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 Tumor Resection in Synchronous Metastatic Colorectal Cancer Patients Treated with Upfront Chemotherapy plus Bevacizumab: A Pooled Analysis of TRIBE and TRIBE2 Studies.

BACKGROUND: The decision to resect or not the primary tumor in asymptomatic patients with synchronous metastatic colorectal cancer (mCRC) is a complex and challenging issue for oncologists, especially when an antiangiogenic-based therapy is planned. METHODS: Patients enrolled in the phase III TRIBE and TRIBE2 studies that compared upfront FOLFOXIRI + bevacizumab to FOLFIRI or FOLFOX + bevacizumab, respectively, were included. We assessed the association of primary tumor resection (PTR) with progression-free survival (PFS), overall survival (OS), response rate (ORR), rate of grade > 2 adverse events (AEs), and serious gastrointestinal and surgical AEs in the overall population and according to the treatment arm. RESULTS: Of the 999 patients included, 513 (51%) underwent PTR at baseline. Longer PFS and OS were observed in resected patients compared to those with unresected primary tumors: 11.2 vs. 10.0 months (p < 0.001) and 26.6 vs. 22.5 (p < 0.001), respectively. In multivariate models, PTR was confirmed as an independent prognostic factor for better PFS (p = 0.032) and OS (p = 0.018). Patients with PTR experienced a higher incidence of grade 3 or 4 diarrhea (p = 0.055) and lower incidence of anemia (p = 0.053), perforation (p = 0.015), and serious gastrointestinal and surgical AEs (p < 0.001). No statistically significant differences were noted in incidence of bleeding (p = 0.39). The benefit of FOLFOXIRI + bevacizumab in terms of PFS (p for interaction: 0.46), OS (p for interaction: 0.80), ORR (p for interaction: 0.36), and incidence of grade 3 or 4 AEs was independent of PTR. CONCLUSIONS: PTR at baseline was independently associated with good prognosis in synchronous mCRC patients and with lower incidence of serious gastrointestinal and surgical AEs during upfront chemotherapy plus bevacizumab. The benefit and toxicity profile of FOLFOXIRI plus bevacizumab was independent of PTR.

Predictive Impact of RNF43 Mutations in Patients With Proficient Mismatch Repair/Microsatellite Stable BRAFV600E-Mutated Metastatic Colorectal Cancer Treated With Target Therapy or Chemotherapy.

PURPOSE: Target therapy (TT) with encorafenib plus cetuximab is a standard option in patients with BRAFV600E-mutated (mut) pretreated metastatic colorectal cancer (mCRC). Recently, mutations in RNF43, encoding a negative regulator of the WNT pathway, were associated with longer progression-free survival (PFS) and overall survival (OS) in patients with proficient mismatch repair/microsatellite stable (pMMR/MSS) BRAFV600E-mut mCRC treated with TT. Here, we explored the effect of RNF43 mutations on the efficacy of second-line TT versus standard chemotherapy (CT). METHODS: A retrospective cohort of patients with pMMR/MSS BRAFV600E-mut tumors, available RNF43 mutational status, and treated with second-line TT or oxaliplatin- and/or irinotecan-based CT was analyzed. RESULTS: One hundred thirty-two patients with pMMR/MSS BRAFV600E-mut mCRC were included. RNF43 was found mut in 34 (26%) cases. Among RNF43 mutants, TT was associated with longer PFS (7.7 v 3.0 months; P = .002) and higher overall response rate (ORR; 45% v 0%; P = .009) compared with CT. Conversely, among RNF43 wild-type (wt) patients, only a trend for longer PFS (4.5 v 3.7 months; P = .064) favoring TT, with no differences in ORR (P = .14), was observed. After excluding 36 patients receiving TT in third line or beyond, a longer OS (19.4 v 10.1 months; P = .022) and a numerically OS advantage (10.6 v 6.6 months; P = .068) were reported for TT both in the RNF43-mut and in the RNF43 wt groups. However, no interaction effect was reported between RNF43 mutational status and treatment in ORR (Pinteraction = .96), PFS (Pinteraction = .13), and OS (Pinteraction = .44). CONCLUSION: Patients with pMMR/MSS BRAFV600E-mut mCRC achieve benefit from TT versus CT independently of RNF43 mutational status, although a higher magnitude of benefit from TT is observed in RNF43-mut tumors. These findings deserve confirmation in concluded and ongoing randomized trials.

The prognostic value of radiomic features in liver-limited metastatic colorectal cancer patients from the TRIBE2 study.

Aims: Evaluating the prognostic role of radiomic features in liver-limited metastatic colorectal cancer treated with first-line therapy at baseline and best response among patients undergoing resection. Patients & methods: Among patients enrolled in TRIBE2 (NCT02339116), the association of clinical and radiomic data, extracted by SOPHiA-DDM™ with progression-free and overall survival (OS) in the overall population and with disease-free survival/postresection OS in those undergoing resection was investigated. Results: Among 98 patients, radiomic parameters improved the prediction accuracy of our model for OS (area under the curve: 0.83; sensitivity: 0.85; specificity: 0.73; accuracy: 0.78), but not progression-free survival. Of 46 resected patients, small-distance high gray-level emphasis was associated with shorter disease-free survival and high gray-level zone emphasis/higher kurtosis with shorter postresection OS. Conclusion: Radiomic features should be implemented as tools of outcome prediction for liver-limited metastatic colorectal cancer.

Transcriptomic Signatures of MSI-High Metastatic Colorectal Cancer Predict Efficacy of Immune Checkpoint Inhibitors.

PURPOSE: Microsatellite instability (MSI) is currently the only predictive biomarker of efficacy of immune checkpoint inhibitors (ICI) in metastatic colorectal cancers (mCRC). However, 10% to 40% of patients with MSI mCRC will experience a primary resistance to ICI. EXPERIMENTAL DESIGN: In two cohorts of patients with MSI mCRC treated with ICI (exploratory, N = 103; validation, N = 35), 3' RNA sequencing was performed from primary tumors. Previously described single-cell transcriptomic signatures of tumor microenvironment (TME) were analyzed. RESULTS: In the exploratory cohort, the unsupervised clustering allowed the identification of three clusters of tumors with distinct transcriptional profiles: cluster A ("stromalHIGH-proliferationLOW"), cluster B ("stromalHIGH-proliferationMED"), and cluster C ("stromalLOW-proliferationHIGH"), with an enrichment of patients progressing at first disease assessment under ICI in cluster A (30% vs. 12% in cluster B and 8.1% in cluster C; P = 0.074). Progression-free survival (PFS) was also significantly shorter in patients belonging to cluster A, compared with clusters B or C (P < 0.001) with 2-year PFS rates of 33.5%, 80.5%, and 78.3%, respectively. In multivariate analysis, PFS was still significantly longer in patients belonging to cluster B [HR, 0.19; 95% confidence interval (CI), 0.08-0.45; P < 0.001] and cluster C (HR, 0.25; 95% CI, 0.10-0.59; P = 0.02), compared with patients belonging to cluster A. The association of this clustering with PFS under ICI was confirmed in the validation cohort. PFS related to non-ICI-based regimens was not significantly different according to cluster. CONCLUSIONS: This unsupervised transcriptomic classification identified three groups of MSI mCRCs with different compositions of TME cells and proliferative capacities of TME/tumor cells. The "stromalHIGH-proliferationLOW" cluster is associated with a poorer prognosis with ICI treatment.

Colorectal neoplastic emergencies in immunocompromised patients: preliminary result from the Web-based International Register of Emergency Surgery and Trauma (WIRES-T trial).

Association of advanced age, neoplastic disease and immunocompromission (IC) may lead to surgical emergencies. Few data exist about this topic. Present study reports the preliminary data from the WIRES-T trial about patients managed for colorectal neoplastic emergencies in immunocompromised patients. The required data were taken from a prospective observational international register. The study was approved by the Ethical Committee with approval n. 17575; ClinicalTrials.gov Identifier: NCT03643718. 839 patients were collected; 753 (80.7%) with mild-moderate IC and 86 (10.3%) with severe. Median age was 71.9 years and 73 years, respectively, in the two groups. The causes of mild-moderate IC were reported such malignancy (753-100%), diabetes (103-13.7%), malnutrition (26-3.5%) and uremia (1-0.1%), while severe IC causes were steroids treatment (14-16.3%); neutropenia (7-8.1%), malignancy on chemotherapy (71-82.6%). Preoperative risk classification were reported as follow: mild-moderate: ASA 1-14 (1.9%); ASA 2-202 (26.8%); ASA 3-341 (45.3%); ASA 4-84 (11.2%); ASA 5-7 (0.9%); severe group: ASA 1-1 patient (1.2%); ASA 2-16 patients (18.6%); ASA 3-41 patients (47.7%); ASA 4-19 patients (22.1%); ASA 5-3 patients (3.5%); lastly, ASA score was unavailable for 105 cases (13.9%) in mild-moderate group and in 6 cases (6.9%) in severe group. All the patients enrolled underwent urgent/emergency surgery Damage control approach with open abdomen was adopted in 18 patients. Mortality was 5.1% and 12.8%, respectively, in mild-moderate and severe groups. Long-term survival data: in mild-moderate disease-free survival (median, IQR) is 28 (10-91) and in severe IC, it is 21 (10-94). Overall survival (median, IQR) is 44 (18-99) and 26 (20-90) in mild-moderate and severe, respectively; the same is for post-progression survival (median, IQR) 29 (16-81) and 28, respectively. Univariate and multivariate analyses showed as the only factor influencing mortality in mild-moderate and severe IC is the ASA score. Colorectal neoplastic emergencies in immunocompromised patients are more frequent in elderly. Sigmoid and right colon are the most involved. Emergency surgery is at higher risk of complication and mortality; however, management in dedicated emergency surgery units is necessary to reduce disease burden and to optimize results by combining oncological and acute care principles. This approach may improve outcomes to obtain clinical advantages for patients like those observed in elective scenario. Lastly, damage control approach seems feasible and safe in selected patients.

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.

Negative Ultraselection of Patients With RAS/BRAF Wild-Type, Microsatellite-Stable Metastatic Colorectal Cancer Receiving Anti-EGFR-Based Therapy.

PURPOSE: Several uncommon genomic alterations beyond RAS and BRAFV600E mutations drive primary resistance to anti-epidermal growth factor receptors (EGFRs) in metastatic colorectal cancer (mCRC). Our PRESSING panel (including PIK3CA exon 20/AKT1/PTEN mutations, ERBB2/MET amplifications, gene fusions, and microsatellite instability-high status) represented a paradigm of negative hyperselection with more precise tailoring of EGFR blockade. However, a modest proportion of hyperselected mCRC has intrinsic resistance potentially driven by even rarer genomic alterations. MATERIALS AND METHODS: A prospective data set at three Italian Academic Hospitals included 650 patients with mCRC with comprehensive genomic profiling by FoundationOne CDx and treated with anti-EGFRs. PRESSING2 panel alterations were selected on the basis of previous clinico-biologic studies and included NTRKs, ERBB3, NF1, MAP2K1/2/4, AKT2 pathogenic mutations; PTEN/NF1 loss; ERBB3, FGFR2, IGF1R, KRAS, ARAF, and AKT1-2 amplification; and EGFR rearrangements. These were collectively associated with outcomes in patients with hyperselected disease, ie, RAS/BRAF wild-type, PRESSING-negative, and microsatellite stable. RESULTS: Among 162 hyperselected patients, 24 (15%) had PRESSING2 alterations, which were mutually exclusive except in two samples and were numerically higher in right-sided versus left-sided cancers (28% v 13%; P = .149). Independently of sidedness and other factors, patients with PRESSING2-positive status had significantly worse progression-free survival and overall survival compared with PRESSING2-negative ones (median progression-free survival 6.4 v 12.8 months, adjusted hazard ratio 4.19 [95% CI, 2.58 to 6.79]; median overall survival: 22.6 v 49.9 months, adjusted hazard ratio 2.98 [95% CI, 1.49 to 5.96]). The combined analysis of primary tumor sidedness and PRESSING2 status allowed us to better stratify outcomes. CONCLUSION: Negative ultraselection warrants further investigation with the aim of maximizing the benefit of EGFR blockade strategies in patients with RAS and BRAF wild-type, microsatellite stable mCRC.

Early modulation of Angiopoietin-2 plasma levels predicts benefit from regorafenib in patients with metastatic colorectal cancer.

BACKGROUND: No biomarkers are currently available to predict the efficacy of trifluridine/tipiracil (FTD/TPI) in chemorefractory metastatic colorectal cancer. The multicohort REGOLAND study aims at exploring and validating circulating markers potentially able to predict benefit from regorafenib in this setting. MATERIAL AND METHODS: In the retrospective 'regorafenib exploratory cohort', including 105 patients treated with regorafenib, baseline (d1) plasma levels of angiogenesis-related biomarkers and their early modulation after 15 days (d15) of treatment were investigated for correlation with clinical outcome. Based on a pre-specified statistical hypothesis, main retrospective findings were prospectively challenged in the 'regorafenib validation cohort', including 100 patients treated with regorafenib. Prospectively validated putative biomarkers were then assessed in the control 'FTD/TPI cohort', including 93 patients treated with FTD/TPI. RESULTS: In the 'regorafenib exploratory cohort', the early (d15) increase of Angiopoietin-2 (Ang-2) was associated with longer progression-free survival (HR:0.57 [95%CI:0.38-0.88], P = 0.004) and a trend towards longer OS (HR:0.74 [95%CI:0.48-1.14], P = 0.165), than the early decrease. Similar results were prospectively confirmed in the 'regorafenib validation cohort' (HR for progression-free survival:0.72 [95%CI:0.48-1.08], P = 0.095; HR for OS:0.77 [95%CI:0.51-1.16], P = 0.204). No predictive impact was shown for the early modulation of Ang-2 in the 'FTD/TPI cohort'. High baseline Ang-2 levels predict poor prognosis in all the investigated cohorts, independently of other clinical prognostic variables. CONCLUSIONS: The early modulation of circulating Ang-2 predicts the efficacy of regorafenib. Baseline Ang-2 plasma levels are an independent prognostic biomarker in chemorefractory metastatic colorectal cancer.