Regorafenib Induces Senescence and Epithelial-Mesenchymal Transition in Colorectal Cancer to Promote Drug Resistance.

Potential intrinsic resistance mechanisms to regorafenib were explored after short exposure (3 days) on five CRC cell lines (HCT-116, SW1116, LS-1034, SW480, Caco-2). The observation of senescence-like features led to the investigation of a drug-initiated phenotype switch. Following long-term exposure (12 months) of HCT-116 and SW480 cell lines to regorafenib, we developed resistant models to explore acquired resistance. SW480 cells demonstrated senescent-like properties, including a cell arrest in the late G2/prophase cell cycle stage and a statistically significant decrease in the expression of G1 Cyclin-Dependent Kinase inhibitors and key cell cycle regulators. A specific senescence-associated secretome was also observed. In contrast, HCT-116 treated cells presented early senescent features and developed acquired resistance triggering EMT and a more aggressive phenotype over time. The gained migration and invasion ability by long-exposed cells was associated with the increased expression level of key cellular and extracellular EMT-related factors. The PI3K/AKT pathway was a significant player in the acquired resistance of HCT-116 cells, possibly related to a PI3KCA mutation in this cell line. Our findings provide new insights into the phenotypic plasticity of CRC cells able, under treatment pressure, to acquire a stable TIS or to use an early senescence state to undergo EMT.

Circulating DNA in the neoadjuvant setting of early stage colon cancer.

BACKGROUND: While circulating tumour (ct)DNA is an indicator of minimal residual disease and negative prognostic factor in stage II-III colon cancer, no study has ever analysed the value of this biomarker in colon cancer patients treated with neoadjuvant chemotherapy. We sought to fill this gap by using prospectively collected plasma samples from 80 stage III colon cancer patients, receiving one cycle of neoadjuvant FOLFOX followed by surgery +/- adjuvant FOLFOX in the PePiTA trial. MATERIAL AND METHODS: Samples were collected at baseline, 2 weeks and surgery. NPY and WIF1 were selected as universal methylation markers for ctDNA, and analysed with ddPCR technology. ROC curves were applied for cut-off points, and outcome measures included 5-year disease-free survival (DFS) and 6-year overall survival (OS). RESULTS: After a median follow-up of 52.5 months, baseline circulating-free (cf) DNA was an independent prognostic factor for DFS (HR 3.35, 95% CI: 1.15-9.77, p = .03), and a trend towards a similar association was observed for relative cfDNA changes between baseline and surgery (HR 2.57, 95% CI: 0.94-7.05, p = .07). Among 60 ctDNA assessable patients, 25 (42%) had detectable ctDNA at baseline. While detection of ctDNA at any pre-operative timepoint was not associated with outcome, patients with ctDNA increase (change of the worst trending methylation marker ≥11%, or mean ctDNA change of NPY and WIF1 ≥ 0%) between baseline and surgery showed a trend towards worse 5-year DFS (HR 3.66, 95% CI: 0.81-16.44, p = .09). CONCLUSION: This is the first study of ctDNA in the neoadjuvant setting of early-stage colon cancer. Results are hypothesis-generating and should be confirmed in larger series.

Sex and Regorafenib Toxicity in Refractory Colorectal Cancer: Safety Analysis of the RegARd-C Trial.

BACKGROUND: Regorafenib is a standard treatment for refractory metastatic colorectal cancer (mCRC). In view of the toxicity burden, significant research efforts have been made to increase the therapeutic ratio of this multikinase inhibitor. Predictive factors for treatment-related adverse events (TRAEs), however, are still lacking. MATERIALS AND METHODS: We assessed the association between a number of baseline clinical, laboratory and imaging parameters and the occurrence of TRAEs in 136 patients who had received regorafenib (160 mg/day, 3-weeks-on/1-week-off) in a prospective phase II clinical trial. RESULTS: Grade ≥ 2 TRAEs during the first cycle of treatment (84% vs. 60%, P = .002) and grade ≥ 3 TRAEs throughout the whole treatment (71% vs. 53%, P = .035) occurred more frequently in females, with sex being the only independent predictive factor of early and any-time toxicity (OR 3.4; 95% CI: 1.2-11.1, P = .02 and OR 2.1; 95% CI: 1.0-4.4, P = .045, respectively). Fatigue, anorexia, hypertension, and rash were reported significantly more frequently by females than males (P < .04). Females were also more likely to suffer early (19% vs. 5%, P = .014) and any-time serious AEs (28% vs. 9%, P = .005), and to require early dose modifications (55% vs. 37%, P = .055). CONCLUSION: This is the first study showing an association between sex and TRAEs during regorafenib treatment for mCRC. If confirmed in larger, independent series, these results could pave the way for the implementation of personalized regorafenib dosing strategies with the potential to optimize oncological outcomes while reducing toxicity and preserving quality of life.

Prognostic Value of the Pace of Tumor Progression as Assessed by Serial 18F-FDG PET/CT Scan and Liquid Biopsy in Refractory Colorectal Cancer: The CORIOLAN Trial.

INTRODUCTION: Decision making in refractory colorectal cancer (rCRC) is challenging, with limited data available to predict patient outcome. We conducted a study to assess the pace of cancer progression as a potential prognostic and decision tool. METHODS: CORIOLAN was a prospective, single-center, single-arm trial recruiting refractory CRC patients with an ECOG performance status of ≤1 and an estimated life expectancy of ≥12 weeks. 18 fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) scan and blood sample collection were carried out at baseline and after 2 weeks with no cancer treatment given between these timepoints. The primary objective was to evaluate the association between pace of cancer progression as defined by changes of the whole-body metabolically active tumor volume (WB-MATV) and overall survival (OS). Exploratory objectives included evaluation of the prognostic value of circulating cell-free DNA (cfDNA), circulating tumor cells (CTCs) and carcinoembryonic antigen (CEA). RESULTS: 47 eligible patients who had received a median number of 5 (range 2-8) prior treatments were enrolled. At the time of analysis, 45 deaths had occurred, with 26% of patients dying within 12 weeks. The median OS was 6.3 months (range 0.4-14.3). The median relative delta between WB-MATV at baseline and 2 weeks was +21%. Changes of WB-MATV, however, failed to predict OS (hazard ratio (HR) 1.3, p = 0.383). Similarly, no association was observed between changes of any of the circulating biomarkers investigated and prognosis. By contrast, high WB-MATV (4.2 versus 9.4 months; HR 3.1, p = 0.003), high CEA (4.4 versus 7.0 months; HR 1.9, p = 0.053), high cfDNA (4.7 versus 7.0 months; HR 2.2, p = 0.015) and high CTC count (3.3 versus 7.5 months; HR 6.5, p < 0.001) at baseline were associated with worse OS. CONCLUSIONS: In this study, approximately 1 out of 4 refractory CRC patients who were judged to have a life expectancy >12 weeks actually died within 12 weeks. Baseline assessment of WB-MATV, cfDNA, CTCs and CEA, but not early change evaluation of the same, may help to refine patient prognostication and guide management decisions.

Feasibility and clinical impact of routine molecular testing of gastrointestinal cancers at a tertiary centre with a multi-gene, tumor-agnostic, next generation sequencing panel.

BACKGROUND: High-throughput sequencing technologies are increasingly used in research but limited data are available on the feasibility and value of these when routinely adopted in clinical practice. MATERIAL AND METHODS: We analyzed all consecutive cancer patients for whom genomic testing by a 48-gene next-generation sequencing (NGS) panel (Truseq Amplicon Cancer Panel, Illumina) was requested as part of standard care in one of the largest Belgian cancer networks between 2014 and 2019. Feasibility of NGS was assessed in all study patients, while the impact of NGS on the decision making was analyzed in the group of gastrointestinal cancer patients. RESULTS: Tumor samples from 1064 patients with varying tumor types were tested, the number of NGS requests increasing over time (p < .0001). Success rate and median turnaround time were 91.4% and 12.5 days, respectively, both significantly decreasing over time (p ≤ .0002). Non-surgical sampling procedure (OR 7.97, p < .0001), tissue from metastatic site (OR 2.35, p = .0006) and more recent year of testing (OR 1.79, p = .0258) were independently associated with NGS failure. Excluding well-known actionable or clinically relevant mutations which are recommended by international guidelines and commonly tested by targeted sequencing, 57/279 (20.4%) assessable gastrointestinal cancer patients were found to have tumors harboring at least one actionable altered gene according to the OncoKB database. NGS results, however, had a direct impact on management decisions by the treating physician in only 3 cases (1.1%). CONCLUSIONS: Our findings confirm that NGS is feasible in the clinical setting with acceptably low failure rates and rapid turnaround time. In gastrointestinal cancers, however, NGS-based multiple-gene testing adds very little to standard targeted sequencing, and in routine practice the clinical impact of NGS panels including genes which are not routinely recommended by international guidelines remains limited.

Combining 18F-FDG PET/CT-Based Metabolically Active Tumor Volume and Circulating Cell-Free DNA Significantly Improves Outcome Prediction in Chemorefractory Metastatic Colorectal Cancer.

Baseline whole-body metabolically active tumor volume (WB-MATV) measured by 18F-FDG PET/CT and circulating cell-free DNA (cfDNA) have been separately validated as predictors of overall and progression-free survival (OS/PFS) in chemorefractory metastatic colorectal cancer (mCRC) patients. This study assessed the correlation between WB-MATV and cfDNA, evaluating the added prognostic value of these in combination, along with clinical parameters. Methods: Of 141 mCRC patients included in a prospective multicenter trial, 132 were evaluable for OS/PFS. cfDNA was extracted from 3 mL of plasma and quantified using a fluorometer. All target lesions were delineated on 18F-FDG PET/CT, and their metabolic volumes were summed to obtain the WB-MATV. Results: Baseline WB-MATV and cfDNA were strongly correlated (r = 0.70; P < 0.001) but showed discordance in 23 of 132 (17%) patients. A multivariate analysis identified 3 independent negative predictors of PFS (high cfDNA, short time since diagnosis, and body mass index < 30) and 5 of OS (high cfDNA, high WB-MATV, body mass index < 30, poor performance status, and short time since diagnosis). Combining WB-MATV and cfDNA increased the overall prognostic value and allowed identification of a subgroup of patients with low cfDNA and high WB-MATV who were associated with intermediate survival (median OS of 8.1 for low-cfDNA/high-MATV patients vs. 12.7 mo for low-cfDNA/low-MATV patients; hazard ratio, 2.04; P = 0.02). Conclusion: This study confirms the added prognostic value of combined circulating cfDNA and PET-based WB-MATV in chemorefractory mCRC patients. The combination of these two biomarkers should provide a firm basis for risk stratification, both in clinical practice and in research trials.

Circulating tumor DNA in early response assessment and monitoring of advanced colorectal cancer treated with a multi-kinase inhibitor.

Predictive biomarkers are eagerly awaited in advanced colorectal cancer (aCRC). Targeted sequencing performed on tumor and baseline plasma samples in 20 patients with aCRC treated with regorafenib identified 89 tumor-specific mutations of which ≥50% are also present in baseline plasma. Droplet digital PCR (ddPCR) assays were optimized to monitor circulating tumor DNA (ctDNA) levels in plasmatic samples collected throughout the treatment course and showed the importance of using the absolute value for ctDNA rather than the mutant/wild type ratio in monitoring the therapy outcome. High baseline cell free DNA (cfDNA) levels are associated with shorter overall survival (OS) (HR 7.38, P=0.001). An early increase (D14) in mutated copies/mL is associated with a significantly worse PFS (HR 6.12, P=0.008) and OS (HR 8.02, P=0.004). These data suggest a high prognostic value for early ctDNA level changes and support the use of blood-born genomic markers as a tool for treatment.

Thyroid follicular adenomas and carcinomas: molecular profiling provides evidence for a continuous evolution.

Non-autonomous thyroid nodules are common in the general population with a proportion found to be cancerous. A current challenge in the field is to be able to distinguish benign adenoma (FA) from preoperatively malignant thyroid follicular carcinoma (FTC), which are very similar both histologically and genetically. One controversial issue, which is currently not understood, is whether both tumor types represent different molecular entities or rather a biological continuum. To gain a better insight into FA and FTC tumorigenesis, we defined their molecular profiles by mRNA and miRNA microarray. Expression data were analyzed, validated by qRT-PCR and compared with previously published data sets. The majority of deregulated mRNAs were common between FA and FTC and were downregulated, however FTC showed additional deregulated mRNA. Both types of tumors share deregulated pathways, molecular functions and biological processes. The additional deregulations in FTC include the lipid transport process that may be involved in tumor progression. The strongest candidate genes which may be able to discriminate follicular adenomas and carcinomas, CRABP1, FABP4 and HMGA2, were validated in independent samples by qRT-PCR and immunohistochemistry. However, they were not able to adequately classify FA or FTC, supporting the notion of continuous evolving tumors, whereby FA and FTC appear to show quantitative rather than qualitative changes. Conversely, miRNA expression profiles showed few dysregulations in FTC, and even fewer in FA, suggesting that miRNA play a minor, if any, role in tumor progression.