Circulating microRNA Analysis in a Prospective Co-clinical Trial Identifies MIR652-3p as a Response Biomarker and Driver of Regorafenib Resistance Mechanisms in Colorectal Cancer.

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.

Primary tumour side as a driver for treatment choice in RAS wild-type metastatic colorectal cancer patients: a systematic review and pooled analysis of randomised trials.

BACKGROUND: Retrospective subgroup analyses of previous trials in the first-line therapy of RAS wt metastatic colorectal cancer (mCRC) suggested a predictive impact of primary tumour side on the efficacy of anti-epidermal growth factor receptor (EGFR) agents. Recently, new head-to-head trials of doublets/bevacizumab versus doublets/anti-EGFR, PARADIGM and CAIRO5 were presented. PATIENTS AND METHODS: We searched for phase II and III trials comparing doublet chemotherapy plus an anti-EGFR or bevacizumab as the first-line treatment for RAS wt mCRC patients. Overall survival (OS), progression-free survival (PFS), overall response rate (ORR) and radical resection rate result in the overall study populations and, according to the primary side, were pooled together in a two-stage analysis with random effects and fixed effect models. The interaction between sidedness and treatment effect was then analysed. RESULTS: We identified five trials (PEAK, CALGB/SWOG 80405, FIRE-3, PARADIGM and CAIRO5), including 2739 patients, 77% left- and 23% right-sided. Among patients with left-sided mCRC, the use of anti-EGFRs was associated with higher ORR (74% versus 62%, OR = 1.77 [95% confidence interval {CI} 1.39-2.26-0.88], p < 0.0001), longer OS (hazard ratio [HR] = 0.77 [95% CI 0.68-0.88], p < 0.0001) and not significantly longer PFS (HR = 0.92, p = 0.19). Among patients with right-sided mCRC, the use of bevacizumab was associated with longer PFS (HR = 1.36 [95% CI 1.12-1.65], p = 0.002) and not significantly longer OS (HR = 1.17, p = 0.14). A subgroup analysis confirmed a significant interaction effect between the primary tumour side and treatment arm in terms of ORR (p = 0.02), PFS (p = 0.0004) and OS (p = 0.001). No differences in the radical resection rate were found according to treatment and sidedness. CONCLUSIONS: Our updated metanalysis corroborates the role of the primary tumour location in the choice of the upfront therapy for RAS wt mCRC patients, leading to strongly recommend anti-EGFRs in left-sided tumours and to prefer bevacizumab in the right-sided.

Molecular screening with liquid biopsy for anti-EGFR retreatment in metastatic colorectal cancer: preliminary data from the randomized phase 2 PARERE trial.

Background: Retreatment with anti-EGFR monoclonal antibodies is a promising strategy in patients with RAS/BRAF wild-type (wt) metastatic colorectal cancer (mCRC) who achieved benefit from previous anti-EGFR exposure upon exclusion of mutations in RAS/BRAF genes according to circulating tumor DNA (ctDNA) analysis by means of liquid biopsy (LB). This treatment approach is now being investigated in the randomized phase II trial PARERE (NCT04787341). We here present preliminary findings of molecular screening. Methods: Patients with RAS/BRAFV600E wt mCRC according to tissue genotyping who benefited from previous anti-EGFR-based treatment (fluoropyrimidines, oxaliplatin, irinotecan, and antiangiogenics) and then experienced disease progression to EGFR targeting were eligible for screening in the PARERE trial. The next-generation sequencing (NGS) panel Oncomine™ was employed for ctDNA testing. Results: A total of 218 patients underwent LB, and ctDNA sequencing was successful in 201 of them (92%). RAS/BRAFV600E mutations were found in 68 (34%) patients and were mainly subclonal (median variant allele fraction [VAF] for KRAS, NRAS, and BRAF mutant clones: 0.52%, 0.62%, and 0.12%, respectively; p = 0.01), with KRASQ61H being the most frequently detected (31%). Anti-EGFR-free intervals did not predict ctDNA molecular status (p = 0.12). Among the 133 patients with RAS/BRAFV600E wt tumors according to LB, 40 (30%) harbored a mutation in at least another gene potentially implied in anti-EGFR resistance, mainly with subclonal expression (median VAF, 0.56%). In detail, alterations in PIK3CA, FBXW7, GNAS, MAP2K, ERBB2, BRAF (class I and II non-BRAFV600E), SMAD, EGFR, AKT1, and CTNNB1 occurred in 13%, 8%, 7%, 3%, 2%, 2%, 1%, 1%, 1%, and 1% cases, respectively. Co-mutations were detected in 13 (33%) out of 40 patients. Conclusions: This is the largest prospective cohort of mCRC patients screened with LB for anti-EGFR retreatment in a randomized study. ctDNA genotyping reveals that at least one out of three patients candidate for retreatment should be excluded from this therapy, and other potential drivers of anti-EGFR resistance are found in approximately one out of three patients with RAS/BRAFV600E wt ctDNA.

Next-Generation Sequencing Comparative Analysis of DNA Mutations between Blood-Derived Extracellular Vesicles and Matched Cancer Tissue in Patients with Grade 4 Glioblastoma.

The biological heterogeneity of glioblastoma, IDH-wildtype (GBM, CNS WHO grade 4), the most aggressive type of brain cancer, is a critical hallmark, caused by changes in the genomic mutational asset and influencing clinical progression over time. The understanding and monitoring of the mutational profile is important not only to reveal novel therapeutic targets in this set of patients, but also to ameliorate the clinical stratification of subjects and the prognostic significance. As neurosurgery represents the primary technique to manage GBM, it is of utmost importance to optimize alternative and less invasive methods to monitor the dynamic mutation profile of these patients. Extracellular vesicles (EVs) are included in the liquid biopsy analysis and have emerged as the biological mirror of escaping and surviving mechanisms by many tumors, including glioblastoma. Very few studies have investigated the technical feasibility to detect and analyze the genomic profile by Next-Generation Sequencing (UMI system) in circulating EVs of patients with grade IV glioblastoma. Here, we attempted to characterize and to compare the corresponding matched tissue samples and potential variants with pathogenic significance of the DNA contained in peripheral-blood-derived EVs. The NGS analysis has revealed that patients with grade IV glioblastoma exhibited lesser DNA content in EVs than controls and that, both in EVs and matched cancer tissues, the NF1 gene was consistently mutated in all patients, with the c.2568C>G as the most common pathogenic variant expressed. This study supports the clinical utility of circulating EVs in glioblastoma as an eligible tool for personalized medicine.