-depleted CTC lysates were used to isolate gDNA by a newly established workflow (AllPrep DNA/mRNA Nano) and libraries were constructed with a customized QIAseq® Targeted DNA Pro Panel, targeting hotspots or all exonic regions of 20 genes (AKT1, AR, BCL11B, CXCR4, EPCAM, ERBB4, FAT1, FGFR1, FGFR2, HLA-A, KRAS, MAPK1, MET, MLH1, NF1, NFKBIA, RB1, STAT1, STAT3, YAP1). All consumables: QIAGEN, Germany. Pooled libraries were analyzed by 150bp paired-end sequencing on an Illumina NovaSeq instrument. Buffy coat DNA samples as germline controls were available from 24/33 patients. Statistical analysis was conducted by log-rank testing, multivariate Cox regression and two-sided exact Fisher test. Non-Responders were defined as patients showing a progression-free survival (PFS) of six months or less (primary resistance). Results: Variants in all of the 20 genes were found by sequencing CTCs and matched buffy coat samples (mean UMI coverage: 665) – in total, 225 different variants were called. Comparing the prevalence of variants in CTCs at baseline and at the progressive disease time point revealed increased variant counts after CDK4/6i TX in MET and RB1, respectively. Patients with at least one variant in BCL11B in their CTCs at baseline showed a significantly decreased PFS (p=.042). In contrast, patients with FAT1 p.S3554A or FAT1 p.I2718V detected in their CTCs at baseline had a significantly prolonged PFS (p=.046/.027). In CTCs at baseline, HLA-A variants [p.T345S (log-rank p=.034, Fisher p=.010), p.K335N (log-rank p=.011, Fisher p=.015), p.I306V (log-rank p=0.011, Fisher p=.015), p.R138H (log-rank p=.002, Fisher p=.010), p.Y123F (log-rank p=.011, Fisher p=.015), p.F33S (log-rank p=.015, Fisher p=.010)] significantly correlated with a reduced PFS and their prevalence was significantly different in Responders (n=19) compared to Non-Responders (n=13). HLA-A p.L180* in CTCs at baseline significantly related to a reduced PFS (p=0.011), but not to response. The prevalence of the latter variant increased after CDK4/6i TX in comparison to the baseline time point. Interestingly, HLA-A variants in the matched buffy coat samples did not significantly correlate with PFS. Patients with at least one variant in NFKBIA in their buffy coat showed a significantly prolonged overall survival (p=.019) – even significant in the multivariate Cox regression analysis (p=.024) accounting for age, number of metastases, therapy line, de novo/recurrent metastatic status and visceral or only non-visceral metastases. Conclusion: BCL11B variants as well as different HLA-A variants in CTCs before therapy initiation might be primary resistance markers for CDK4/6i TX and thus, predictive markers for TX stratification. Acquired resistance could be related to variants in MET and RB1 as well as specific HLA-A variants. We further suggest two FAT1 variants in CTCs at baseline as markers to indicate sensitivity to CDK4/6i TX. Citation Format: Sabine Kasimir-Bauer, Stefanos Moukas, Hans-Christian Kolberg, Mitra Tewes, Markus Storbeck, Peter Hahn, Siegfried Hauch, Oliver Hoffmann, Rainer Kimmig, Corinna Keup. Gene variants in circulating tumor cells as markers of resistance in patients with metastatic breast cancer receiving CDK4/6 inhibitors [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-04-19.

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