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Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing™ is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient's cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.

The number of molecularly stratified treatment options available to patients with colorectal cancer (CRC) is increasing, with a parallel rise in the use of biomarkers to guide prognostication and treatment decision-making. The increase in both the number of biomarkers and their use has resulted in a progressively complex situation, evident both from the extensive interactions between biomarkers and from their sometimes complex associations with patient prognosis and treatment benefit. Current and emerging biomarkers also reflect the genomic complexity of CRC, and include a wide range of aberrations such as point mutations, amplifications, fusions and hypermutator phenotypes, in addition to global gene expression subtypes. In this Review, we provide an overview of current and emerging clinically relevant biomarkers and their role in the management of patients with CRC, illustrating the intricacies of biomarker interactions and the growing treatment opportunities created by the availability of comprehensive molecular profiling.

Key Points Molecular alterations fostering the progression of colorectal cancers are acquired early in the carcinogenesis process, and there is inter-connectivity among genomic drivers (gene mutations and chromosomal instability), transcriptomic subtypes (microsatellite instability immune, canonical, metabolic or mesenchymal) and immune signatures (highly immunogenic, poorly immunogenic or inflamed and immune tolerant). Primary and metastatic samples display major similarities at the genomic level: novel gene alterations are usually related to chemotherapy or targeted therapy pressure. More studies on inter-metastatic spatial heterogeneity, molecular shifts at the transcriptomic level and changes in microenvironment markers are needed. Until recently, the evolution of biomarkers for targeted therapies in colorectal cancer has been restrictive, with the identification of multiple negative predictive factors determining the response to epidermal growth factor receptor (EGFR) monoclonal antibodies. At progression to these agents, there is convergent reactivation of MAPK pathway Emerging positive predictive markers for targeted therapies include infrequent genomic events, such as BRAF V600E mutations, ERBB2 amplifications, anaplastic lymphoma kinase ( ALK ) and neurotrophic receptor tyrosine kinase ( NTRK ) fusions and alterations in upstream nodes of the WNT pathway, such as ring finger protein 43 ( RNF43 ), zinc and ring finger 3 ( ZNRF3 ) and R-spondin ( RSPO ) genes. For immune checkpoint inhibitors, promising biomarkers include microsatellite instability and DNA polymerase-ε ( POLE ) mutations Biomarker–drug co-development has evolved to accommodate a 'multi-molecular, multi-drug' perspective of precision medicine. Novel contexts of vulnerability are likely to be identified, leading to drug-repurposing strategies and combination therapies to halt tumour evolution and tackle minimal residual disease. In this Review, Dienstmann et al . analyse the complex nature of colorectal cancer and the different subtypes in which this disease can be classified, advocating for a 'multi-molecular' perspective for the development of therapies to treat it. Critical driver genomic events in colorectal cancer have been shown to affect the response to targeted agents that were initially developed under the 'one gene, one drug' paradigm of precision medicine. Our current knowledge of the complexity of the cancer genome, clonal evolution patterns under treatment pressure and pharmacodynamic effects of target inhibition support the transition from a one gene, one drug approach to a 'multi-gene, multi-drug' model when making therapeutic decisions. Better characterization of the transcriptomic subtypes of colorectal cancer, encompassing tumour, stromal and immune components, has revealed convergent pathway dependencies that mandate a 'multi-molecular' perspective for the development of therapies to treat this disease.

One of the most unexpected discoveries in molecular oncology, in the last decades, was the identification of a new layer of protein coding gene regulation by transcripts that do not codify for proteins, the non-coding RNAs. These represent a heterogeneous category of transcripts that interact with many types of genetic elements, including regulatory DNAs, coding and other non-coding transcripts and directly to proteins. The final outcome, in the malignant context, is the regulation of any of the cancer hallmarks. Non-coding RNAs represent the most abundant type of hormones that contribute significantly to cell-to cell communication, revealing a complex interplay between tumour cells, tumour microenvironment cells and immune cells. Consequently, profiling their abundance in bodily fluids became a mainstream of biomarker identification. Therapeutic targeting of non-coding RNAs represents a new option for clinicians that is currently under development. This review will present the biology and translational value of three of the most studied categories on non-coding RNAs, the microRNAs, the long non-coding RNAs and the circular RNAs. We will also focus on some aspirational concepts that can help in the development of clinical applications related to non-coding RNAs, including using pyknons to discover new non-coding RNAs, targeting human-specific transcripts which are expressed specifically in the tumour cell and using non-coding RNAs to increase the efficiency of immunotherapy.

Metastatic DNA mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal cancer has a poor prognosis after treatment with conventional chemotherapy and exhibits high levels of tumour neoantigens, tumour-infiltrating lymphocytes, and checkpoint regulators. All of these features are associated with the response to PD-1 blockade in other tumour types. Therefore, we aimed to study nivolumab, a PD-1 immune checkpoint inhibitor, in patients with dMMR/MSI-H metastatic colorectal cancer. In this ongoing, multicentre, open-label, phase 2 trial, we enrolled adults (aged ≥18 years) with histologically confirmed recurrent or metastatic colorectal cancer locally assessed as dMMR/MSI-H from 31 sites (academic centres and hospitals) in eight countries (Australia, Belgium, Canada, France, Ireland, Italy, Spain, and the USA). Eligible patients had progressed on or after, or been intolerant of, at least one previous line of treatment, including a fluoropyrimidine and oxaliplatin or irinotecan. Patients were given 3 mg/kg nivolumab every 2 weeks until disease progression, death, unacceptable toxic effects, or withdrawal from study. The primary endpoint was investigator-assessed objective response as per Response Evaluation Criteria in Solid Tumors (version 1.1). All patients who received at least one dose of study drug were included in all analyses. This trial is registered with ClinicalTrials.gov, number NCT02060188. Of the 74 patients who were enrolled between March 12, 2014, and March 16, 2016, 40 (54%) had received three or more previous treatments. At a median follow-up of 12·0 months (IQR 8·6–18·0), 23 (31·1%, 95% CI 20·8–42·9) of 74 patients achieved an investigator-assessed objective response and 51 (69%, 57–79) patients had disease control for 12 weeks or longer. Median duration of response was not yet reached; all responders were alive, and eight had responses lasting 12 months or longer (Kaplan-Meier 12-month estimate 86%, 95% CI 62–95). The most common grade 3 or 4 drug-related adverse events were increased concentrations of lipase (six [8%]) and amylase (two [3%]). 23 (31%) patients died during the study; none of these deaths were deemed to be treatment related by the investigator. Nivolumab provided durable responses and disease control in pre-treated patients with dMMR/MSI-H metastatic colorectal cancer, and could be a new treatment option for these patients. Bristol-Myers Squibb.

ObjectiveEnhancer aberrations are beginning to emerge as a key epigenetic feature of colorectal cancers (CRC), however, a comprehensive knowledge of chromatin state patterns in tumour progression, heterogeneity of these patterns and imparted therapeutic opportunities remain poorly described.DesignWe performed comprehensive epigenomic characterisation by mapping 222 chromatin profiles from 69 samples (33 colorectal adenocarcinomas, 4 adenomas, 21 matched normal tissues and 11 colon cancer cell lines) for six histone modification marks: H3K4me3 for Pol II-bound and CpG-rich promoters, H3K4me1 for poised enhancers, H3K27ac for enhancers and transcriptionally active promoters, H3K79me2 for transcribed regions, H3K27me3 for polycomb repressed regions and H3K9me3 for heterochromatin.ResultsWe demonstrate that H3K27ac-marked active enhancer state could distinguish between different stages of CRC progression. By epigenomic editing, we present evidence that gains of tumour-specific enhancers for crucial oncogenes, such as ASCL2 and FZD10, was required for excessive proliferation. Consistently, combination of MEK plus bromodomain inhibition was found to have synergistic effects in CRC patient-derived xenograft models. Probing intertumour heterogeneity, we identified four distinct enhancer subtypes (EPIgenome-based Classification, EpiC), three of which correlate well with previously defined transcriptomic subtypes (consensus molecular subtypes, CMSs). Importantly, CMS2 can be divided into two EpiC subgroups with significant survival differences. Leveraging such correlation, we devised a combinatorial therapeutic strategy of enhancer-blocking bromodomain inhibitors with pathway-specific inhibitors (PARPi, EGFRi, TGFβi, mTORi and SRCi) for EpiC groups.ConclusionOur data suggest that the dynamics of active enhancer underlies CRC progression and the patient-specific enhancer patterns can be leveraged for precision combination therapy.

Sotorasib, a specific, irreversible KRASG12C protein inhibitor, has shown monotherapy clinical activity in KRASG12C-mutated solid tumours, including colorectal cancer, in the CodeBreaK100 phase 1 trial. We aimed to investigate the activity and safety of sotorasib in phase 2 of the trial. In this single-arm, phase 2 trial, adult patients with KRASG12C-mutated advanced solid tumours were enrolled, from 59 medical centres in 11 countries, if they were aged 18 years or older, had at least one measurable lesion according to the Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1, and had an Eastern Cooperative Oncology Group performance status of 1 or lower. Only data for patients with colorectal cancer, enrolled at 33 medical centres in nine countries, are presented from this basket trial. To be enrolled, the patients had to have progressed after receiving fluoropyrimidine, oxaliplatin, and irinotecan treatment. These patients were administered 960 mg sotorasib orally once per day until disease progression, development of unacceptable side-effects, withdrawal of consent, or death. The primary endpoint was objective response (complete or partial response) as assessed by blinded independent central review. Response was evaluated in patients who received at least one dose of sotorasib and had at least one measurable lesion at baseline; safety was evaluated in patients who received at least one dose of sotorasib. This analysis is a prespecified analysis triggered by the phase 2 colorectal cancer cohort. This study is registered with ClinicalTrials.gov, NCT03600883, and is active but no longer recruiting. On March 1, 2021, at data cutoff, 62 patients with KRASG12C-mutant colorectal cancer had been enrolled between Aug 14, 2019, and May 21, 2020, and had received at least one dose of sotorasib monotherapy. Objective response was observed in six (9·7%, 95% CI 3·6–19·9) of 62 patients, all with partial response. Treatment-related adverse events at grade 3 occurred in six (10%) patients, the most common of which was diarrhoea (two [3%] of 62 patients), and at grade 4 occurred in one (2%) patient (blood creatine phosphokinase increase); no fatal events were recorded. Serious treatment-related adverse events occurred in two (3%) patients (back pain and acute kidney injury). Although the 9·7% overall response rate did not reach the benchmark, oral administration of sotorasib once per day showed modest anti-tumour activity and manageable safety in these heavily pretreated chemorefractory patients. Sotorasib is under evaluation in combination with other therapeutics to increase potential activity and overcome potential resistance mechanisms. Amgen.

Immune checkpoint therapy with anti-CTLA-4 and anti-PD-1/PD-L1 has revolutionized the treatment of many solid tumors. However, the clinical efficacy of immune checkpoint therapy is limited to a subset of patients with specific tumor types 1 , 2 . Multiple clinical trials with combinatorial immune checkpoint strategies are ongoing; however, the mechanistic rationale for tumor-specific targeting of immune checkpoints is elusive. To garner an insight into tumor-specific immunomodulatory targets, we analyzed 94 patients representing five different cancer types, including those that respond relatively well to immune checkpoint therapy and those that do not, such as glioblastoma multiforme, prostate cancer and colorectal cancer. Through mass cytometry and single-cell RNA sequencing, we identified a unique population of CD73 hi macrophages in glioblastoma multiforme that persists after anti-PD-1 treatment. To test if targeting CD73 would be important for a successful combination strategy in glioblastoma multiforme, we performed reverse translational studies using CD73 −/− mice. We found that the absence of CD73 improved survival in a murine model of glioblastoma multiforme treated with anti-CTLA-4 and anti-PD-1. Our data identified CD73 as a specific immunotherapeutic target to improve antitumor immune responses to immune checkpoint therapy in glioblastoma multiforme and demonstrate that comprehensive human and reverse translational studies can be used for rational design of combinatorial immune checkpoint strategies. Analysis of a mass cytometry dataset for different human solid tumors coupled with murine reverse translational experiments suggests that targeting CD73 could enhance the efficacy of checkpoint inhibitor therapy in glioblastoma.

Background In patients undergoing resection of colorectal liver metastases (CLM), resection margin status is a significant predictor of survival, particularly in patients with suboptimal response to preoperative therapy. RAS mutations have been linked to more invasive and migratory tumor biology and poor response to modern chemotherapy. Objective The aim of this study was to evaluate the relationship between RAS mutation and resection margin status in patients undergoing resection of CLM. Methods Patients who underwent curative resection of CLM from 2005 to 2013 with known RAS mutation status were identified from a prospectively maintained database. A positive margin was defined as tumor cells <1 mm from the parenchymal transection line. Results The study included 633 patients, of whom 229 (36.2 %) had mutant RAS . The positive margin rate was 11.4 % (26/229) for mutant RAS and 5.4 % (22/404) for wild-type RAS ( p  = 0.007). In multivariate analysis, the only factors associated with a positive margin were RAS mutation (hazard ratio [HR] 2.439; p  = 0.005) and carcinoembryonic antigen level 4.5 ng/mL or greater (HR 2.060; p  = 0.026). Among patients presenting with liver-first recurrence during follow-up, those with mutant RAS had narrower margins at initial CLM resection (median 4 mm vs. 7 mm; p  = 0.031). A positive margin (HR 3.360; p  < 0.001) and RAS mutation (HR 1.629; p  = 0.044) were independently associated with worse overall survival. Conclusion RAS mutations are associated with positive margins in patients undergoing resection of CLM. Tumors with RAS mutation should prompt careful efforts to achieve negative resection margins.

Although circulating tumor cells (CTCs) have potential as diagnostic biomarkers for cancer, determining their prognostic role in cancer patients undergoing treatment is a challenge. We evaluated the prognostic value of programmed death-ligand 1 (PD-L1) expression in CTCs in colorectal and prostate cancer patients undergoing treatment. Peripheral blood samples were collected from 62 metastatic colorectal cancer patients and 30 metastatic prostate cancer patients. CTCs were isolated from the samples using magnetic separation with the cell-surface vimentin(CSV)-specific 84-1 monoclonal antibody that detects epithelial-mesenchymal transitioned (EMT) CTCs. CTCs were enumerated and analyzed for PD-L1 expression using confocal microscopy. PD-L1 expression was detectable in CTCs and was localized in the membrane and/or cytoplasm and nucleus. CTC detection alone was not associated with poor progression-free or overall survival in colorectal cancer or prostate cancer patients, but nuclear PD-L1 (nPD-L1) expression in these patients was significantly associated with short survival durations. These results demonstrated that nPD-L1 has potential as a clinically relevant prognostic biomarker for colorectal and prostate cancer. Our data thus suggested that use of CTC-based models of cancer for risk assessment can improve the standard cancer staging criteria and supported the incorporation of nPD-L1 expression detection in CTCs detection in such models.