MicroRNAs as biomarkers for trastuzumab-based therapy in HER2-positive advanced oesophago-gastric cancer patients.

Background: 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. Methods: 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). Results: 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). Conclusion: Normalised miR-148a-3p may be a relevant biomarker for trastuzumab-based therapy in advanced HER2-positive oesophago-gastric cancer patients.

New molecular mechanisms in cholangiocarcinoma: signals triggering interleukin-6 production in tumor cells and KRAS co-opted epigenetic mediators driving metabolic reprogramming.

BACKGROUND: Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA. METHODS: Cholangiocarcinogenesis was induced in rats (TAA) and mice (JnkΔhepa + CCl4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRASG12D cells. Cell signaling, growth, gene regulation and [U-13C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model. RESULTS: Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRASG12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRASG12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRASG12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression. CONCLUSIONS: In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA.

KRAS and BRAF mutations in circulating tumour DNA from locally advanced rectal cancer.

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.

Functional imaging and circulating biomarkers of response to regorafenib in treatment-refractory metastatic colorectal cancer patients in a prospective phase II study.

OBJECTIVE: 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. DESIGN: Patients 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. RESULTS: 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). 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). CONCLUSIONS: Combining DCE-MRI and ctDNA predicts duration of anti-angiogenic response to regorafenib and may improve patient management with potential health/economic implications.