Ongoing Clinical Trials and Future Research Scenarios of Circulating Tumor DNA for the Treatment of Metastatic Colorectal Cancer.

Liquid biopsy using circulating tumor DNA (ctDNA) has emerged as a minimally invasive, timely approach to provide molecular diagnosis and monitor tumor evolution in patients with cancer. Since the molecular landscape of metastatic colorectal cancer (mCRC) is substantially heterogeneous and dynamic over space and time, ctDNA holds significant advantages as a biomarker for this disease. Numerous studies have demonstrated that ctDNA broadly recapitulates the molecular profile of the primary tumor and metastases, and have mainly focused on the genotyping of RAS and BRAF, that is propaedeutic for anti-EGFR treatment selection. However, ctDNA soon broadened its scope towards the assessment of early tumor response, as well as the identification of drug resistance biomarkers to drive potential molecular actionability. In this review article, we provide an overview of the current state-of-the-art of this methodology and its applications, focusing on ongoing clinical trials that employ ctDNA to prospectively guide treatment in patients with mCRC.

Clinicopathological characterisation of MTAP alterations in gastrointestinal cancers.

BACKGROUND: Methylthioadenosine phosphorylase (MTAP) is an essential metabolic enzyme in the purine and methionine salvage pathway. In cancer, MTAP gene copy number loss (MTAP loss) confers a selective dependency on the related protein arginine methyltransferase 5. The impact of MTAP alterations in gastrointestinal (GI) cancers remains unknown although hypothetically druggable. Here, we aim to investigate the prevalence, clinicopathological features and prognosis of MTAP loss GI cancers. METHODS: Cases with MTAP alterations were retrieved from The Cancer Genome Atlas (TCGA) and a real-world cohort of GI cancers profiled by next-generation sequencing. If MTAP alterations other than loss were found, immunohistochemistry was performed. Finally, we set a case-control study to assess MTAP loss prognostic impact. RESULTS: Findings across the TCGA dataset (N=1363 patients) and our cohort (N=508) were consistent. Gene loss was the most common MTAP alteration (9.4%), mostly co-occurring with CDKN2A/B loss (97.7%). Biliopancreatic and gastro-oesophageal cancers had the highest prevalence of MTAP loss (20.5% and 12.7%, respectively), being mostly microsatellite stable (99.2%). In colorectal cancer, MTAP loss was rare (1.1%), while most MTAP alterations were mutations (5/7, 71.4%); among the latter, only MTAP-CDKN2B truncation led to protein loss, thus potentially actionable. MTAP loss did not confer worse prognosis. CONCLUSIONS: MTAP alterations are found in 5%-10% of GI cancers, most frequently biliopancreatic and gastro-oesophageal. MTAP loss is the most common alteration, identified almost exclusively in MSS, CDKN2A/B loss, upper-GI cancers. Other MTAP alterations were found in colorectal cancer, but unlikely to cause protein loss and drug susceptibility.

Divarasib plus cetuximab in KRAS G12C-positive colorectal cancer: a phase 1b trial.

KRAS G12C mutation is prevalent in ~4% of colorectal cancer (CRC) and is associated with poor prognosis. Divarasib, a KRAS G12C inhibitor, has shown modest activity as a single agent in KRAS G12C-positive CRC at 400 mg. Epidermal growth factor receptor has been recognized as a major upstream activator of RAS-MAPK signaling, a proposed key mechanism of resistance to KRAS G12C inhibition in CRC. Here, we report on divarasib plus cetuximab (epidermal growth factor receptor inhibitor) in patients with KRAS G12C-positive CRC (n = 29) from arm C of an ongoing phase 1b trial. The primary objective was to evaluate safety. Secondary objectives included preliminary antitumor activity. The safety profile of this combination was consistent with those of single-agent divarasib and cetuximab. Treatment-related adverse events led to divarasib dose reductions in four patients (13.8%); there were no treatment withdrawals. The objective response rate was 62.5% (95% confidence interval: 40.6%, 81.2%) in KRAS G12C inhibitor-naive patients (n = 24). The median duration of response was 6.9 months. The median progression-free survival was 8.1 months (95% confidence interval: 5.5, 12.3). As an exploratory objective, we observed a decline in KRAS G12C variant allele frequency associated with response and identified acquired genomic alterations at disease progression that may be associated with resistance. The manageable safety profile and encouraging antitumor activity of divarasib plus cetuximab support the further investigation of this combination in KRAS G12C-positive CRC.ClinicalTrials.gov identifier: NCT04449874.

Circulating Tumor DNA to Drive Treatment in Metastatic Colorectal Cancer.

In the evolving molecular treatment landscape of metastatic colorectal cancer (mCRC), the identification of druggable alterations is pivotal to achieve the best therapeutic opportunity for each patient. Because the number of actionable targets is expanding, there is the need to timely detect their presence or emergence to guide the choice of different available treatment options. Liquid biopsy, through the analysis of circulating tumor DNA (ctDNA), has proven safe and effective as a complementary method to address cancer evolution while overcoming the limitations of tissue biopsy. Even though data are accumulating regarding the potential for ctDNA-guided treatments applied to targeted agents, still major gaps in knowledge exist as for their application to different areas of the continuum of care. In this review, we recapitulate how ctDNA information could be exploited to drive different targeted treatment strategies in mCRC patients, by refining molecular selection before treatment by addressing tumor heterogeneity beyond tumor tissue biopsy; longitudinally monitoring early-tumor response and resistance mechanisms to targeted agents, potentially leading to tailored, molecular-driven, therapeutic options; guiding the molecular triage towards rechallenge strategies with anti-EGFR agents, suggesting the best time for retreatment; and providing opportunities for an "enhanced rechallenge" through additional treatments or combos aimed at overcoming acquired resistance. Besides, we discuss future perspectives concerning the potential role of ctDNA to fine-tune investigational strategies such as immuno-oncology.

Corrigendum: Case report: MAP2K1 K57N mutation is associated with primary resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer.

[This corrects the article DOI: 10.3389/fonc.2022.1030232.].

Case Report: MAP2K1 K57N mutation is associated with primary resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer.

Background: We aim to identify the prevalence and the role of the MAP2K1 K57N mutation in predicting resistance to anti-EGFR agents in metastatic colorectal cancer (mCRC) patients. Methods: We retrospectively reviewed tumor-based next generation sequencing (NGS) results from mCRC patients screened for enrollment in the GO40872/STARTRK-2 clinical trial between July 2019 and March 2021. Then, in patients harboring microsatellite stable (MSS) RAS and BRAF wild-type MAP2K1 mutant mCRC, we reviewed outcome to treatment with anti-EGFR monoclonal antibodies. Results: A total of 246 mCRC patients were screened. Most of them, 215/220 (97.7%), were diagnosed with MSS mCRC and 112/215 (52.1%) with MSS, RAS and BRAF wild-type mCRC. Among the latter, 2/112 (1.8%) had MAP2K1 K57N mutant mCRC and both received anti-EGFR monotherapy as third line treatment. In both patients, MAP2K1 K57N mutant tumors proved primary resistant to anti-EGFR agent panitumumab monotherapy. Of interest, one of these patients was treated with anti-EGFR agents three times throughout his course of treatment, achieving some clinical benefit only when associated with other cytotoxic agents (FOLFOX or irinotecan). Conclusion: We verified in a clinical real-world setting that MAP2K1 K57N mutation is a resistance mechanism to anti-EGFR agents in mCRC. Thus, we suggest avoiding the administration of these drugs to MSS RAS and BRAF wild-type MAP2K1 N57K mutant mCRC.

Preclinical Characterization and Phase I Study of an Anti-HER2-TLR7 Immune-Stimulator Antibody Conjugate in Patients with HER2+ Malignancies.

Immune-stimulator antibody conjugates (ISAC) combining tumor-targeting monoclonal antibodies with immunostimulatory agents allow targeted delivery of immune activators into tumors. NJH395 is a novel, first-in-class ISAC comprising a Toll-like receptor 7 (TLR7) agonist conjugated to an anti-HER2 antibody via a noncleavable linker payload. Preclinical characterization showed ISAC-mediated activation of myeloid cells in the presence of antigen-expressing cancer cells, with antigen targeting and TLR7 agonism contributing to antitumor activity. Safety, efficacy, immunogenicity, pharmacokinetics, and pharmacodynamics were investigated in a phase I, multicenter, open-label study in patients with HER2+ non-breast advanced malignancies (NCT03696771). Data from 18 patients enrolled in single ascending dose escalation demonstrated delivery of the TLR7-agonist payload in HER2+ tumor cells and induction of type I IFN responses, which correlated with immune modulation in the tumor microenvironment. Cytokine release syndrome was a common, but manageable, drug-related adverse event. Antidrug antibodies and neuroinflammation at high doses represented significant clinical challenges. Data provide proof-of-mechanism and critical insights for novel immunotherapies.

Circulating tumor DNA to guide rechallenge with panitumumab in metastatic colorectal cancer: the phase 2 CHRONOS trial.

Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies are approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC), but the emergence of resistance mutations restricts their efficacy. We previously showed that RAS, BRAF and EGFR mutant alleles, which appear in circulating tumor DNA (ctDNA) during EGFR blockade, decline upon therapy withdrawal. We hypothesized that monitoring resistance mutations in blood could rationally guide subsequent therapy with anti-EGFR antibodies. We report here the results of CHRONOS, an open-label, single-arm phase 2 clinical trial exploiting blood-based identification of RAS/BRAF/EGFR mutations levels to tailor a chemotherapy-free anti-EGFR rechallenge with panitumumab (ClinicalTrials.gov: NCT03227926 ; EudraCT 2016-002597-12). The primary endpoint was objective response rate. Secondary endpoints were progression-free survival, overall survival, safety and tolerability of this strategy. In CHRONOS, patients with tissue-RAS WT tumors after a previous treatment with anti-EGFR-based regimens underwent an interventional ctDNA-based screening. Of 52 patients, 16 (31%) carried at least one mutation conferring resistance to anti-EGFR therapy and were excluded. The primary endpoint of the trial was met; and, of 27 enrolled patients, eight (30%) achieved partial response and 17 (63%) disease control, including two unconfirmed responses. These clinical results favorably compare with standard third-line treatments and show that interventional liquid biopsies can be effectively and safely exploited in a timely manner to guide anti-EGFR rechallenge therapy with panitumumab in patients with mCRC. Further larger and randomized trials are warranted to formally compare panitumumab rechallenge with standard-of-care therapies in this patient setting.

Temozolomide Treatment Alters Mismatch Repair and Boosts Mutational Burden in Tumor and Blood of Colorectal Cancer Patients.

The majority of metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient and unresponsive to immunotherapy, whereas MMR-deficient (MMRd) tumors often respond to immune-checkpoint blockade. We previously reported that the treatment of colorectal cancer preclinical models with temozolomide (TMZ) leads to MMR deficiency, increased tumor mutational burden (TMB), and sensitization to immunotherapy. To clinically translate these findings, we designed the ARETHUSA clinical trial whereby O6-methylguanine-DNA-methyltransferase (MGMT)-deficient, MMR-proficient, RAS-mutant mCRC patients received priming therapy with TMZ. Analysis of tissue biopsies and circulating tumor DNA (ctDNA) revealed the emergence of a distinct mutational signature and increased TMB after TMZ treatment. Multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes, and the p.T1219I MSH6 variant was detected in ctDNA and tissue of 94% (16/17) of the cases. A subset of patients whose tumors displayed the MSH6 mutation, the TMZ mutational signature, and increased TMB achieved disease stabilization upon pembrolizumab treatment. SIGNIFICANCE: MMR-proficient mCRCs are unresponsive to immunotherapy. We provide the proof of concept that inactivation of MMR genes can be achieved pharmacologically with TMZ and molecularly monitored in the tissue and blood of patients with mCRC. This strategy deserves additional evaluation in mCRC patients whose tumors are no longer responsive to standard-of-care treatments. See related commentary by Willis and Overman, p. 1612. This article is highlighted in the In This Issue feature, p. 1599.

Efficacy of Retreatment with Oxaliplatin-Based Regimens in Metastatic Colorectal Cancer Patients: The RETROX-CRC Retrospective Study.

BACKGROUND: oxaliplatin with fluoropyrimidine is a "mainstay" regarding the upfront treatment of metastatic colorectal cancer (mCRC). In contrast, the efficacy and safety of oxaliplatin-based regimens in late-care settings have been poorly reported. METHODS: we identified a real-world mCRC patient cohort who were re-treated with oxaliplatin, and in which clinicopathological features were retrospectively analyzed to identify efficacy-predictive determinants (RETROX-CRC study). RESULTS: of 2606 patients, 119 fulfilled the eligibility criteria. Oxaliplatin retreatment response rate (RR) and disease control rate (DCR) were 21.6% (CI 14.4-31.0%), and 57.8% (CI 47.7-67.4). A trend towards better RR and DCR was observed among patients who had first oxaliplatin in an adjuvant setting; a poorer outcome was observed if two or more intervening treatments were delivered. Median progression-free survival (PFS) was 5.1 months (95%CI 4.3-6.1), reducing to 4.0 months (95%CI 3.07-5.13) if oxaliplatin was readministered beyond third-line (HR 2.02; 1.25-3.25; p = 0.004). Safety data were retrieved in 65 patients (54.6%); 18.5% (12/65) and 7.7% (5/65) had G3-4 toxicities. Toxicities led to discontinuation in 34/119 (28.6%). CONCLUSIONS: oxaliplatin retreatment produced further RR in around one-fifth of patients and DCR 57.8%. Efficacy decreased in more pre-treated patients and around one-third of patients discontinued treatment due to adverse events. Translational studies improving patient selection are warranted.

The evolving panorama of HER2-targeted treatments in metastatic urothelial cancer: A systematic review and future perspectives.

PURPOSE: HER2 alterations are potential candidates for targeted treatments in metastatic urothelial/bladder cancer (mUC). ERBB2 gene amplification and mutations are found in around 6% and 4% of mUC, respectively. METHODS: This is a systematic review of clinical trials evaluating HER2-targeting (amplification and mutations) in mUC. We assigned each study to one of the following strategies: HER2-targeting with single agents, anti-HER2 agents in combination with cytotoxic chemotherapy, dual HER2 blockade, HER2-targeted antibody-drug conjugates (ADCs), and other novel therapeutic approaches. RESULTS: 36 clinical trials (17 with results and 19 ongoing) were included. As for ERBB2 amplification, anti-HER2 single agents (5 studies) and combinations with chemotherapy (4 studies) failed to provide any benefit, whereas dual HER2 blockade through monoclonal antibodies proved active in one trial in pretreated patients. Two studies assessed single-agent targeting for ERBB2 mutations with negative results. Most promising data come from 2 studies with ADCs in ERBB2 amplified tumors (disitamab-vedotin and trastuzumab-duocarmazine), while 2 other studies with TDM-1 and ADCT-502 was discontinued due to toxicity. In this category, trastuzumab-deruxtecan and other ADCs are still under investigation for either ERBB2-amplified or mutated mUC. Novel approaches include ADCs with immunotherapy (1 study with results), CAR-T cells, and HER2-sensitising vaccines. CONCLUSIONS: ERBB2 amplification could become a novel target in mUC, although the magnitude of clinical benefit remains to be clarified. To this regard, novel ADCs are the most promising strategy. ERBB2 mutations are still at very early stage of clinical study.

Impaired seroconversion after SARS-CoV-2 mRNA vaccines in patients with solid tumours receiving anticancer treatment.

BACKGROUND: Patients with solid tumours have high COVID-19 mortality. Limited and heterogeneous data are available regarding the immunogenicity of SARS-CoV-2 mRNA vaccines in this population. METHODS AND FINDINGS: This is a prospective, single-centre cohort study aiming at evaluating seroconversion in terms of anti-spike antibodies in a population of patients with solid tumours undergoing cancer therapy within 2 months before the second vaccine dose, as compared with a cohort of controls. Subjects who were not SARS-CoV-2 naïve were excluded, and 171 patients were included in the final study population (150 vaccinated with BNT162b2, 87.7%; 21 with mRNA-1273, 12.3%) and compared with 2406 controls. The median follow-up time from the second dose of vaccination was 30 days (12-42; IQR: 26-34). Most patients had metastatic disease (138, 80.7%). Seroconversion rate was significantly lower in cancer patients than in controls (94.2% versus 99.8%, p < 0.001). At univariate logistic regression analysis, Odds ratio (OR) for seroconversion was also reduced in older individuals (>70 years). A multivariate logistic model confirmed cancer as the only significant variable in impairing seroconversion (OR 0.03, p < 0.001). In the cancer population, a multivariate analysis among clinical variables, including the type of cancer treatment, showed ECOG PS > 2 as the only one of impact (OR 0.07, p = 0.012). CONCLUSIONS: There is a fraction of 6% of patients with solid tumours undergoing cancer treatment, mainly with poorer performance status, who fail to obtain seroconversion after SARS-CoV-2 mRNA vaccines. These patients should be considered for enhanced vaccination strategies and carefully monitored for SARS-CoV-2 infection during cancer treatment.

Liquid Biopsy for Small Cell Lung Cancer either De Novo or Transformed: Systematic Review of Different Applications and Meta-Analysis.

BACKGROUND: The potential added value of liquid biopsy (LB) is not well determined in the case of small cell lung cancer (SCLC), an aggressive tumor that can occur either de novo or from the histologic transformation of non-small cell lung cancer (NSCLC). METHODS: A systematic review of studies adopting LB in patients with SCLC have been performed to assess the clinical utility of circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs). RESULTS: After a screening of 728 records, 62 studies (32 evaluating CTCs, 27 ctDNA, and 3 both) met predetermined eligibility criteria. Only four studies evaluated LB in the diagnostic setting for SCLC, while its prognostic significance was evaluated in 38 studies and prominently supported by both ctDNA and CTCs. A meta-analysis of 11 studies as for CTCs enumeration showed an HR for overall survival of 2.63 (1.71-4.05), with a potential publication bias. The feasibility of tumor genomic profiling and the predictive role of LB in terms of response/resistance to chemotherapy was assessed in 11 and 24 studies, respectively, with greater consistency for those regarding ctDNA. Intriguingly, several case reports suggest that LB can indirectly capture the transition to SCLC in NSCLC treated with EGFR tyrosine kinase inhibitors. CONCLUSIONS: While dedicated trials are needed, LB holds potential clinical roles in both de novo and transformed SCLC. CtDNA analysis appears the most valuable and practicable tool for both disease monitoring and genomic profiling.

Clonally expanded EOMES+ Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

Liquid Biopsy for Prognosis and Treatment in Metastatic Colorectal Cancer: Circulating Tumor Cells vs Circulating Tumor DNA.

Liquid biopsy recently gained widespread attention as a noninvasive alternative/complementary technique to tissue biopsy in patients with cancer. As technological advances have improved both feasibility and turnaround time, liquid biopsy has expanded tumor molecular analysis with acknowledgement of both spatial and temporal heterogeneity, overcoming many limitations of traditional tissue biopsy. Because of its diagnostic, prognostic, and predictive value, liquid biopsy has been extensively studied also in metastatic colorectal cancer. Indeed, as personalized medicine establishes its role in cancer treatment, genetic biomarkers unveiling the emergence of early resistance are needed. Among the wide variety of tumor analytes amenable to collection, circulating DNA and circulating tumor cells are the most adopted approaches, and both carry clinical relevance in colorectal cancer. However, few studies focused on comparing feasibility between these two approaches. In this review, we discuss the potential implications of liquid biopsy in metastatic colorectal cancer, assessing the advantages and drawbacks of circulating DNA and circulating tumor cells, and highlighting the most relevant trials for clinical practice.

The Evolutionary Landscape of Treatment for BRAFV600E Mutant Metastatic Colorectal Cancer.

The BRAFV600E mutation is found in 8-10% of metastatic colorectal cancer (mCRC) patients and it is recognized as a poor prognostic factor with a median overall survival inferior to 20 months. At present, besides immune checkpoint inhibitors (CPIs) for those tumors with concomitant MSI-H status, recommended treatment options include cytotoxic chemotherapy + anti-VEGF in the first line setting, and a combination of EGFR and a BRAF inhibitor (cetuximab plus encorafenib) in second line. However, even with the latter targeted approach, acquired resistance limits the possibility of more than an incremental benefit and survival is still dismal. In this review, we discuss current treatment options for this subset of patients and perform a systematic review of ongoing clinical trials. Overall, we identified six emerging strategies: targeting MAPK pathway (monotherapy or combinations), targeting MAPK pathway combined with cytotoxic agents, intensive cytotoxic regimen combinations, targeted agents combined with CPIs, oxidative stress induction, and cytotoxic agents combined with antiangiogenic drugs and CPIs. In the future, the integration of new therapeutic strategies targeting key players in the BRAFV600E oncogenic pathways with current treatment approach based on cytotoxic chemotherapy and surgery is likely to redefine the treatment landscape of these CRC patients.

Systemic doxycycline for pre-emptive treatment of anti-EGFR-related skin toxicity in patients with metastatic colorectal cancer receiving first-line panitumumab-based therapy: a post hoc analysis of the Valentino study.

INTRODUCTION: The combination of anti-EGFRs and doublet chemotherapy is considered the optimal upfront option for patients with RAS/BRAF wild-type left-sided metastatic colorectal cancer (mCRC). The prophylactic or reactive treatment with tetracyclines for EGFR inhibitor-induced skin toxicity is currently clinical practice, though non-conclusive results are available. METHODS: We performed a post hoc analysis of the Valentino study that randomized RAS wild-type mCRC patients to two panitumumab-based maintenance regimens after the first-line induction, aimed at assessing the safety and efficacy of the administration of a pre-emptive doxycycline prophylaxis for anti-EGFR-related skin toxicity. We assessed the rate of treatment-related and panitumumab-related adverse events (AEs), treatment intensity, progression-free survival (PFS), and overall survival (OS). RESULTS: A total of 226 patients, out of the 229 enrolled in the Valentino study, were eligible for the analysis. Overall, 143 (63%) and 83 (37%) patients received or not the antibiotic prophylaxis for skin toxicity. Any grade and G3/4 panitumumab-related AEs were reported in 89% versus 92% (p = 0.650) and 27% versus 27% (p = 1.000) patients who received or not the pre-emptive prophylaxis, respectively. Any grade and G3/4 skin rash occurred in 81% versus 90% (p = 0.085) and 27% versus 25% (p = 0.876) patients receiving or not the prophylaxis, respectively. No significant differences in terms of treatment duration, treatment delays or dose reductions, PFS, and OS were observed in the two sub-populations. CONCLUSION: The adequate management of anti-EGFR-related skin toxicity is fundamental to optimize the outcome of mCRC patients, balancing the survival benefit with patients' quality of life, especially in the first-line setting.