Abstract PD13-03: Genomic profiling of breast cancer leptomeningeal metastasis (BCLM) reveals a divergent evolution and therapeutic targets
Authors: Amanda Fitzpatrick, Marjan Iravani, Adam Mills, Eleanor Knight, Thanussuyah Alaguthurai, Vandna Shah, Alicia Okines, Nick Turner, Mark Harries, Syed Haider, Andrew Tutt, Clare Isacke
Journal: Cancer Research (2021)

Abstract Background: Leptomeningeal metastasis remains a devastating development in breast cancer, with a median survival of 3-4 months, no widely accepted standard treatment, and limited access to trials of novel therapies. In addition, lack of access to leptomeningeal metastatic material hampers the pre-clinical investigation of the disease process and molecular drivers. This project uses CSF as a liquid biopsy to characterise BCLM, through genomic analysis of the cell-free DNA (cfDNA), and the development of pre-clinical BCLM models by the expansion of CSF disseminated tumour cells. Methods: CSF (surplus to clinical requirements) and blood were collected from patients undergoing evaluation of leptomeningeal metastasis. cfDNA was extracted from CSF and plasma, and subjected to ultra-low pass whole genome sequencing (ulpWGS) to assess tumour-derived cfDNA fraction. Samples with &amp;gt;10% tumour fraction underwent whole exome sequencing, along with matched archival primary tumour, archival extra-cranial metastatic site(s) and germline DNA. CSF cells were expanded in vitro (to establish 3D patient-derived organoids (PDOs). Results: Cohort demographics are shown in Table 1. Whole exome sequencing (WES) in 21 patients reveals that 65.2% of variants found in CSF cfDNA were not shared with the primary tumour or other matched samples. Phylogenetic analysis shows a divergent evolution from extra-cranial metastatic sites, represented by plasma cfDNA (n = 12) and/or metastatic site tissue DNA (n = 7). The most frequently mutated cancer-associated genes in CSF were MUC16 (12/21), TP53 (11/21), CDH1 (10/21), and KMT2D (7/21). The common occurrence of CDH1 loss-of-function mutations was in keeping with the large number of lobular cases, however were also discovered in CSF of two cases with E-cadherin positive ductal primary tumours. Furthermore, mutations (including frameshift indels) in JAK family proteins (JAK1, JAK3 and TYK2) were present in 5/21 cases, and were private to CSF in 4/21. Potential actionable gene alterations private to CSF include; IDH2 (3/21), GLI1 (3/21), PIK3CA (2/21) and PTCH1 (2/21). Further, there was an enrichment for somatic BRCA1/2 mutations (5/21, 2 private to CSF) indicating potential for platinum and/or PARP inhibitor therapy in these individuals. Patient-derived organoids (PDOs) were established using CSF tumour cells from 3 ER+/HER2- and 2 TNBC BCLM cases. WES of PDOs revealed high concordance with genomic variants identified in the matched CSF cfDNA. Therapeutic compound testing revealed 3/5 PDOs did not display sensitivity to methotrexate, the most commonly used BCLM intrathecal treatment. Patient-derived xenograft (PDX) models have been established by mammary fat pad, intraductal, intracardiac and intracerebroventricular injection routes. Conclusion/future plans: WES of CSF cfDNA provides insight into genomic changes in BCLM, including the divergent evolution, and BCLM specific alterations, some of which are potentially targetable. Parallel PDO and PDX models are being used to validate potential drivers and therapeutic targets. Treatment options beyond intrathecal methotrexate are urgently needed and in future might be molecularly tailored based on alterations discovered by CSF cfDNA sequencing. Table 1. Clinical demographicsDemographicsMedian, years (range)Age at BC diagnosis45 (24 – 66)Time from primary BC to BCLM4.7 (0.7 – 14.7)Time from first metastasis to BCLM1.2 (0.0 – 6.5)Histological Typen (%)Lobular10 (48)Ductal8 (38)Mixed ductal/lobular3 (14)Immuno-histochemical phenotypen (%)ER+ HER2-13 (62)TNBC5 (24)ER+ HER2+2 (9)ER- HER2+1 (5)Metastatic sitesn (%)No other metastatic sites (BCLM only)3 (14)Bone12 (57)Serosal8 (38)Brain6 (29)Liver4 (19)Ovary4 (19) Citation Format: Amanda Fitzpatrick, Marjan Iravani, Adam Mills, Eleanor Knight, Thanussuyah Alaguthurai, Vandna Shah, Alicia Okines, Nick Turner, Mark Harries, Syed Haider, Andrew Tutt, Clare Isacke. Genomic profiling of breast cancer leptomeningeal metastasis (BCLM) reveals a divergent evolution and therapeutic targets [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD13-03.