Despite biomarker stratification, the anti-EGFR antibody cetuximab is only effective against a subgroup of colorectal cancers (CRCs). This genomic and transcriptomic analysis of the cetuximab ...resistance landscape in 35 RAS wild-type CRCs identified associations of NF1 and non-canonical RAS/RAF aberrations with primary resistance and validated transcriptomic CRC subtypes as non-genetic predictors of benefit. Sixty-four percent of biopsies with acquired resistance harbored no genetic resistance drivers. Most of these had switched from a cetuximab-sensitive transcriptomic subtype at baseline to a fibroblast- and growth factor-rich subtype at progression. Fibroblast-supernatant conferred cetuximab resistance in vitro, confirming a major role for non-genetic resistance through stromal remodeling. Cetuximab treatment increased cytotoxic immune infiltrates and PD-L1 and LAG3 immune checkpoint expression, potentially providing opportunities to treat cetuximab-resistant CRCs with immunotherapy.
•NF1 and non-canonical KRAS and BRAF aberrations associate with cetuximab resistance•Genetic resistance drivers are absent in most biopsies that acquired resistance•Stromal remodeling is an alternative non-genetic mechanism of cetuximab resistance•Cetuximab-mediated immune modulation may sensitize CRCs to immunotherapy
Woolston et al. show that in metastatic colorectal cancer cetuximab resistance can be conferred by genetic mechanisms, such as NF1 loss or RAS/RAF alterations, or by transcriptomic changes that induce a stroma-rich phenotype. They also provide a rationale for combining cetuximab with immunotherapy.
Response to preoperative chemo-radiotherapy (CRT) varies. We assessed whether circulating tumor DNA (ctDNA) might be an early indicator of tumor response or progression to guide therapy adaptation in ...rectal cancer.
A total of 243 serial plasma samples were analyzed from 47 patients with localized rectal cancer undergoing CRT. Up to three somatic variants were tracked in plasma using droplet digital PCR. RECIST and MRI tumor regression grade (mrTRG) evaluated response. Survival analyses applied Kaplan-Meier method and Cox regression.
ctDNA detection rates were: 74% (
= 35/47) pretreatment, 21% (
= 10/47) mid CRT, 21% (
= 10/47) after completing CRT, and 13% (
= 3/23) after surgery. ctDNA status after CRT was associated with primary tumor response by mrTRG (
= 0.03). With a median follow-up of 26.4 months, metastases-free survival was shorter in patients with detectable ctDNA after completing CRT HR 7.1; 95% confidence interval (CI), 2.4-21.5;
< 0.001, persistently detectable ctDNA pre and mid CRT (HR 3.8; 95% CI, 1.2-11.7;
= 0.02), and pre, mid, and after CRT (HR 11.5; 95% CI, 3.3-40.4;
< 0.001) compared with patients with undetectable or nonpersistent ctDNA. In patients with detectable ctDNA, a fractional abundance threshold of ≥0.07% mid CRT or ≥0.13% after completing CRT predicted for metastases with 100% sensitivity and 83.3% specificity for mid CRT and 66.7% for CRT completion. All 3 patients with detectable ctDNA post-surgery relapsed compared with none of the 20 patients with undetectable ctDNA (
= 0.001).
ctDNA identified patients at risk of developing metastases during the neoadjuvant period and post-surgery, and could be used to tailor treatment.
Sequential profiling of plasma cell-free DNA (cfDNA) holds immense promise for early detection of patient progression. However, how to exploit the predictive power of cfDNA as a liquid biopsy in the ...clinic remains unclear. RAS pathway aberrations can be tracked in cfDNA to monitor resistance to anti-EGFR monoclonal antibodies in patients with metastatic colorectal cancer. In this prospective phase II clinical trial of single-agent cetuximab in
wild-type patients, we combine genomic profiling of serial cfDNA and matched sequential tissue biopsies with imaging and mathematical modeling of cancer evolution. We show that a significant proportion of patients defined as
wild-type based on diagnostic tissue analysis harbor aberrations in the RAS pathway in pretreatment cfDNA and, in fact, do not benefit from EGFR inhibition. We demonstrate that primary and acquired resistance to cetuximab are often of polyclonal nature, and these dynamics can be observed in tissue and plasma. Furthermore, evolutionary modeling combined with frequent serial sampling of cfDNA allows prediction of the expected time to treatment failure in individual patients. This study demonstrates how integrating frequently sampled longitudinal liquid biopsies with a mathematical framework of tumor evolution allows individualized quantitative forecasting of progression, providing novel opportunities for adaptive personalized therapies.
Liquid biopsies capture spatial and temporal heterogeneity underpinning resistance to anti-EGFR monoclonal antibodies in colorectal cancer. Dense serial sampling is needed to predict the time to treatment failure and generate a window of opportunity for intervention.
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Cholangiocarcinomas (CCA) are resistant to chemotherapy, so new therapeutic agents are needed. We performed a screen to identify small-molecule compounds that are active against CCAs. Levels of ...microRNA 21 (MIR21 or miRNA21) are increased in CCAs. We investigated whether miRNA21 mediates resistance of CCA cells and organoids to HSP90 inhibitors.
We performed a high-throughput screen of 484 small-molecule compounds to identify those that reduced viability of 6 human CCA cell lines. We tested the effects of HSP90 inhibitors on cells with disruption of the MIR21 gene, cells incubated with MIR21 inhibitors, and stable cell lines with inducible expression of MIR21. We obtained CCA biopsies from patients, cultured them as organoids (patient-derived organoids). We assessed their architecture, mutation and gene expression patterns, response to compounds in culture, and when grown as subcutaneous xenograft tumors in mice.
Cells with IDH1 and PBRM1 mutations had the highest level of sensitivity to histone deacetylase inhibitors. HSP90 inhibitors were effective in all cell lines, irrespective of mutations. Sensitivity of cells to HSP90 inhibitors correlated inversely with baseline level of MIR21. Disruption of MIR21 increased cell sensitivity to HSP90 inhibitors. CCA cells that expressed transgenic MIR21 were more resistant to HSP90 inhibitors than cells transfected with control vectors; inactivation of MIR21 in these cells restored sensitivity to these agents. MIR21 was shown to target the DnaJ heat shock protein family (Hsp40) member B5 (DNAJB5). Transgenic expression of DNAJB5 in CCA cells that overexpressed MIR21 re-sensitized them to HSP90 inhibitors. Sensitivity of patient-derived organoids to HSP90 inhibitors, in culture and when grown as xenograft tumors in mice, depended on expression of miRNA21.
miRNA21 appears to mediate resistance of CCA cells to HSP90 inhibitors by reducing levels of DNAJB5. HSP90 inhibitors might be developed for the treatment of CCA and miRNA21 might be a marker of sensitivity to these agents.
There are limited data on circulating, cell-free, tumour (ct)DNA analysis in locally advanced rectal cancer (LARC). Digital droplet (dd)PCR was used to investigate KRAS/BRAF mutations in ctDNA from ...baseline blood samples of 97 LARC patients who were treated with CAPOX followed by chemoradiotherapy, surgery and adjuvant CAPOX ± cetuximab in a randomised phase II trial. KRAS mutation in G12D, G12V or G13D was detected in the ctDNA of 43% and 35% of patients with tumours that were mutant and wild-type for these hotspot mutations, respectively, according to standard PCR-based analyses on tissue. The detection rate in the ctDNA of 10 patients with less common mutations was 50%. In 26 cases ctDNA analysis revealed KRAS mutations that were not previously found in tissue. Twenty-two of these (84.6%) were detected following repeat tissue testing by ddPCR. Overall, the ctDNA detection rate in the KRAS mutant population was 66%. Detection of KRAS mutation in ctDNA failed to predict prognosis or refine patient selection for cetuximab. While this study confirms the feasibility of ctDNA analysis in LARC and the high sensitivity of ddPCR, larger series are needed to better address the role of ctDNA as a prognostic or predictive tool in this setting.
Regorafenib demonstrated efficacy in patients with metastatic colorectal cancer (mCRC). Lack of predictive biomarkers, potential toxicities and cost-effectiveness concerns highlight the unmet need ...for better patient selection.
Patients with
mutant mCRC with biopsiable metastases were enrolled in this phase II trial. Dynamic contrast-enhanced (DCE) MRI was acquired pretreatment and at day 15 post-treatment. Median values of volume transfer constant (K
), enhancing fraction (EF) and their product KEF (summarised median values of K
× EF) were generated. Circulating tumour (ct) DNA was collected monthly until progressive disease and tested for clonal
mutations by digital-droplet PCR. Tumour vasculature (CD-31) was scored by immunohistochemistry on 70 sequential tissue biopsies.
Twenty-seven patients with paired DCE-MRI scans were analysed. Median KEF decrease was 58.2%. Of the 23 patients with outcome data, >70% drop in KEF (6/23) was associated with higher disease control rate (p=0.048) measured by RECIST V. 1.1 at 2 months, improved progression-free survival (PFS) (HR 0.16 (95% CI 0.04 to 0.72), p=0.02), 4-month PFS (66.7% vs 23.5%) and overall survival (OS) (HR 0.08 (95% CI 0.01 to 0.63), p=0.02). KEF drop correlated with CD-31 reduction in sequential tissue biopsies (p=0.04).
mutant clones decay in ctDNA after 8 weeks of treatment was associated with better PFS (HR 0.21 (95% CI 0.06 to 0.71), p=0.01) and OS (HR 0.28 (95% CI 0.07-1.04), p=0.06).
Combining DCE-MRI and ctDNA predicts duration of anti-angiogenic response to regorafenib and may improve patient management with potential health/economic implications.
Circulating tumour DNA (ctDNA) to detect minimal residual disease (MRD) is emerging as a biomarker to predict recurrence in patients with curatively treated early stage colorectal cancer (CRC). ctDNA ...risk stratifies patients to guide adjuvant treatment decisions. We are conducting the UK's first multi-centre, prospective, randomised study to determine whether a de-escalation strategy using ctDNA to guide adjuvant chemotherapy (ACT) decisions is non-inferior to standard of care (SOC) chemotherapy, as measured by 3-year disease free survival (DFS) in patients with resected CRC with no evidence of MRD (ctDNA negative post-operatively). In doing so we may be able to spare patients unnecessary chemotherapy and associated toxicity and achieve significant cost savings for the National Health Service (NHS).
We are recruiting patients with fully resected high risk stage II and stage III CRC who are being considered for ACT into the study which uses results from a plasma-only ctDNA assay to guide treatment decisions. Eligible patients are randomised 1:1 to receive ctDNA-guided chemotherapy versus SOC chemotherapy. The primary endpoint is the difference in DFS at 3 years between the trial arms. Secondary endpoints include the proportion of patients in the ctDNA-guided arm who are ctDNA negative post-operatively and receive de-escalated ACT compared to the standard arm, the difference in overall survival (OS), neurotoxicity and quality of life between the arms, and the cost-effectiveness of ctDNA-guided therapy compared to SOC treatment. We hypothesise that using a ctDNA-guided approach to ACT decisions is non-inferior to SOC. Target accrual is 1621 patients over 4 years, which will provide a power of 80% with an alpha of 0.1 to demonstrate non-inferiority with a margin of 1.25 in survival of the ctDNA-guided approach compared to SOC. We anticipate approximately 50 UK centres will participate. The study opened with the Guardant Reveal plasma-only ctDNA assay in August 2022.
The trial will determine whether ctDNA guided ACT is non-inferior to SOC ACT in patients with fully resected high risk stage II and stage III resected CRC, with the potential to significantly reduce unnecessary ACT and the toxicity associated with it.
NCT04050345.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Previously, we classified colorectal cancers (CRCs) into five CRCAssigner (CRCA) subtypes with different prognoses and potential treatment responses, later consolidated into four consensus molecular ...subtypes (CMS). Here we demonstrate the analytical development and validation of a custom NanoString nCounter platform-based biomarker assay (NanoCRCA) to stratify CRCs into subtypes. To reduce costs, we switched from the standard nCounter protocol to a custom modified protocol. The assay included a reduced 38-gene panel that was selected using an in-house machine-learning pipeline. We applied NanoCRCA to 413 samples from 355 CRC patients. From the fresh frozen samples (n = 237), a subset had matched microarray/RNAseq profiles (n = 47) or formalin-fixed paraffin-embedded (FFPE) samples (n = 58). We also analyzed a further 118 FFPE samples. We compared the assay results with the CMS classifier, different platforms (microarrays/RNAseq) and gene-set classifiers (38 and the original 786 genes). The standard and modified protocols showed high correlation (> 0.88) for gene expression. Technical replicates were highly correlated (> 0.96). NanoCRCA classified fresh frozen and FFPE samples into all five CRCA subtypes with consistent classification of selected matched fresh frozen/FFPE samples. We demonstrate high and significant subtype concordance across protocols (100%), gene sets (95%), platforms (87%) and with CMS subtypes (75%) when evaluated across multiple datasets. Overall, our NanoCRCA assay with further validation may facilitate prospective validation of CRC subtypes in clinical trials and beyond.
FOLFIRINOX, a combination of chemotherapy drugs (Fluorouracil, Oxaliplatin, Irinotecan -FOI), provides the best clinical benefit in pancreatic ductal adenocarcinoma (PDAC) patients. In this study we ...explore the role of miRNAs (MIR) as modulators of chemosensitivity to identify potential biomarkers of response. We find that 41 and 84 microRNA inhibitors enhance the sensitivity of Capan1 and MiaPaCa2 PDAC cells respectively. These include a MIR1307-inhibitor that we validate in further PDAC cell lines. Chemotherapy-induced apoptosis and DNA damage accumulation are higher in MIR1307 knock-out (MIR1307KO) versus control PDAC cells, while re-expression of MIR1307 in MIR1307KO cells rescues these effects. We identify binding of MIR1307 to CLIC5 mRNA through covalent ligation of endogenous Argonaute-bound RNAs cross-linking immunoprecipitation assay. We validate these findings in an in vivo model with MIR1307 disruption. In a pilot cohort of PDAC patients undergoing FOLFIRONX chemotherapy, circulating MIR1307 correlates with clinical outcome.
This study aimed to identify microRNAs (miRs) as circulating biomarkers of resistance to first-line trastuzumab-based therapy in advanced HER2-positive oesophago-gastric cancer patients.
A ...high-throughput 1015 Exiqon miRCURY LNA™ microRNA inhibitor library screen was performed in trastuzumab-treated HER2-positive NCI-N87 and HER2-negative FLO-1 oesophago-gastric cancer cell lines. NanoString nCounter
miR analysis was performed in NCI-N87, FLO-1, and MAGIC trial (ISRCTN93793971) formalin-fixed paraffin-embedded (FFPE) oesophago-gastric cancer patient samples. MiR-148a-3p copies in plasma samples were quantified using digital droplet polymerase chain reaction (ddPCR) from HER2-positive oesophago-gastric cancer patients treated with standard-of-care trastuzumab-based therapy within the FOrMAT (NCT02112357) and PLATFORM (NCT02678182) clinical trials. The primary endpoints were overall survival (OS) for plasma miR-148a-3p HIGH (>median) versus LOW (≤median). The secondary endpoints were progression-free survival (PFS) and 3-month progression-free rates (PFRs) miR-148a-3p HIGH versus LOW. PLATFORM sensitivity analysis normalised miR-148a-3p (NmiR-148a-3p).
The inhibition of miR-148a-3p reduced NCI-N87 relative cell viability (<0.6) and expression was high (>242) in NCI-N87 and HER2-positive MAGIC trial patients (n=5). Normalised-miR-148a-3p (NmiR-148a-3p) LOW versus HIGH demonstrated a statistically significant difference in 3-month PFRs (n=23; OR, 0.11 0.02-0.78; p=0.027; aOR, 0.03 0.001-0.71, p=0.029) but no difference in OS or PFS. There was no statistically significant relationship between miR-148-3p LOW versus HIGH for OS (PLATFORM, n=62; hazard ratio HR, 0.98 0.57-1.66; p=0.933; FOrMAT, n=8; HR, 0.54 0.13-2.31; p=0.322), PFS (n=62; HR, 1.08 0.65-1.81; p=0.759; FOrMAT, n=8; HR, 1.26 0.31-5.07; p=0.714), or PFRs (PLATFORM, n=31; odds ratio OR, 0.67 0.2-2.8; p=0.577).
Normalised miR-148a-3p may be a relevant biomarker for trastuzumab-based therapy in advanced HER2-positive oesophago-gastric cancer patients.
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