Background The T cell bispecific antibody cibisatamab (CEA-TCB) binds Carcino-Embryonic Antigen (CEA) on cancer cells and CD3 on T cells, which triggers T cell killing of cancer cell lines expressing moderate to high levels of CEA at the cell surface. Patient derived colorectal cancer organoids (PDOs) may more accurately represent patient tumors than established cell lines which potentially enables more detailed insights into mechanisms of cibisatamab resistance and sensitivity. Methods We established PDOs from multidrug-resistant metastatic CRCs. CEA expression of PDOs was determined by FACS and sensitivity to cibisatamab immunotherapy was assessed by co-culture of PDOs and allogeneic CD8 T cells. Results PDOs could be categorized into 3 groups based on CEA cell-surface expression: CEA_hi (n = 3), CEA_lo (n = 1) and CEA_mixed PDOs (n = 4), that stably maintained populations of CEA_hi and CEA_lo cells, which has not previously been described in CRC cell lines. CEA_hi PDOs were sensitive whereas CEA_lo PDOs showed resistance to cibisatamab. PDOs with mixed expression showed low sensitivity to cibisatamab, suggesting that CEA_lo cells maintain cancer cell growth. Culture of FACS-sorted CEA_hi and CEA_lo cells from PDOs with mixed CEA expression demonstrated high plasticity of CEA expression, contributing to resistance acquisition through CEA antigen loss. RNA-sequencing revealed increased WNT/β-catenin pathway activity in CEA_lo cells. Cell surface CEA expression was up-regulated by inhibitors of the WNT/β-catenin pathway. Conclusions Based on these preclinical findings, heterogeneity and plasticity of CEA expression appear to confer low cibisatamab sensitivity in PDOs, supporting further clinical evaluation of their predictive effect in CRC. Pharmacological inhibition of the WNT/β-catenin pathway may be a rational combination to sensitize CRCs to cibisatamab. Our novel PDO and T cell co-culture immunotherapy models enable pre-clinical discovery of candidate biomarkers and combination therapies that may inform and accelerate the development of immuno-oncology agents in the clinic.

Purpose: The multi-kinase inhibitor (mKi) regorafenib has demonstrated efficacy in chemorefractory patients with metastatic colorectal cancer (mCRC). However, lack of predictive biomarkers and concerns over significant toxicities hamper the use of regorafenib in clinical practice. Experimental Design: Serial liquid biopsies were obtained at baseline and monthly until disease progression in chemorefractory patients with mCRC treated with regorafenib in a phase II clinical trial (PROSPECT-R n = 40; NCT03010722) and in a multicentric validation cohort (n = 241). Tissue biopsies collected at baseline, after 2 months and at progression in the PROSPECT-R trial were used to establish patient-derived organoids (PDO) and for molecular analyses. MicroRNA profiling was performed on baseline bloods using the NanoString nCounter platform and results were validated by digital-droplet PCR and/or ISH in paired liquid and tissue biopsies. PDOs co-cultures and PDO-xenotransplants were generated for functional analyses. Results: Large-scale microRNA expression analysis in longitudinal matched liquid and tissue biopsies from the PROSPECT-R trial identified MIR652–3p as a biomarker of clinical benefit to regorafenib. These findings were confirmed in an independent validation cohort and in a “control” group of 100 patients treated with lonsurf. Using ex vivo co-culture assays paired with single-cell RNA-sequencing of PDO established pre- and post-treatment, we modeled regorafenib response observed in vivo and in patients, and showed that MIR652–3p controls resistance to regorafenib by impairing regorafenib-induced lethal autophagy and by orchestrating the switch from neo-angiogenesis to vessel co-option. Conclusions: Our results identify MIR652–3p as a potential biomarker and as a driver of cell and non–cell-autonomous mechanisms of resistance to regorafenib.

Objectives: Percutaneous liver biopsy (PLB) poses specific challenges in oncological patients such as bleeding and tumour seeding. This study’s aim was to compare a coaxial (C-PLB) and non-coaxial (NC-PLB) biopsy technique in terms of diagnostic yield, safety and seeding risk of image-guided PLB techniques in an oncological setting. Methods: Local research committee approval was obtained for this single-site retrospective study. Patients who underwent a PLB between November 2011 and December 2017 were consecutively included. Medical records were reviewed to determine diagnostic yield and complications. Follow-up imaging was re-reviewed for seeding, defined as visible tumour deposits along the PLB track. Mann-Whitney U and chi-squared tests were performed to investigate differences between biopsy techniques in sample number, complications and seeding rate. Results: In total, 741 patients (62 ± 13 years, 378 women) underwent 932 PLB (C-PLB 72.9% (679/932); NC-PLB 27.1% (253/932)). More tissue cores (p < 0.001) were obtained with C-PLB (median 4 cores; range 1–12) compared with NC-PLB (2 cores; range 1–4) and diagnostic yield was similar for both techniques (C-PLB 92.6% (629/679); NC-PLB 92.5% (234/253); p = 0.940). Complication rate (9.3%; 87/932) using C-PLB (8.2% (56/679)) was lower compared with NC-PLB (12.3% (31/253); p = 0.024). Major complications were uncommon (C-PLB 2.7% (18/679); NC-PLB 2.8% (7/253)); bleeding developed in 1.2% (11/932; C-PLB 1.2% (8/679); NC-PLB 1.2% (3/253)). Seeding was a rare event, occurring significantly less in C-PLB cases (C-PLB 1.3% (7/544); NC-PLB 3.1% (6/197); p = 0.021). Conclusions: C-PLB allows for high diagnostic tissue yield with a lower complication and seeding rate than a NC-PLB and should be the preferred method in an oncological setting. Key Points: • A coaxial percutaneous liver biopsy achieves a significant higher number of cores and fewer complications than a non-coaxial biopsy technique. • The risk of tumour seeding is very low and is significantly lower using the coaxial biopsy technique. • In this study, a larger number of cores (median = 4) could be safely acquired using the coaxial technique, providing sufficient material for advanced molecular analysis.

Purpose: Radiofrequency ablation (RFA) is a curative treatment option for small lung metastases, which conventionally involves multiple freehand manipulations until the treating electrode is satisfactorily positioned. Stereotactic and robotic guidance has been gaining popularity for liver ablation, although has not been established in lung ablation. The purpose of this study is to determine the feasibility, safety, and accuracy of robotic RFA for pulmonary metastases, and compare procedures with a conventional freehand cohort. Methods: A single center study with prospective robotic cohort, and retrospective freehand cohort. RFA was performed under general anesthesia using high frequency jet ventilation and CT guidance. Main outcomes were (i) feasibility/technical success (ii) safety using Common Terminology Criteria for Adverse Events (iii) targeting accuracy (iv) number of needle manipulations for satisfactory ablation. Robotic and freehand cohorts were compared using Mann–Whitney U tests for continuous variables, and Fisher’s exact for categorical variables. Results: Thirty-nine patients (mean age 65 ± 13 years, 20 men) underwent ablation of 44 pulmonary metastases at single specialist cancer center between July 2019 and August 2022. 20 consecutive participants underwent robotic ablation, and 20 consecutive patients underwent freehand ablation. All 20/20 (100%) robotic procedures were technically successful, and none were converted to freehand procedures. There were 6/20 (30%) adverse events in the robotic cohort, and 15/20 (75%) in the freehand cohort (P = 0.01). Robotic placement was highly accurate with 6 mm tip-to-target distance (range 0–14 mm) despite out-of-plane approaches, with fewer manipulations than freehand placement (median 0 vs. 4.5 manipulations, P < 0.001 and 7/22, 32% vs. 22/22, 100%, P < 0.001). Conclusions: Robotic radiofrequency ablation of pulmonary metastases with general anesthesia and high frequency jet ventilation is feasible and safe. Targeting accuracy is high, and fewer needle/electrode manipulations are required to achieve a satisfactory position for ablation than freehand placement, with early indications of reduced complications.