ObjectiveRegorafenib 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.DesignPatients with RAS 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 (Ktrans), enhancing fraction (EF) and their product KEF (summarised median values of Ktrans× EF) were generated. Circulating tumour (ct) DNA was collected monthly until progressive disease and tested for clonal RAS mutations by digital-droplet PCR. Tumour vasculature (CD-31) was scored by immunohistochemistry on 70 sequential tissue biopsies.ResultsTwenty-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). RAS 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).ConclusionsCombining DCE-MRI and ctDNA predicts duration of anti-angiogenic response to regorafenib and may improve patient management with potential health/economic implications.
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.
Trastuzumab in combination with chemotherapy represents the standard of care in HER2-positive advanced gastro-esphageal cancer (GOC), but development of resistance limits response. MicroRNAs (miRs) modulate key pathways in GOC. Identification of miRs responsible for resistance to HER2 inhibition may help stratify patients, predict response and define novel strategies to restore drug sensitivity. A high-throughput LNA™ miR-inhibitor screen in HER2-positive NCI-N87 and HER2-negative FLO-1 GOC cell lines was performed in order to identify potential miRs involved in trastuzumab sensitivity/resistance. Cells were treated with a combination of cisplatin, 5FU, and trastuzumab. MiRs were considered significant if LNAs caused >40% reduction in cell viability 72 hours post treatment relative to controls with a t-test p-value of <0.001 across 3 biological replicates. Putative hits with higher baseline miR levels on nCounter® NCI-N87 cell line analysis were validated in two HER2-positive cell lines by calculating the difference (Δ) in cell viability between cells treated with trastuzumab+chemotherapy versus ‘no drug' control. MiR levels in translational samples from ten HER2-positive GOC patients recruited to the FOrMAT trial (NCT02112357) were analysed by ddPCR. MiR levels were categorized as ‘low' or ‘high' relative to median expression and correlated with clinical response. Kaplan-Meier curves for PFS and OS were generated. In NCI-N87 and FLO-1 cell lines, 59 and 37 LNAs respectively caused >40% reduction in cell viability compared to controls (p<0.001). Eighteen miRs were significant exclusively in the NCI-N87 cell line screen, suggesting potential involvement in the HER2-signaling pathway. nCounter® analysis showed 6 of these were elevated at baseline in NCI-N87: miR-1260, miR-148-3p, miR-28-3p, miR-331-3p, miR-7-5p and miR-365. Inhibition of miR-148a-3p increased sensitivity to trastuzumab + 5FU+cisplatin in both the NCI-N87 cell line (Δ 67% versus Δ 55% in the control) and OACP4C cell line (Δ 50% versus Δ 47% in the control). Patients who responded to trastuzumab + chemotherapy in FOrMAT had a trend towards lower average miR-148a-3p plasma levels than those who failed to respond. Median PFS for plasma miR-148a-3p ‘low' subgroup was 146 days versus 154 days in ‘high' subgroup. Median OS for plasma miR-148a-3p ‘low' subgroup was 357 days versus 226 days for miR-148a-3p ‘high' subgroup. Our screen identified a panel of miRs associated with GOC resistance to HER2 inhibitors in combination with chemotherapy. Inhibition of these miRs affects GOC cell viability and restores sensitivity. MiR-148a-3p emerged as a potential biomarker of resistance and further evaluation is underway in the PLATFORM trial (NCT02678182). Citation Format: Hazel Lote, Andrea Lampis, George Vlachogiannis, Jens Hahne, Sing-Yu Moorcraft, Michael Davidson, Carolin Fong, Ruwaida Begum, Matteo Fassan, Sheela Rao, David Watkins, Naureen Starling, Ian Chau, David Cunningham, Nicola Valeri. MicroRNAs as biomarkers of resistance to HER2 inhibition in combination with chemotherapy in gastro-esophageal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 258.
MicroRNAs (miRs) are small non-coding RNAs involved in cell homeostasis and carcinogenesis and control multiple oncogenic pathways. Numerous miRs are aberrantly expressed in colorectal cancer (CRC) and their deregulation is associated with clinical outcome and cancer progression. Chemo-refractory metastatic CRC (mCRC) patients are often treated with regorafenib, a multi-tyrosine kinase inhibitor with anti-angiogenic effect. Given the limited clinical benefits of regorafenib in unselected patient populations, there is an unmet need for better patient selection and identification of mechanisms of resistance. miRs are highly stable and have shown encouraging value as potential biomarkers for CRC detection and prognosis. Here we aimed to identify circulatory miRs that might be exploited for the upfront selection of patients’ candidate to regorafenib treatment. We ran a translational phase II trial of regorafenib in chemo-refractory mCRC patients. Serum, plasma and tissue biopsies were obtained at baseline (BL), every four weeks and at disease progression (PD). Patient Derived Organoids (PDOs) and PDO-xenotransplants were generated to study primary and acquired resistance to regorafenib. MiR profiling was performed in baseline serum of all patients by NanoString nCounter platform of 800 genes and validated with digital droplet (dd)PCR in serum, plasma, PDOs and by In Situ Hybridization (ISH) in matching tissue biopsies. Progression Free Survival (PFS) was measured from date of registration to date of first progression/relapse or death from cancer progression. Overall Survival (OS) was measured from date of randomisation to death from cancer. Further validation was performed by ddPCR in 97 patients from an independent patient’s cohort. MiR expression was tested in 43 BL sera and dysregulation in 28 miRs was associated with PFS and OS. Up-regulation of miR-652-3p was associated with worse PFS and OS. These results were validated by ddPCR on the same serum samples, and matching plasmas. ISH confirmed upregulation of this miR in sequential tissues biopsies, PDOs and PDO-xenotransplants of patients with primary and acquired resistance to regorafenib. Validation in an independent patient’s cohort confirmed direct association of miR-652-3p dysregulation with PFS and OS. Functional experiments to define miR652-3p mediated resistance have shown that there is a decrease of tumour growth and migration in miR-652-3p inhibition in PDOs. We provide initial evidence suggesting that circulating miR-652-3p might work as a negative predictive biomarker for the upfront selection of patients’ candidate to regorafenib treatment. Citation Format: Somaieh Hedayat, Khurum Khan, David Cunningham, George Vlachogiannis, Andrea Lampis, Silvia Marchetti, Matteo Fassan, Ruwaida Begum, Marta Schirripa, Fotios Fotios Loupakis, Nicola Valeri. Circulating miR-652-3p as a biomarker of resistance to regorafenib in metastatic colorectal cancer patients [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 LB-305.
Background: MicroRNAs (miRs) are small non-coding RNAs involved in cell homeostasis. miR dysregulation has been linked with activation of oncogenic pathways, cancer progression and clinical outcome in mCRC. Chemo-refractory mCRC patients are often treated with regorafenib, a multi-tyrosine kinase inhibitor with anti-angiogenic effect. Given the limited clinical benefits of regorafenib in unselected patient populations, there is an unmet need for better patient stratification and identification of mechanisms of resistance. Methods: Serial liquid biopsies were obtained at baseline (BL) and monthly until disease progression (PD) in 43 patients treated with regorafenib for chemo-refractory mCRC in the context of a phase II clinical trial (PROSPECT-R). Tissue biopsies were obtained at BL, after 2 months and at PD within the same trial and used to establish Patient-Derived Organoids (PDOs) and for molecular analyses. PDOs co-cultures and PDO-xenotransplants were generated to study primary and acquired resistance to regorafenib. Liquid biopsies were also obtained from an additional cohort (n=97) of mCRC patients treated with regorafenib. MiR profiling was performed on baseline seras using NanoString nCounter platform and significant miRs were validated with digital droplet (dd)PCR in serum, plasma, PDOs and by In Situ Hybridization (ISH) in matching tissue biopsies. Functional experiments were performed in PDOs, PDO co-cultures and PDO-xenotransplants. Results: MiR expression was tested in 43 BL in the PROSPECT-R trial. Up-regulation of miR-652-3p was associated with poor PFS and OS. These results were validated by ddPCR on the same serum samples, matching plasmas and organoids. ISH confirmed upregulation of this miR in sequential tissues biopsies, PDOs and PDO-xenotransplants of patients with primary and acquired resistance to regorafenib. The same findings were confirmed in the validation cohort. Functional experiments showed that miR-652-3p upregulation has significant effects on cancer cell migration. Up and down-regulation of miR-652-3p upon regorafenib treatment translated in a significant effect on cell viability in PDO co-cultures and liver PDO-xenotransplants. RNA-sequencing analysis of miR-652-3p over-expressing organoids showed downregulation of several components of the serine synthesis pathway. Among them, phosphoserine aminotransferase (PSAT1) was validated as a miR-652-3p direct target. Rescue experiments confirmed that PSAT1 over-expression and silencing lead to increase sensitivity and resistance to regorafenib respectively via cell and non-cell autonomous regulation of autophagy. Conclusions: Our data suggest that miR-652-3p may be uses as a prognostic/predictive biomarker for the selection of treatment and provide mechanics insight on regorafenib resistance. Citation Format: Somaieh Hedayat, Andrea Lampis, George Vlachogiannis, khurum Khan, Silvia Marchetti, Matteo Fassan, Michele Ghidini, Ruwaida Begum, Marta Schirripa, Rodolfo Passalacqua, David Cunningham, Fotios Loupakis, Nicola Valeri. MicroRNA deregulation of the serine synthesis pathway controls intrinsic and non-cell autonomous mechanism of resistance to Regorafenib in metastatic colorectal cancer (mCRC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5720.
Despite showing clinical activity in BRAF-mutant melanoma, the MEK inhibitor (MEKi) trametinib failed to show clinical benefit in KRAS-mutant colorectal cancer. To identify mechanisms of resistance to MEK inhibition we identified gene expression differences between MEKi-sensitive and MEKi-resistant colorectal cancer cell lines. Strikingly, inflammation-related gene sets were the most significantly enriched in cell lines exhibiting intrinsic or acquired resistance to MEK inhibition. The bromodomain inhibitor JQ1 suppressed inflammatory gene expression and in combination with MEK inhibitors displayed synergistic anti-proliferative activity, inhibited colony formation and induced apoptosis in colorectal cancer cell lines. NFkB activation was greater in cell lines resistant to MEK inhibition and JQ1 treatment suppressed TNF-induced translocation of NFkB to the nucleus. Resistance to MEK inhibition could be induced by inflammatory cytokines or by conditioned medium from macrophage cultures and was associated with greater NFkB activation. In 2-dimensional cell culture and in 3-dimensional spheroid models of colorectal cancer, resistance to trametinib was readily established; however, co-treatment of cells with JQ1 and trametinib suppressed the emergence of resistant populations. Notably, high inflammatory gene expression was confirmed in patient-derived organoid (PDO) models of colorectal cancer, which displayed resistance to trametinib. JQ1 treatment of PDOs suppressed inflammatory gene expression and showed synergistic anti-proliferative activity in combination with trametinib. Combination treatment of in vivo models of KRAS-mutant colorectal cancer significantly suppressed tumor growth. Our findings provide a potential explanation for the limited response to MEK inhibitors in KRAS-mutant colorectal cancer, where a highly inflammatory environment may prime cells to be resistant to MEK inhibition. Moreover, the high expression of inflammatory genes was associated with significantly reduced survival of colorectal cancer patients. Excitingly, this opens novel therapeutic opportunities to overcome intrinsic and acquired resistance to MEK inhibition in colorectal cancer. Citation Format: Steve Wagner, George Vlachogiannis, Alexis de Haven Brandon, Melanie Valenti, Gary Box, Liam Jenkins, Caterina Mancusi, Annette Self, Ritika Chauhan, Alistair Rust, Nik Matthews, Kate Eason, Anguraj Sadanandam, Clare Isacke, Vladimir Kirkin, Nicola Valeri, Steven R. Whittaker. Suppression of inflammatory gene expression overcomes resistance to MEK inhibition in KRAS-mutant 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-094.
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