Kasabwala, Dimpal M.

e14548 Background: Micronuclei (MN) are small, excised DNA fragments resulting from biological DNA repair mechanisms in response to internal chromosomal instability, such as those caused by genotoxic events (i.e. chemotherapy and radiation), and whose formation in tumor stromal cells in blood circulation is prognostic for worse clinical outcomes (Fenech 2020). Further, mutagenic events occurring during the formation of MN are associated with the development of cancer neoantigens, which are a major element in the programmed cell death ligand (PD-L1) pathway that influences response to immunotherapies. We hypothesized that MN can be detected in metastatic breast cancer (mBC) patients (pts) within circulating stromal cells (CStCs) that prognosticates for clinical outcomes and may associate with PD-L1 cell expression. Methods: We initiated a prospective pilot study using a compiled set of n=124 mBC samples, from multiple institutions, enrolled prior to starting new lines of treatment for progressive mBC. We enumerated MN formation found in CStCs from whole peripheral blood (7.5mL), filtered for CStCs, then stained for PD-L1 and DAPI. DAPI was used to identify MN, defined by small (<3 µm) DAPI+ circular formations within the cytoplasm, separate from primary nuclei. In conjunction, all cells were stained with PD-L1 with expression normalized to background and evaluated against MN by T-test analysis. MN presence was compared to all available clinical variables, including induction of PD-L1 immunotherapy. Pts’ progression-free survival (PFS) and overall survival (OS) hazard ratios (HRs) were analyzed by censored univariate and multivariate analyses based on RECIST v1.1 over 24 months. Results: MN were identified in 56% (n=70/124) of CStCs and their presence was significantly prognostic for worse PFS (HR=2.4, CI= 1.53-3.76, p<0.001) & OS (HR= 1.89 CI=1.07-3.34, p=0.039) over 24 months. Overall, mPFS of pts with MN was 2.8 months and without MN was 12.1 months. The mOS of pts with MN was 12.7 months and without MN was 20.8 months. Additionally, MN positive CStCs had a significantly higher PD-L1 expression than MN negative CStCs (p<0.001), with 67% of MN positive pts (n=46/66) having high PD-L1 expression while 30% of MN positive pts (n=20/66) having low PD-L1. Further, evaluation of PD-L1 in CStCs showed a linear relationship (R=0.9806) between MN number and PD-L1 in cells, with 0 MN average=0.016, 1 MN=0.200, 2 MN=0.318, and ≥3 MN=0.400. Conclusions: These initial findings suggest that MN are detected in pts with advanced mBC and may identify pts with faster progression and worse survival outcomes. Further, the number of MN in CStCs appears to correlate with higher PD-L1 expression within cells. Follow up clinical information and subtyping studies are ongoing to also evaluate of the effect of MN positive CStCs on PD-L1 immunotherapies in these pts.

Abstract Background: Micronuclei (MN) are a result of biological DNA repair mechanisms which form due to internal chromosomal aberrations indicating sub-clonal cancer populations with higher cell survivability and drug therapy resistance. MN are often observed as small fragments of nucleic acids excised from a primary nucleus in Circulating Stomal Cells (CStCs) in response to DNA damage. CStCs with damaged DNA undergoing repair mechanisms, such as those that form MN, are likely to have upregulated expression of programmed cell death ligand (PD-L1) which can be abnormally high on cancer cells that have high mutational burdens. We evaluated CStCs in metastatic breast cancer (mBC) patients for presence of MN and the cell's PD-L1 expression, to determine its prognostic significance to clinical outcomes. Methods: We enumerated MN formation in CStCs in a prospective pilot study using n=76 mBC patients starting new lines of treatment. Whole peripheral blood (7.5mL) was procured and filtered for CStCs and then stained for PD-L1. DAPI was used to identify MN, defined by small (&lt;3 µm) DAPI+ circular formations within the cytoplasm, separate from the primary nucleus. We compared number of MN to PD-L1 expression of all cells, and the MN presence to all available clinical variables. Patients’ progression-free survival (PFS) and overall survival (OS) hazard ratios (HRs) were analyzed by censored univariate analysis based on RECIST v1.1 over two-years. Results: MN were identified in 62% (n=46/76) of CStCs and MN positive CStCs had a significantly higher PD-L1 expression than MN negative CStCs (p=0.0082). A linear relationship between increasing MN number and higher PDL-1 expression within cells was identified, R=0.9411. Further, presence of MN with in CStCs was significantly prognostic for worse PFS (HR=2.62 95%CI 1.50-4.59, p=0.0012) and worse OS (HR= 3.07 95%CI 1.56-6.03, p = 0.002) over 24 months. Conclusion: MN formations in mBC are observable biomarkers of an underlying repair mechanism which suggest the survivability of sub-clonal cancer populations associated with more aggressive cancer subtypes with worsening progression rates. Further studies to evaluate the effect of PD-1 immunotherapies in MN positive patients is ongoing. Citation Format: Dimpal M. Kasabwala, Massimo Cristofanilli, R. Katherine Alpaugh, Saranya Chumsri, Carolina Reduzzi, Cha-Mei Tang, Rena G. Lapidus, Giuseppe Del Priore, William V. Williams, Daniel L. Adams. Micronuclei in circulating stromal cells correlates with PD-L1 expression and predicts progression in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 304.