Background: Circulating tumor cells (CTCs) in breast cancer (BC) are commonly defined as epithelial cells (EPCAM and cytokeratin (CK) positive), lacking the universal blood cell marker CD45. Nonetheless, CTCs expressing both CK and CD45 (= dual-positive, DP cells) can be observed in the blood of cancer patients. Early evidence suggests that DP cells might derive from the fusion of tumor cells and macrophages, and we have previously demonstrated that they present aberrant genomes and are associated with worse prognosis in BC [1,2]. Here, to further investigate the mechanisms/pathways underlying their presence, we analyzed the association between DP cells and circulating tumor DNA (ctDNA) alterations. Methods: Blood samples were collected from patients with advanced BC (aBC), before starting a new line of therapy. All patients were enrolled in a prospective clinical trial. For CTC and DP cells analysis, 7.5 ml of blood collected in CellSave® tubes was processed with the FDA-approved CellSearch® platform (positivity cutoffs were ≥1 cell for DPcells and ≥5 cells for CTCs). For ctDNA analysis, plasma was collected from Streck stabilizing tubes and analyzed with the Guardant360™ NGS platform for the detection of somatic single nucleotide variants (SNVs), insertions/deletions (indels), gene fusions/rearrangements and copy number variations (CNVs), which were then classified into pathways based on previously defined profiles generated on the Cancer Genome Atlas database (RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, P53, cell cycle, notch, PI3K). Associations between ctDNA-detected gene alterations and circulating cell types were analyzed through chi square test, while mutant allele frequency (MAF) and number of detected alterations (NDA) were tested by Mann Whitney test. Results: We analyzed blood samples from 169 patients with luminal-like (n=80), HER2+ (n=34) and triple-negative (n=52) aBC. DPcells were detected in 85 patients (50.3 %, range 0-53), of which 40 (47 %) were CTC-positive and 45 (53%) CTC-negative. Somatic ctDNA alterations were detected in all analyzed samples. In the overall population, the presence of ≥1 DPcell was associated with SNVs in the cell cycle pathway (p = 0.043), a numerically higher incidence was also observed for CNVs in this pathway. SNVs and CNVs in the cell cycle pathway were associated with CTCs ≥ 5 as well (p = 0.005 and p = 0.003, respectively). Moreover, associations with CTCs ≥ 5 were observed for RTK SNVs and CNVs (p = 0.041 and p = 0.046, respectively), PI3K SNVs and CNVs (p = 0.006 and p = 0.007, respectively), MYC SNVs and CNVs (p = 0.042). No associations were observed in terms of MAF and NDA. In the luminal-like subgroup an association was highlighted for CNVs in the cell cycle pathway, p = 0.038. CTCs ≥ 5 were associated with PI3K SNVs (p = 0.031). In the triple-negative subgroup DPcells were associated with SNVs in the RAF pathway (p = 0.041), whereas CTCs ≥ 5 were associated with PI3K SNVs and CNVs (p = 0.044 and p = 0.024, respectively) and RTK SNVs (p = 0.008). In the HER2 positive subgroup, a higher MAF and number of detected SNVs was observed for samples with ≥1 DPcell (p = 0.0286 and p = 0.0099, respectively). Conclusions: The study analyzed ctDNA features associated with canonical and CK+/CD45+ CTCs, showing differential gene alteration profiles. Cell cycle pathway SNVs were common in both CTC populations, while other pathways (RTK, PI3K, MYC and RAF) were significantly altered in a mutually exclusive pattern. These results suggest that DPcells might have a different biological meaning compared to canonical CTCs. More studies need to be conducted to better characterize this understudied CTC subpopulation and understand their specific contribution to cancer progression. References: 1) Reduzzi C. et al., Semin Cancer Biol. 2020;60:344-350. DOI:10.1016/j.semcancer.2019.10.008 2) Reduzzi C. et al., Journal of Clinical Oncology 40, no. 16_suppl (June 01, 2022) 1093-1093. DOI: 10.1200/JCO.2022.40.16_suppl.1093 Citation Format: Carolina REDUZZI, Lorenzo Gerratana, Youbin Zhang, Maroua MANAI, Paolo D’Amico, Andrew A. Davis, Jeannine Donahue, Ami N. Shah, Massimo Cristofanilli. Association between CK+/CD45+ circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) alterations in advanced breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-06.
Background. Because of its high metastatic potential, inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer. We previously demonstrated that Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein overexpression was associated with shorter metastasis-free survival (MFS) in IBC patients, but not in non-IBC (nIBC) patients. However, the mechanism of action of MARCKS and its particular association to poorer outcome in IBC compared to nIBC are poorly understood. Methods. We evaluated in vitro the inhibitory effect of MPS (MARCKS phosphorylation site domain), a peptide targeting MARCKS phosphorylation site domain (PSD) in single and in combination with paclitaxel treatment, on cell proliferation and cell motility in two cell lines of different phenotype (SUM149 for IBC and MDA-MB-231 for nIBC), as well as its distinct molecular mechanisms of action. We also assessed the clinical relevance of the protein expression of MARCKS and phosphatase and tensin homolog (PTEN) in a large series of IBC vs. nIBC patients. Results. In vitro, the treatment with MPS peptide impaired cell proliferation, migration, and invasion in SUM149 compared to MDA-MB-231 cells. More important, MARCKS inhibition increased paclitaxel sensitivity when using combination therapy in SUM149 cells compared to MDA-MB-231 cells. Interestingly, we could partially explain this specific inhibitory effect in IBC cells using western blot: MARCKS inhibition in single and in combination induced up and downregulation of the PTEN/AKT signaling pathway respectively in IBC compared to nIBC cells. Importantly, a negative correlation of MARCKS and PTEN was only found in the clinical IBC samples (180 patients) compared to nIBC samples (355 patients). More importantly, the group of patients with negative MARCKS and positive PTEN protein expression was associated to better 5-year MFS only in IBC patients. Conclusion. These results indicate two major findings: first, the important prognostic value of the negative correlation of MARCKS and PTEN expression in IBC patients, and second the specific role of MARCKS in regulating different downstream pathways and increasing the paclitaxel response in combination treatment in IBC compared to non-IBC. They suggest a potential IBC-specific targetable biomarker, the inhibition of which might impair disease aggressiveness and perhaps enhance patients’ survival. Citation Format: Maroua Manai, Ines Bini, Pascal Finetti, Haifa Bichiou, Carolina Reduzzi, Naziha Ben Hamida, Marc Lopez, Khaled Rahal, Karima Mrad, Mohamed Manai, Massimo Cristofanilli, Hamouda Boussen, Raoudha Doghri, Maher Kharrat, François Bertucci. Targeting MARCKS in inflammatory breast cancer increased paclitaxel sensitivity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-15.
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