Search

Background Pegylated arginine deiminase (ADI-PEG20; pegargiminase) depletes arginine and improves survival outcomes for patients with argininosuccinate synthetase 1 (ASS1)-deficient malignant pleural mesothelioma (MPM). Optimisation of ADI-PEG20-based therapy will require a deeper understanding of resistance mechanisms, including those mediated by the tumor microenvironment. Here, we sought to reverse translate increased tumoral macrophage infiltration in patients with ASS1-deficient MPM relapsing on pegargiminase therapy. Methods Macrophage-MPM tumor cell line (2591, MSTO, JU77) co-cultures treated with ADI-PEG20 were analyzed by flow cytometry. Microarray experiments of gene expression profiling were performed in ADI-PEG20-treated MPM tumor cells, and macrophage-relevant genetic “hits” were validated by qPCR, ELISA, and LC/MS. Cytokine and argininosuccinate analyses were performed using plasma from pegargiminase-treated patients with MPM. Results We identified that ASS1-expressing macrophages promoted viability of ADI-PEG20-treated ASS1-negative MPM cell lines. Microarray gene expression data revealed a dominant CXCR2-dependent chemotactic signature and co-expression of VEGF-A and IL-1α in ADI-PEG20-treated MPM cell lines. We confirmed that ASS1 in macrophages was IL-1α-inducible and that the argininosuccinate concentration doubled in the cell supernatant sufficient to restore MPM cell viability under co-culture conditions with ADI-PEG20. For further validation, we detected elevated plasma VEGF-A and CXCR2-dependent cytokines, and increased argininosuccinate in patients with MPM progressing on ADI-PEG20. Finally, liposomal clodronate depleted ADI-PEG20-driven macrophage infiltration and suppressed growth significantly in the MSTO xenograft murine model. Conclusions Collectively, our data indicate that ADI-PEG20-inducible cytokines orchestrate argininosuccinate fuelling of ASS1-deficient mesothelioma by macrophages. This novel stromal-mediated resistance pathway may be leveraged to optimize arginine deprivation therapy for mesothelioma and related arginine-dependent cancers.

PDF file - 315K, Immunostaining of ASS1, TS, and DHFR in representative urothelial carcinomas.

PDF file - 408K, Pyrosequencing data of bladder cancer cell lines and primary bladder cancer samples.

PDF file - 177K, Kaplan-Meier plot and log-rank test for disease-specific and metastasis-free survival.

PDF file - 65K

PDF file - 198K, Table S1 Correlation between ASS1 expression and various clinicopathological factors. Table S2 Univariate log-rank analysis for disease-specific survival and metastasis-free survival. Table S3 Correlation between ASS1 IHC and pyrosequencing of primary bladder cases. Table S4 Combination index (CI) analysis.

PDF file - 802K, Metabolomics of ADI-PEG20 in a corroborative cell line panel (mesothelioma).

Background: Detection of circulating tumour DNA (ctDNA) in patients (pts) who have completed treatment for early-stage breast cancer is associated with a high risk of future relapse. Identifying those at high risk of subsequent relapse may allow tailoring of further therapy to delay or prevent recurrence. Previous analysis of this cohort showed that tools capable of detecting ctDNA at lower concentrations are needed to increase sensitivity and lengthen the lead time between ctDNA detection and relapse. We compared ctDNA detection via a personalised sequencing assay to dPCR in patients from the cTRAK TN clinical trial. Methods: The cTRAK-TN trial recruited 161 pts into prospective ctDNA surveillance with dPCR, with ctDNA positive pts randomised to 1) CT staging plus pembrolizumab therapy for patients without relapse or 2) observation. Pts had serial post-treatment surveillance plasma samples collected every 3 months for up to 2 years. Whole exome sequencing (WES) was performed on tumor DNA from FFPE samples to design personalised Residual Disease and Recurrence (RaDaR®) multiplex PCR based NGS assays. Retrospectively, plasma DNA extracted from a minimum of 2mls banked plasma, was sequenced with personalised RaDaR assays, and ctDNA detection identified with a proprietary algorithm. dPCR assays tracked 1-2 mutations, as previously described. Primary endpoint was rate of positive ctDNA detection by 12 months from start of surveillance in both assays. Secondary endpoints were agreement in ctDNA detection between RaDaR and dPCR assays and lead-time between ctDNA detection and disease recurrence. Results: Overall, 147 pts and 241 tissue samples were subject to WES, and RaDaR assays were developed for 142 pts with sufficient plasma for testing. RaDaR assays tracked a median of 47 variants (range 33-56) per patient, and a total of 907 timepoints were analysed (median 6 timepoints per pt, range 1-11). With RaDaR, 39.4% (56/142) patients tested ctDNA positive during follow-up, with a median ctDNA detected level of 0.081% estimated variant allele fraction (eVAF). With dPCR, 35.2% (50/142) pts tested ctDNA positive. The ctDNA detection rate by 12 months from the start of ctDNA surveillance was 36.2% (95% CI; 27.6% – 43.7%) with RaDaR and 29.9% (95%CI; 21.6% – 37.3%) with dPCR. The overall test agreement between RaDaR and dPCR assays was 92.7% (95%CI; 90.7% – 94.4%). From a patient perspective, 58.7% pts were ctDNA negative for both assays, 32.9% ctDNA were positive for both assays and 8.6% presented discrepancies. ctDNA was detected by RaDaR but not by dPCR in 9 pts and it was detected by dPCR but not by RaDaR in 3 pts. Among ctDNA positive pts, 55.2% were first detected positive by RaDaR, 5.2% by dPCR, and 39.6% were detected at the same time-point (test of proportions, p< 0.001). The median lead time from ctDNA detection to relapse was 7.1 months (95% CI 5.9 – 15.9%) with RaDaR and 5.7 months (95% CI 3.2% – 7.4%) with dPCR. Conclusion: The RaDaR personalised multi-mutation sequencing assay detected MRD with a longer median lead time prior to relapse, and with higher sensitivity, than dPCR mutation tracking assays. These findings have implications for the choice of ctDNA assay in clinical trials designed to treat patients at the point of MRD detection. Citation Format: Maria Coakley, Prithika Sritharan, Guillermo Villacampa, Claire Swift, Kathryn Dunne, Lucy Kilburn, Katie Goddard, Patricia Rojas, Andy Joad, Warren Emmett, Charlene Knape, Karen Howarth, Peter S. Hall, Catherine Harper-Wynne, Tamas Hickish, Iain Macpherson, Alicia F. Okines, Andrew M. Wardley, Duncan Wheatley, Simon Waters, Rosalind Cutts, Isaac Garcia-Murillas, Judith Bliss, Nicholas Turner. PD5-03 Comparison of a personalized sequencing assay and digital PCR for circulating tumor DNA based Molecular Residual Disease detection in early-stage triple negative breast cancer in the cTRAK-TN trial [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 PD5-03.

Introduction: Identification of Molecular Residual Disease (MRD) in patients with breast cancer with circulating tumor DNA (ctDNA) presents a strategy to identify patients at high risk of relapse. Approaches that detect ctDNA at lower concentrations are required to increase sensitivity and improve on the lead time between ctDNA detection and clinical relapse. Here we present results using novel highly sensitive tumor-informed sequencing assays for ctDNA detection of MRD based on detection of multiple patient specific mutations in ctDNA. Methods: 62 stage II-III breast cancer patients (23 hormone receptor positive HER2 negative (HR+HER2-), 20 HER2+, 15 triple negative breast cancer (TNBC) and 4 unknown receptor status) enrolled in the ChemoNEAR sample collection study were included. All patients received neoadjuvant chemotherapy, followed up by surgery, with samples taken at diagnosis, and post-surgery every 3 months for the first two years, followed by every 6 months for up to five years. Tumor DNA from FFPE samples and germline was Whole Exome Sequenced to identify patient specific mutations and design anchored-multiplex PCR (AMP™) Personalized Cancer Monitoring (PCMTM) assays to track mutations in plasma. Cell free DNA was extracted from 613 plasma samples (median volume 4ml, range 0.5-4.5ml) and sequenced with PCMTM assays, with 37-177 variants (median 52) per panel, to a depth of 100,000x per locus. A proprietary algorithm was used to identify ctDNA. Results: At a median follow-up of 52.7 months post-surgery (range 15.3-96.4 months), ctDNA was detected in 25.8% (16/62) of patients, with detected ctDNA levels ranging from allele frequency (AF) of 0.01%, to 32.5%) (median 0.24% AF). Detection of ctDNA was associated with a high risk of future relapse (HR 65.4, 95% CI 14.5-293.7), with a median lead-time from ctDNA detection to clinical relapse of 13.7 months (range 3.9-58.9). MRD was identified in 76.9% (10/13) of patients who relapsed. ctDNA was detected prior to relapse in both patients with brain only relapse, but with a reduced lead time over clinical relapse (5.73 and 3.90 months), which was previously not achievable with digital PCR MRD-detection assays. Of patients with assessable baseline samples, 81% (39/48) had ctDNA detected. No patients with undetected ctDNA, or detectable ctDNA with AF< 0.1%, relapsed during follow-up, whereas ctDNA was detected at baseline in all 10 patients who relapsed during follow-up (p=0.1). Conclusions: PCMTM detected breast cancer relapse with a long lead-time over clinical relapse, and strong association with relapse free survival, an advancement over previously published data with digital PCR MRD detection. Prospective, interventional trials are now required to assess whether treatment on the basis of MRD detection improves outcome, including the TRAK ER Trial (NCT04985266). Citation Format: Isaac Garcia-Murillas, Giselle Walsh-Crestani, Edward Phillips, Rosalind Cutts, Sarah Hrebien, Kathryn Dunne, Kally Sidhu, Robert Daber, Amber C. Carter, Lorena De La Peña, Stephen Johnston, Alistair Ring, Simon Russell, Abigail Evans, Anthony Skene, Duncan Wheatley, Ian Smith, Nicholas Turner. Personalized Cancer Monitoring (PCM): a novel ctDNA tool to detect molecular residual disease in patients with early-stage breast cancer [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 P5-05-01.