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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.

Predictive factors for adverse events in metastatic colorectal cancer patients treated with regorafenib are currently lacking. In this safety analysis of a prospective phase II clinical trial, we assess the association between several clinical, laboratory, and imaging parameters and the occurrence of adverse events. Our results show that female sex is an independent risk factor for increased toxicity. 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

SCOPUS: le.j, info:eu-repo/semantics/published

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 &le, 1 and an estimated life expectancy of &ge, 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&ndash, 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&ndash, 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 &lt, 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 &gt, 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.

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.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.Tumor samples from 1064 patients with varying tumor types were tested, the number of NGS requests increasing over time (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.

Introduction Circulating tumour (ct)DNA is an indicator of minimal residual disease and negative prognostic factor in stage II-III CC treated with surgery +/- adjuvant chemotherapy. No study, however, has ever analysed the prognostic value of this biomarker in CC patients (pts) treated with neoadjuvant chemotherapy. We sought to evaluate the prognostic value of baseline and early, on-treatment changes of cfDNA and ctDNA in stage II-III CC pts who were treated with one cycle of neoadjuvant FOLFOX chemotherapy followed by surgery +/- adjuvant FOLFOX chemotherapy in the PePiTA trial. Methods PePITA was a multicentre, single-arm, prospective phase II trial aiming to test in vivo tumour chemosensitivity as assessd by metabolic response using 18F-FDG PET/CT scan of early stage CC and to evaluate its association with survival outcome (NCT00994864). Plasma samples were prospectively collected at baseline and 2 weeks (ie, after one cycle of neoadjuvant FOLFOX chemotherapy). cfDNA was isolated with the QIAmp circulating nucleic acid kit (Qiagen), and quantified with the Qubit fluorometer (Life-Technologies). cfDNA samples were bisulfite converted using the EZ DNA Methylation-Gold™ Kit (Zymo Research), with NPY and WIF1 being selected as universal methylation markers for ctDNA and analysed with digital droplet (dd)PCR technology. Data from ddPCR were processed with the QuantaSoft V1.6 software (BioRad). The primary outcome measure was 3-year disease-free survival (DFS). Receiver operating characteristics curve analyses, Kaplan-Meier method, cox proportional hazards models and log-rank tests were used. Statistical analyses were carried out with the SPSS for MacOS version 25.0 (SPSS Inc). Results 80 pts were included (ypStage I-II 56%, ypStage III 44%). After a median follow-up of 52.5 months, 3-year DFS was 80% (95%CI 71.2-90.8) and 5-year OS 84% (95%CI 75.2-94.9). Pts with high (≥1.2 ng/µl) baseline cfDNA level had worse 3-year DFS (48% vs 80%; HR 2.72, 95%CI 1.02-7.25; p=0.036) and 5-year OS (71% vs 90%; HR 5.36, 95%CI 1.14-25.28; p=0.017) than those with low baseline cfDNA level. In a multivariable analysis (including sex, ypStage and CEA), baseline cfDNA was the only factor showing a trend towards statistical significance (HR DFS 2.6, 95%CI 0.96-7.01; p=0.059; HR OS 4.65, 95%CI 0.97-22.32; p=0.055). Early changes of cfDNA (Δ ≥11%) after one cycle of neoadjuvant FOLFOX chemotherapy failed to predict survival (HR DFS 1.08, 95%CI 0.42-2.81; p=0.873; HR OS 0.68, 95%CI 0.19-2.39; p=0.543). ctDNA analyses are ongoing and will be presented at the meeting. Conclusions For the first time, we have shown that baseline cfDNA may predict survival outcome in early stage CC pts treated with neoadjuvant chemotherapy. Pending confirmation in larger series, testing for cfDNA at baseline could help select high-risk pts who may benefit from neoadjuvant, FOXTROT-like, treatment strategies. Citation Format: Giacomo Bregni, Elena Trevisi, Chiara Senti, Andrea Pretta, Caroline Vandeputte, Pashalina Kehagias, Elena Acedo Reina, Amélie Deleporte, Paraskevas Gkolfakis, Jean-Luc Van Laethem, Philippe Vergauwe, Marc Van den Eynde, Guido Deboever, Jos Janssens, Gauthier Demolin, Stephane Holbrechts, Marylene Clausse, Thierry De Grez, Marc Peeters, Lionel D'Hondt, Karen Geboes, Tatiana Besse-Hammer, Alexis Buggenhout, Françoise Rothé, Patrick Flamen, Alain Hendlisz, Francesco Sclafani. Prognostic value of baseline and early changes of circulating cell-free (cf)DNA in the neoadjuvant setting of early stage colon cancer (CC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 28.

The introduction of the multi-targeted tyrosine kinase inhibitor (TKI) regorafenib in clinical routine improve patients' survival with metastatic CRC who failed to respond to standard therapies, however at the cost of significant toxicities. Moreover, patients' tumor exhibit heterogeneous responses and become resistant. Its mechanism of action remains largely unknown, challenging the selection of patients through the identification of clinically useful predictive biomarkers. This study aimed to explore the effects on human CRC cell lines of short- and long-term exposure to regorafenib and investigate resistance-related mechanisms. Two representative CRC cell lines were used, HCT-116 and SW480. Short-term (3 days) and long-term exposure to IC50 values (HCT-116, 3µM and 6µM; SW480, 5µM and 6µM respectively) were intended to explore intrinsic and acquired resistance. The observation of early morphological changes (increased cell volume) in SW480 cells after regorafenib exposure led us to investigate a drug-initiated senescence-like phenotype commonly associated with resistance to treatment allowing cell death escape. These cells acquired stable senescent-like properties (slow-cycling cells, high β-galactosidase activity) and the majority of them were arrested in the G2/M cell cycle phase after long treatment exposure. A specific senescence-associated secretome (high sIL6Rα in cell supernatants) was also observed. In contrast, HCT-116 cells exposed to regorafenib presented early senescent features (high β-galactosidase activity) but developed over time an acquired resistance triggering EMT (high migration activity, upregulation of EMT-related factors) also known to contribute to TKI resistance. Moreover, the investigation of MAPK and PI3K/AKT pathways pointed to the latter as a significant player in acquired resistance of HCT-116 cells (increase of AKT phosphorylation) possibly related to the presence of a PI3KCA mutation in this cell line. Our findings provide new insights into the phenotypic plasticity of CRC cells under treatment pressure able to (1) either acquire stable senescent-like properties or (2) use early senescence state to undergo EMT. Our findings need further investigations of the major determinants involved in regorafenib-induced phenotype-switching and may help to develop new therapeutic strategies to overcome related resistances. Citation Format: Pashalina Kehagias, Mohammad Krayem, Nadège Kindt, Ahmad Najem, Caroline Vandeputte, Fabrice Journé, Ahmad Awada, Ghanem E. Ghanem, Alain Hendlisz. Regorafenib resistance is associated with senescence-like phenotype and EMT in colorectal cancer (CRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1082.

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 5 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 5 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., SCOPUS: ar.j, info:eu-repo/semantics/published

Baseline whole-body metabolically active tumor volume (WB-MATV) measured by

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., SCOPUS: ar.j, info:eu-repo/semantics/published

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., SCOPUS: ar.j, info:eu-repo/semantics/published

Purpose: Circulating tumor DNA (ctDNA) monitoring based on an individual mutation profile during therapy is under intense investigation in modern oncology. We previously reported that the increase of ≥50% of at least one somatic mutation among multiple monitored mutations per patient is associated with a significantly worse outcome1. This study investigates whether the ctDNA monitoring of one driver gene mutation, provides enough information as compared to multiple mutations to assess response to regorafenib in advanced chemorefractory colorectal cancer (aCRC) at an early timepoint. Experimental procedures: Archival tumor tissue and plasma samples (PL) at baseline (BL) and 14 days (D14) after treatment initiation in aCRC pts (n=141) were prospectively collected in the RegARd-C multicenter clinical trial (NCT01929616). Somatic mutations were identified based on a CRC-oriented targeted gene sequencing of tumor tissue. All available (median 2 (1-4)) driver gene mutations were monitored per patient in PL at BL and D14 via droplet digital PCR (Bio-Rad QX200 ddPCR system) to assess ctDNA dynamics. Results: In 96 evaluable patients, the most frequently monitored mutated genes were APC (73%), TP53 (72%), KRAS (66%), and PI3KCA (23%). Among patients with ≥2 monitored mutations (73/96), one was selected at random and compared to previous methodology taking in account dynamics of all followed mutations. Optimal cutoff (CO) evaluation (Contal & O’Quigley method) separated patients (n=96) according to a ctDNA increase of ≥50% versus an increase of Conclusion: The monitoring of ctDNA dynamics based on only one randomly selected driver gene mutation versus multiple is equally informative to describe adequately aCRC patients’ outcome under regorafenib after 14 days of treatment onset. Especially, combined with pre-treatment ctDNA levels, this simplifies a personalized patient monitoring. 1 P. Kehagias, et al. Circulating tumor DNA detects early response to regorafenib in advanced colorectal cancer [abstract]. AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-221 Citation Format: Pashalina Kehagias, Caroline Vandeputte, Lieveke Ameye, Hakim El Housni, Amélie Deleporte, Karen Geboes, Thierry Delaunoit, Gauthier Demolin, Marc Peeters, Lionel D'Hondt, Jos Janssens, Javier Carrasco, Maria Gomez Galdon, Pierre Heimann, Marianne Paesmans, Patrick Flamen, Alain Hendlisz. Is a single driver gene mutation sufficient for monitoring early response in advanced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2293.

Purpose: To demonstrate that an early increase in ctDNA plasmatic levels after 14 days may identify patients (pts) with advanced chemorefractory colorectal cancer (aCRC) unlikely to benefit from regorafenib. Experimental procedures: The RegARd-C prospective multicentric clinical trial (NCT01929616) collected archival tumor tissue and plasma samples at baseline (BL), 14 days (D14) and every 2 months after treatment initiation in aCRC pts (n=141). A targeted gene sequencing was performed on pts' tumor tissue to identify tumor-specific mutations using a panel of 47 genes commonly mutated in CRC. To monitor circulating tumor DNA (ctDNA) levels in plasma samples collected at BL and D14 via droplet digital PCR (Bio-Rad QX200 ddPCR system), we selected tumor-specific mutations based on their allelic frequency. Results: As foreseen, in 96 pts the most common mutated genes were APC (73%), TP53 (72%), KRAS (66%), and PI3KCA (23%). Among these pts, 24 (25%), 49 (51%), 18 (19%) and 5 (5%) had respectively one, two, three and four tumor-specific mutations followed via ddPCR. On average 2 (range 1-4) tumor-specific mutations were followed per patient. In total, 65 specific paired PrimePCRTM ddPCRTM Mutation Detection Assays were optimized following digital MIQE guidelines to assess ctDNA levels. As previously described1, ctDNA levels were assessed in terms of absolute values of mutated copies per mL of plasma to avoid bias in the mutated/mutated + wild-type copies ratio. We considered an increase in ctDNA level of at least 12% as significant2. Pts were separated according to the presence (n=53) or absence (n=43) of at least one increasing mutation between BL and D14. An increase of at least one tumor-specific mutation as compared to none is associated with a significantly worse clinical outcome with a median PFS of 1.3 months versus 3.0 months (HR 1.88, P=0.002, 95% CI 1.24-2.85) and a median OS of 3.9 months versus 8.5 months (HR 2.04, P Conclusion: Early (14 days) change in ctDNA levels under regorafenib therapy describes adequately the outcome of patients and may be used as a reliable tool for treatment personalization. Additionally, we confirmed the importance of using the absolute value of ctDNA instead of the mutated/total copies ratio usually reported in the literature for monitoring the outcome of a toxic treatment such as regorafenib. 1Kehagias P. et al. Oral presentation. LKI symposium, 2017; Leuven 2Whale AS. et al. Anal Chem. 2017 Citation Format: Pashalina Kehagias, Caroline Vandeputte, Lieveke Ameye, Hakim El Housni, Jean-François Laes, Amélie Deleporte, Karen Geboes, Thierry Delaunoit, Gauthier Demolin, Marc Peeters, Lionel D'Hondt, Jos Janssens, Javier Carrasco, Maria Gomez Galdon, Ghanem Ghanem, Pierre Heimann, Marianne Paesmans, Patrick Flamen, Alain Hendlisz. Circulating tumor DNA detects early response to regorafenib in advanced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-221.

3532Background: BL-cfDNA combined with plasmatic changes in tumor-specific mutations after 14 days of therapy are explored as independent determinants of outcome in pts with aCRC. Methods: Archival...

11569 Background: No validated prognostic biomarker is currently available for mCRC. This trial assessed cfDNA and MTV before treatment with regorafenib as prognostic biomarkers for progression-free survival (PFS) and overall survival (OS) in mCRC. Methods: After signed informed consent, mCRC patients were enrolled in a prospective non-randomized trial aiming to define unlikelihood to benefit from regorafenib (EudraCT number: 2012-005655-16) and assessed for cfDNA and FDG PET/CT MTV at baseline. cfDNA was extracted from 3mL of plasma and quantified using the Qubit 2.0 fluorometer. All target lesions were delineated on FDG PET/CT using a PERCIST-based threshold and their volumes were summed to obtain total MTV. MTV and cfDNA optimal cutoffs for OS and PFS prediction were determined by the Contal and O’Quigley’s method. MTV, cfDNA, age, gender, Body Mass Index (low, normal, high, obese), ECOG PS, number of chemotherapy lines (NCL), previous use of bevacizumab and presence of a KRAS mutation were included in a multivariate analysis. Results: MTV and cfDNA of 132 evaluable/141 eligible patients were well correlated (Spearman’s correlation coefficient = 0.70; p < 0.001) and risk groups for both PFS and OS were identified on the basis of cfDNA (cfDNA < 1 µg/mL; cfDNA≥1 µg/mL) and MTV (MTV < 100 cm³; 100-300 cm³; > 300 cm³). The multivariate analysis retained cfDNA, MTV, NCL, and obesity as independent parameters for PFS prediction, and cfDNA, MTV, NCL, BMI, and previous use of bevacizumab as independent parameters for OS prediction. Prognostic scores for PFS and OS were developed based on regression coefficients from the final Cox proportional hazards models. Prognostic scores for PFS (1.8 vs 5.3 months, HR: 3.15 for score ≥-3 vs < -3, (95% CI, 2.08-4.76); p < 0.001) and for OS (4.2 vs 13.9 months, HR: 4.59 for score ≥-6 vs < -6: (95% CI, 2.92-7.21); p < 0.001) both identified patients with much contrasted outcomes. Conclusions: Baseline cfDNA and MTV along with BMI parameters predict outcome in patients with mCRC before regorafenib onset. These parameters not related to treatment should be considered, if validated in further studies, as stratification factors in future clinical trials. Clinical trial information: 2012-005655-16.