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Introduction Patients with gastrointestinal (GI) cancers have an increased risk of serious complications and death from SARS-CoV-2 infection. The immunogenicity of vaccines in patients with GI cancers receiving anti-cancer therapies is unclear. We conducted a prospective study to evaluate the prevalence of neutralizing antibodies in a cohort of GI cancer patients receiving chemotherapy following SARS-CoV-2 vaccination. Materials and Methods Between September 2020 and April 2021, patients with cancer undergoing chemotherapy were enrolled. At baseline (day 0), days 28, 56, and 84, we assessed serum antibodies to SARS-CoV-2 spike (anti-S) and anti-nucleocapsid (anti-NP) and concomitantly assessed virus neutralization using a pseudovirus neutralization assay. Patients received either the Pfizer/BioNTech BNT162b2, or the Oxford/AstraZeneca ChAdOx1 vaccine. Results All 152 patients enrolled had a prior diagnosis of cancer; colorectal (n = 80, 52.6%), oesophagogastric (n = 38, 25.0%), and hepato pancreatic biliary (n = 22, 12.5%). Nearly all were receiving systemic anti-cancer therapy (99.3%). Of the 51 patients who did not receive a vaccination prior to, or during the study, 5 patients had detectable anti-NP antibodies. Ninety-nine patients received at least one dose of vaccine prior to, or during the study. Within 19 days following the first dose of vaccine, 30.0% had anti-S detected in serum which increased to 70.2% at days 20-39. In the 19 days following a second dose, anti-S positivity was 84.2% (32/38). However, pseudovirus neutralization titers (pVNT80) decreased from days 20 to 39. Conclusion Despite the immunosuppressive effects of chemotherapy, 2 doses of SARS-CoV-2 vaccines are able to elicit a protective immune response in patients’ ongoing treatment for gastrointestinal cancers. Decreases in pseudoviral neutralization were observed after 20-39 days, re-affirming the current recommendation for vaccine booster doses. Clinical Trial Registration Number NCT04427280.

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Patients with cancer are currently prioritized in coronavirus disease 2019 (COVID-19) vaccination programs globally, which includes administration of mRNA vaccines. Cytokine release syndrome (CRS) has not been reported with mRNA vaccines and is an extremely rare immune-related adverse event of immune checkpoint inhibitors. We present a case of CRS that occurred 5 d after vaccination with BTN162b2 (tozinameran)—the Pfizer-BioNTech mRNA COVID-19 vaccine—in a patient with colorectal cancer on long-standing anti-PD-1 monotherapy. The CRS was evidenced by raised inflammatory markers, thrombocytopenia, elevated cytokine levels (IFN-γ/IL-2R/IL-18/IL-16/IL-10) and steroid responsiveness. The close temporal association of vaccination and diagnosis of CRS in this case suggests that CRS was a vaccine-related adverse event; with anti-PD1 blockade as a potential contributor. Overall, further prospective pharmacovigillence data are needed in patients with cancer, but the benefit–risk profile remains strongly in favor of COVID-19 vaccination in this population., A rare case of cytokine release syndrome in a patient on anti-PD-1 blockade that was likely related to BNT162b2 vaccination supports prospective monitoring of patients with cancer after COVID-19 vaccine administration.

Introduction: Gastric and esophageal adenocarcinoma is a leading cause of cancer-related death globally. Surgery is the cornerstone modality for cure where feasible. Clinical studies over the past two decades have provided evidence for the use of perioperative chemotherapy and chemoradiotherapy to improve patient outcomes. However, there remains no global consensus in the optimal use of these therapies.Areas covered: In this review, the authors summarize the latest evidence for perioperative multimodality therapy in resectable esophagogastric adenocarcinoma including the use of combination chemotherapy and targeted therapy containing regimens. In addition, the authors discuss some of the clinical and molecular biomarkers, such as PET imaging and microsatellite instability which can inform future practice and further clinical investigation.Expert opinion: A multimodal approach has been proven to improve survival outcomes over surgery alone. Whilst there is no global standard of care for multi-modality therapies in resectable OG cancer, clinical trials are refining the use of chemotherapy and radiotherapy in the neoadjuvant and adjuvant settings. Further investigation is on-going to further optimize therapy and the integration of molecular targeted agents.

Amplification or overexpression of the FGFR family of receptor tyrosine kinases occurs in a significant proportion of gastric cancers. Regorafenib is a multikinase inhibitor of angiogenic and oncogenic kinases, including FGFR, which showed activity in the randomized phase II INTEGRATE clinical trial in advanced gastric cancer. There are currently no biomarkers that predict response to this agent, and whether regorafenib is preferentially active in FGFR-driven cancers is unknown. Through screening 25 gastric cancer cell lines, we identified five cell lines that were exquisitely sensitive to regorafenib, four of which harbored amplification or overexpression of FGFR family members. These four cell lines were also sensitive to the FGFR-specific inhibitors, BGJ398, erdafitinib, and TAS-120. Regorafenib inhibited FGFR-driven MAPK signaling in these cell lines, and knockdown studies confirmed their dependence on specific FGFRs for proliferation. In the INTEGRATE trial cohort, amplification or overexpression of FGFRs 1–4 was detected in 8%–19% of cases, however, this was not associated with improved progression-free survival and no objective responses were observed in these cases. Further preclinical analyses revealed FGFR-driven gastric cancer cell lines rapidly reactivate MAPK/ERK signaling in response to FGFR inhibition, which may underlie the limited clinical response to regorafenib. Importantly, combination treatment with an FGFR and MEK inhibitor delayed MAPK/ERK reactivation and synergistically inhibited proliferation of FGFR-driven gastric cancer cell lines. These findings suggest that upfront combinatorial inhibition of FGFR and MEK may represent a more effective treatment strategy for FGFR-driven gastric cancers.

Despite recent advances in the systemic treatment of gastroesophageal cancers, prognosis remains poor. Comprehensive molecular analyses have characterized the genomic landscape of gastroesophageal cancer that has established therapeutic targets such as human epidermal growth factor receptor 2 (HER2), vascular endothelial growth factor receptor (VEGFR) and programmed death ligand 1 (PD-L1). The aberrant fibroblast growth factor receptor 2 (FGFR2) pathway is attractive for targetable therapy with FGFR inhibition based on preclinical data showing a pivotal role in the progression of gastric cancer (GC).

Introduction: Outcomes in metastatic colorectal cancer are improving, due to the tailoring of therapy enabled by better understanding of clinical behavior according to molecular subtype. Areas covered: A review of the literature and recent conference presentations was undertaken on the topic of systemic treatment of metastatic colorectal cancer. This review summarizes expert discussion of the current evidence for therapies in metastatic colorectal cancer (mCRC) based on molecular subgrouping. Expert opinion: EGFR-targeted and VEGF-targeted antibodies are now routinely incorporated into treatment strategies for mCRC. EGFR-targeted antibodies are restricted to patients with extended RAS wild-type profiles, with evidence that they should be further restricted to patients with left-sided tumors. Clinically distinct treatment pathways based on tumor RAS, BRAF, HER2 and MMR status, are now clinically applicable. Evidence suggests therapy for additional subgroups will soon be defined; the most advanced being for patients with KRAS G12 C mutation and gene TRK fusion defects.

Anti-angiogenic drugs are an efficacious class of therapy in the treatment of patients with metastatic colorectal cancer (mCRC). Aflibercept, a vascular endothelial growth factor (VEGF) trap which binds the angiogenic factors VEGF-A, VEGF-B, and placental growth factor (PIGF) is approved in combination with FOLFIRI chemotherapy following progression after an oxaliplatin-containing regimen.This report provides a review of the practice-changing clinical studies which have established the use of anti-angiogenic therapy as second-line therapy in mCRC including aflibercept with FOLFIRI (5FU, leucovorin, irinotecan). This review also evaluates aflibercept with other chemotherapy regimens as well as efficacy and safety data from real-world studies.Aflibercept in combination with FOLFIRI chemotherapy is an established safe and efficacious regimen for the treatment of mCRC as second-line chemotherapy. Although several toxicities have been described, the majority are either low grade or manageable by drug cessation and supportive therapies. For optimal outcomes, patient selection and close observation of toxicities is essential.

Background Mutations and fusions in Fibroblast Growth Factor Receptor 3 (FGFR3) occur in 10–20% of metastatic urothelial carcinomas and confer sensitivity to FGFR inhibitors. However, responses to these agents are often short-lived due to the development of acquired resistance. The objective of this study was to identify mechanisms of resistance to FGFR inhibitors in two previously uncharacterised bladder cancer cell lines harbouring FGFR3 fusions and assess rational combination therapies to enhance sensitivity to these agents. Methods Acquired resistance to FGFR inhibitors was generated in two FGFR3 fusion harbouring cell lines, SW780 (FGFR3-BAIAP2L1 fusion) and RT4 (FGFR3-TACC3 fusion), by long-term exposure to the FGFR inhibitor BGJ398. Changes in levels of receptor tyrosine kinases were assessed by phospho-RTK arrays and immunoblotting. Changes in cell viability and proliferation were assessed by the Cell-Titre Glo assay and by propidium iodide staining and FACS analysis. Results Long term treatment of FGFR3-fusion harbouring SW780 and RT4 bladder cancer cell lines with the FGFR inhibitor BGJ398 resulted in the establishment of resistant clones. These clones were cross-resistant to the clinically approved FGFR inhibitor erdafitinib and the covalently binding irreversible FGFR inhibitor TAS-120, but remained sensitive to the MEK inhibitor trametinib, indicating resistance is mediated by alternate activation of MAPK signalling. The FGFR inhibitor-resistant SW780 and RT4 lines displayed increased expression of pERBB3, and strikingly, combination treatment with an FGFR inhibitor and the ATP-competitive pan-ERBB inhibitor AZD8931 overcame this resistance. Notably, rapid induction of pERBB3 and reactivation of pERK also occurred in parental FGFR3 fusion-driven lines within 24 h of FGFR inhibitor treatment, and combination treatment with an FGFR inhibitor and AZD8931 delayed the reactivation of pERBB3 and pERK and synergistically inhibited cell proliferation. Conclusions We demonstrate that increased expression of pERBB3 is a key mechanism of adaptive resistance to FGFR inhibitors in FGFR3-fusion driven bladder cancers, and that this also occurs rapidly following FGFR inhibitor treatment. Our findings demonstrate that resistance can be overcome by combination treatment with a pan-ERBB inhibitor and suggest that upfront combination treatment with FGFR and pan-ERBB inhibitors warrants further investigation for FGFR3-fusion harbouring bladder cancers.

We present a case of cytokine release syndrome (CRS) that occurred five days after vaccination with BTN162b2 (tozinameran), an mRNA COVID-19 vaccine, in a patient with colorectal cancer on long-standing anti-PD-1 monotherapy. The CRS was evidenced by raised inflammatory markers, thrombocytopenia, elevated cytokine levels (IFN-y/IL-2R/IL-18/IL-16/IL-10), and steroid responsiveness.

BackgroundMutations and fusions in Fibroblast Growth Factor Receptor 3 (FGFR3) occur in 10-20% of metastatic urothelial carcinomas, and can confer sensitivity to FGFR inhibitors such as BGJ398 and erdafitinib. However, responses to these agents are often short-lived due to the development of acquired resistance. The objective of this study was to identify mechanisms of acquired resistance to FGFR inhibitors in two previously uncharacterized bladder cancer cell lines harbouring FGFR3 fusions, and assess rational combination therapies to enhance their activity. MethodsAcquired resistance to FGFR inhibitors was generated in two FGFR3 fusion harbouring cell lines, SW780 (FGFR3-BAIAP2L1 fusion) and RT4 (FGFR3-TACC3 fusion), by either long-term exposure to increasing concentrations of BGJ398 or sustained exposure to high concentrations of drug. Alterations in levels of key cell signalling regulators was assessed in resistant lines by phospho-RTK arrays and immunoblotting. Synergy between BGJ398 and alternate targeted therapies was explored using cell viability and apoptosis assays.ResultsAcquired resistance to BGJ398 in SW780 and RT4 cells was associated with increased expression of members of ERBB family of receptor tyrosine kinases and pAXL. Combination treatment of resistant cells with an FGFR inhibitor and either a pan-ERBB or an AXL inhibitor overcame this resistance. We also noted rapid reactivation of pERK in parental FGFR3 fusion-driven lines within 4-72 hours of BGJ398 treatment, with concomitant increase in pERBB3. Up-front combination treatment with BGJ398 and the pan-ERBB inhibitor AZD8931 delayed the reactivation of pERK, and induced a synergistic inhibition of cell viability and a concomitant increase in apoptosis.ConclusionsWe identify increased activation of AXL and ERBB family receptors as mechanisms of resistance to FGFR inhibition. Our findings suggest that upfront combination treatment with FGFR and pan-ERBB inhibitors warrants further investigation for FGFR3-fusion harbouring bladder cancers.

Despite recent advances in the treatment of metastatic colorectal cancer (mCRC), 5 years survival rates remain low. Chemotherapy remains as the mainstay of treatment with only few available targeted therapies. Human epidermal growth factor receptor 2 (HER2) amplification occurs in approximately 5% of metastatic colorectal cancer and it has been studied as a mechanism of resistance for anti-epidermal growth factor receptor (EGFR) therapy. Furthermore, several studies such as HERACLES-A, MyPathway and the DESTINY-CRC01 trials have shown significant clinical benefit of HER2 blockade in patients with HER2 amplified mCRC. In this review, we provide an overview of the clinicopathological features of HER2 amplification and mutations in mCRC. In addition, we review HER2 as a biomarker of intrinsic and acquired anti-EGFR resistance as well as the preclinical, clinical and translational studies investigating the use of HER2 targeted therapies and future studies.

Background 10%–15% of early-stage colon cancers harbour either deficient mismatch repair (dMMR), microsatellite instability high (MSI-H) or POLE exonuclease domain mutations, and are characterised by high tumour mutational burden and increased lymphocytic infiltrate. Metastatic dMMR colon cancers are highly sensitive to immune checkpoint inhibition, and recent data show POLE-mutant tumours are similarly responsive. We are conducting a phase III randomised trial to determine if the addition of the anti-PD-L1 antibody avelumab following adjuvant chemotherapy improves disease-free survival (DFS) in patients with stage III dMMR/MSI-H or POLE mutant colon cancer and is a cost-effective approach for the UK National Health Service (NHS). Methods We are recruiting patients with completely resected, stage III colon cancer confirmed to have dMMR/MSI-H, locally or POLE exonuclease domain mutation on central testing. Eligible patients are randomised in a 1:1 ratio to standard fluoropyrimidine-based chemotherapy (capecitabine, oxaliplatin for 12 weeks or capecitabine for 24 weeks) or chemotherapy, followed by avelumab (10 mg/kg, 2 weekly for 24 weeks). Stratification is by chemotherapy received and MMR/MSI-H status. The primary endpoint is DFS. Secondary endpoints include overall survival, toxicity, quality of life and health resource use. The 3-year DFS rate in the control arm is expected to be ~75%. Avelumab is expected to improve the 3-year DFS rate by 12% (ie, 87%). Target accrual is 402 patients, which provides 80% power to detect an HR of 0.48 for DFS at a two-sided alpha of 0.05. This national, multicentre phase III trial is sponsored by the Royal Marsden NHS Foundation Trust and it is anticipated that approximately 40 centres in the UK will participate. This study opened to recruitment in August 2018. Trial registration number NCT03827044

BACKGROUND: Advanced biliary tract cancers (BTCs) have a poor prognosis and limited treatment options. This exploratory phase II study aimed to evaluate the activity of the mTOR inhibitor everolimus in advanced BTC and explore prognostic biomarkers. METHODS: Patients with advanced BTCs, who had not received chemotherapy for advanced disease, were enroled to receive everolimus (10 mg daily). The primary endpoint was disease control rate (DCR) at 12 weeks. Secondary endpoints included overall response rate, progression-free survival (PFS), overall survival (OS) and adverse events. Activation status of the RAS and phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathways was assessed by DNA sequencing and immunohistochemistry on archival tumour tissue. RESULTS: The study enroled 27 patients and the DCR at 12 weeks was 48%. Median PFS was 5.5 months (95% confidence interval (CI): 2.1–10.0 months) and median OS was 9.5 months (95% CI: 5.5–16.6 months). DCR at 12 weeks was significantly worse for gall bladder carcinoma compared to other anatomical sites, and there was a trend towards a worsened PFS and OS. Treatment was well tolerated. KRAS (12%) and PIK3CA mutations (12%) were uncommon. Immunohistochemical staining for PI3K/AKT/mTOR pathways did not significantly correlate with outcome. CONCLUSION: In unselected patients, everolimus demonstrated clinical activity as first-line monotherapy in advanced BTC.

Advanced biliary tract cancer (BTC) has a poor prognosis and limited treatment options. The PI3K/Akt/mTOR signalling pathway is hyperactivated in a subset of BTCs, and clinical activity of the mTOR inhibitor everolimus has been observed in some patients with BTC. The goal of this study was to identify biomarkers predictive of everolimus response. Twenty BTC cell lines were assessed for everolimus sensitivity with a spectrum of growth inhibitory responses observed. Molecular biomarkers of sensitivity and resistance were identified by interrogation of the activation status of the Ras/MAPK and PI3K/Akt/mTOR pathways. K-Ras mutations and/or amplifications were identified in 45% of cell lines and were associated with resistance to everolimus. Activating mutations in PIK3CA or loss of PTEN was not predictive of everolimus response; however, high basal levels of pAKT were associated with sensitivity, independent of Ras/MAPK pathway activation status. Notably, everolimus inhibited mTOR signalling to a similar extent in sensitive and resistant cell lines, suggesting that relative dependence on the mTOR pathway rather than the magnitude of pathway inhibition determines everolimus response. Consistent with the known limitations of rapalogs, everolimus induced feedback-mediated activation of AKT in BTC cell lines, which could be overcome by cotreatment with an AKT inhibitor or ATP-competitive mTORC1/mTORC2 inhibitors. However, both approaches failed to induce greater apoptosis compared to everolimus, and mTORC1/mTORC2 kinase inhibitors induced compensatory activation of pERK, identifying an inherent limitation of these agents in BTC cell lines. These findings suggest that future trials of everolimus in BTC would benefit from preselecting patients based on their K-Ras and PI3K/mTOR pathway activation status. The study also identifies strategies for enhancing inhibition of the PI3K/mTOR pathway in BTC cell lines.

Summary Biliary tract cancers (BTCs) currently have no approved targeted therapies. Although genomic profiling of primary BTCs has identified multiple potential drug targets, accurate models are needed for their evaluation. Genomic profiling of 22 BTC cell lines revealed they harbor similar mutational signatures, recurrently mutated genes, and genomic alterations to primary tumors. Transcriptomic profiling identified two major subtypes, enriched for epithelial and mesenchymal genes, which were also evident in patient-derived organoids and primary tumors. Interrogating these models revealed multiple mechanisms of MAPK signaling activation in BTC, including co-occurrence of low-activity BRAF and MEK mutations with receptor tyrosine kinase overexpression. Finally, BTC cell lines with altered ERBB2 or FGFRs were exquisitely sensitive to specific targeted agents, whereas surprisingly, IDH1-mutant lines did not respond to IDH1 inhibitors in vitro. These findings establish BTC cell lines as robust models of primary disease, reveal specific molecular disease subsets, and highlight specific molecular vulnerabilities in these cancers., Graphical Abstract, Highlights • BTC cell lines harbor similar genomic alterations to primary tumors • Transcriptomic profiling of BTC cell lines identified two molecular subtypes • MAPK signaling is activated in BTC via multiple mechanisms • BTC lines with deregulated ERBB2 or FGFRs respond to specific targeted therapies, Biological Sciences; Genetics; Genomics; Cancer

Bacteria of the genus Shigella, consisting of 4 species and >50 serotypes, cause shigellosis, a foodborne disease of significant morbidity, mortality, and economic loss worldwide. Classical Shigella identification based on selective media and serology is tedious, time-consuming, expensive, and not always accurate. A molecular diagnostic assay does not distinguish Shigella at the species level or from enteroinvasive Escherichia coli (EIEC). We inspected genomic sequences from 221 Shigella isolates and observed low concordance rates between conventional designation and molecular serotyping: 86.4% and 80.5% at the species and serotype levels, respectively. Serotype determinants for 6 additional serotypes were identified. Examination of differentiation gene markers commonly perceived as characteristic hallmarks in Shigella showed high variability among different serotypes. Using this information, we developed ShigaTyper, an automated workflow that utilizes limited computational resources to accurately and rapidly determine 59 Shigella serotypes using Illumina paired-end whole-genome sequencing (WGS) reads. Shigella serotype determinants and species-specific diagnostic markers were first identified through read alignment to an in-house curated reference sequence database. Relying on sequence hits that passed a threshold level of coverage and accuracy, serotype could be unambiguously predicted within 1 min for an average-size WGS sample of ∼500 MB. Validation with WGS data from 380 isolates showed an accuracy rate of 98.2%. This pipeline is the first step toward building a comprehensive WGS-based analysis pipeline of Shigella spp. in a field laboratory setting, where speed is essential and resources need to be more cost-effectively dedicated. IMPORTANCEShigella causes diarrheal disease with serious public health implications. However, conventional Shigella identification methods are laborious and time-consuming and can be erroneous due to the high similarity between Shigella and enteroinvasive Escherichia coli (EIEC) and cross-reactivity between serotyping antisera. Further, serotype interpretation is complicated for inexperienced users. To develop an easier method with higher accuracy based on whole-genome sequencing (WGS) for Shigella serotyping, we systematically examined genomic information of Shigella isolates from 53 serotypes to define rules for differentiation and serotyping. We created ShigaTyper, an automated pipeline that accurately and rapidly excludes non-Shigella isolates and identifies 59 Shigella serotypes using Illumina paired-end WGS reads. A serotype can be unambiguously predicted at a data processing speed of 538 MB/min with 98.2% accuracy from a regular laptop. Once it is installed, training in bioinformatics analysis and Shigella genetics is not required. This pipeline is particularly useful to general microbiologists in field laboratories.

Biliary tract cancers (BTC) comprise a group of rare and heterogeneous poor-prognosis tumours with the incidence of intrahepatic cholangiocarcinoma increasing over recent years. Combination chemotherapy with gemcitabine and cisplatin is the established first-line treatment for advanced BTC with a significant but modest survival advantage over monotherapy. There remains no accepted standard treatment in the second-line setting, although recent results from a randomised study have shown a survival benefit with 5-fluorouracil and oxaliplatin chemotherapy. Historically, clinical trials investigating targeted therapies in unselected BTC have failed to demonstrate significant clinical benefit. More recently, advancement in molecular exploration of BTC has shed light on the complex biological heterogeneity within these tumours and has also identified actionable genomic aberrations, such as fibroblast growth factor receptor 2 (FGFR2) gene fusions, isocitrate dehydrogenase (IDH) and BRAF mutations, which offer promise with the anticipation of increased responses and durable clinical benefit in selected patients. Several targeted drugs have now entered clinical development with some encouraging results being seen. Here we review the current and rapidly evolving therapeutic landscape of advanced BTC, including targeted therapies and immunotherapy. We also discuss how recent efforts and successes in BTC are overcoming the obstacles typically associated with precision medicine in rare cancers. Ultimately, the management of advanced BTC is likely to become molecularly selected in the near future with the hope of finally improving the bleak prognosis of patients with this disease.

499 Background: Regorafenib was the first treatment to demonstrate a survival benefit in patients with HCC after progression on sorafenib. The RESORCE trial found that regorafenib improved overall survival with acceptable toxicity, in patients with disease progression on sorafenib who tolerated ≥400mg sorafenib daily and had Child-Pugh A liver function. Methods: We performed a multicentre, retrospective, observational study of patients with HCC receiving regorafenib in the UK, following its availability in April 2018. Results: Data on a total of 104 patients were included from April 2018–August 2019, and 80.8% were male. Age was collected in 85 patients, with a median of 68 years (range 22–86). 23.5% had NAFLD, 21.2% had ALD, 12.9% had HBV, and 3.5% had HCV. Prior management included sorafenib (100%), TACE (30.8%), resection (12.9%). Duration of sorafenib treatment was evaluable in 99/104 patients, and reported a median of 8.7 months (range 1.8–76.6). Duration of regorafenib treatment was evaluable in 92/104 patients, and reported a median of 3.9 months (range 0.0–15.7). Following treatment with regorafenib, 6 patients (5.8%) achieved partial response, 37 (35.6%) achieved stable disease and 45 (43.3%) had progressive disease as the best response. 15 (14.4%) were not assessed and 1 (1.1%) had mixed response. Survival data is immature with 62/101 (61.4%) patients alive at the time of census with median survival currently 6.5 months. Fatigue was the most frequent AE, with 69/88 patients (85.2%) for all grades. 12/88 patients (14.8%) had Grade 3 fatigue. Other significant AEs include hand-foot syndrome (6/85 patients [7.3%] had Grade 3) and diarrhoea (4/83 patients [4.9%] had Grade 3). Conclusions: The population in our real-world experience of regorafenib for HCC had a similar duration of prior sorafenib to those in the RESORCE trial. However, there was different balance of aetiologies with a lower proportion of patients with HBV and HCV. The rate of partial response is similar to the RESORCE trial with fewer patients achieving stable disease. The incidence of fatigue was higher, but the incidence of hand-foot syndrome and diarrhoea were lower. Further expansion and follow-up of this population is warranted.

TPS3615 Background: Colon cancer with deficient mismatch repair (dMMR) and POLE mutations are characterised by a high tumor mutational burden (TMB) and an immunogenic lymphocyte infiltrate. Approximately 12% of stage III colon cancer have dMMR. POLE mutations occur in 1% of colon cancers, but is enriched in patients

Shiga toxin-producing Escherichia coli (STEC) is a significant foodborne pathogen with great economic consequences. There has been an increased food safety concern with this organism since outbreaks of human illnesses caused by this pathogen were first reported in 1982. Therefore, developing a reliable, sensitive, and rapid assay capable of detecting E. coli O157 and the main toxins produced by STEC (i.e., Shiga toxins 1 [Stx(1)] and 2 [Stx(2)]) will directly benefit regulatory agencies by minimizing analysis time. Here, we use Luminex technology to detect multiple analytes in a single 50-ml sample. Using commercially available monoclonal antibodies coupled to carboxylated magnetic microbeads, we developed an immunoassay capable of simultaneously serotyping E. coli O157 and detecting Stx(1) and/or Stx(2). The specificity and sensitivity of this immunoassay was tested against a collection of 34 E. coli isolates belonging to various O serogroups phenotypically different for Stx. The results were compared with microplate sandwich enzyme-linked immunosorbent assay (ELISA), and no cross-reactivity was observed for any of the monoclonal antibodies used. An increased sensitivity up to 1,000 times was observed in the microbead-based immunoassay when compared with the microplate sandwich ELISA. The results indicate that Luminex technology has the potential to simultaneously detect multiple targets without loss of specificity and/or sensitivity. A blind experiment was conducted with 48 samples of ground beef, lettuce, and milk spiked with ≤2 CFU/g E. coli. All the samples were correctly identified, with no false positives or false negatives. This microbead-based immunoassay could be extended to simultaneously detect additional foodborne pathogens and their toxic markers.

Summary What is known and objective The management of metastatic melanoma has changed significantly in the past decade with the development of immunotherapies and targeted molecular therapies. Trials of targeted therapies have focused mainly on patients with the most common BRAF V600 mutations, namely V600E/K substitutions, with very little information available on the benefit of targeted therapies on less commonly occurring mutations such as V600R/D and M. Case summary We present a 54-year-old man with metastatic melanoma harbouring a rare BRAF V600M mutation, who experienced clinical and radiological response to combined therapy with the BRAF inhibitor dabrafenib and MEK inhibitor trametinib. What is new and conclusion As our understanding of these therapies evolves and an increasing number of patients have mutational testing performed, there is a clear imperative – as highlighted by this case – to test for rarer mutations and facilitate their inclusion both in everyday practice and in future clinical trials.

Background Case reports suggest that voriconazole 1% eye drops are effective in treating refractory fungal keratitis. There are no data available on the stability and shelf-life of extemporaneously prepared voriconazole 1% eye drops. Aim To determine the stability and shelf-life of extemporaneous voriconazole 1% eye drops when stored at 2 to 8 °C. Method Voriconazole 1% solution was manufactured using voriconazole injection and preserved with 0.01% benzalkonium chloride. Aliquots (2.5 mL) were dispensed into pre-sterilised plastic eye dropper bottles. Samples from 3 different batches were stored at 2 to 8 °C and analysed using high performance liquid chromatography to measure voriconazole concentrations on Days 0, 15, 30, 45, 60 and 75 after manufacture. Results After 75 days at 2 to 8 °C there was minimal reduction in the potency of the voriconazole 1% solution. Extrapolating the stability data beyond 75 days using linear regression suggest that it may take up to 4 months for voriconazole concentrations to drop below 90% of the nominal content. Conclusion Voriconazole 1% eye drops stored at 2 to 8 °C display negligible chemical degradation after 75 days.

We investigated involvement of feral swine in contamination of agricultural fields and surface waterways with Escherichia coli O157:H7 after a nationwide outbreak traced to bagged spinach from California. Isolates from feral swine, cattle, surface water, sediment, and soil at 1 ranch were matched to the outbreak strain.

The objective of this study was to develop a rapid, simple method for enhanced detection and isolation of low levels of Escherichia coli O157:H7 from leafy produce and surface water using recirculating immunomagnetic separation (RIMS) coupled with real-time PCR and a standard culture method. The optimal enrichment conditions for the method also were determined. Analysis of real-time PCR data (C(T) values) suggested that incubation of lettuce and spinach leaves rather than rinsates provides better enrichment of E. coli O157:H7. Enrichment of lettuce or spinach leaves at 42 degrees C for 5 h provided better detection than enrichment at 37 degrees C. Extended incubation of surface water for 20 h at 42 degrees C did not improve the detection. The optimized enrichment conditions were also employed with modified Moore swabs, which were used to sample flowing water sites. Positive isolation rates and real-time PCR results indicated an increased recovery of E. coli O157:H7 from all samples following the application of RIMS. Under these conditions, the method provided detection and/or isolation of E. coli O157:H7 at levels as low as 0.07 CFU/g of lettuce, 0.1 CFU/g of spinach, 6 CFU/100 ml of surface water, and 9 CFU per modified Moore swab. During a 6-month field study, modified Moore swabs yielded high isolation rates when deployed in natural watershed sites. The method used in this study was effective for monitoring E. coli O157:H7 in the farm environment, during postharvest processing, and in foodborne outbreak investigations.

We studied the efficacy, tolerability and clinical courses of dabrafenib in patients with metastatic melanoma who were ineligible for enrolment into a clinical trial. Between July 2011 and May 2013, patients with unresectable stage III or stage IV, V600-mutated metastatic melanoma who were not eligible for inclusion into clinical trials were offered treatment with dabrafenib through a named patient programme. Routine efficacy and toxicity data were collected throughout treatment and studied retrospectively. The endpoints were progression-free survival (PFS), overall survival and best overall response. Thirty-one patients commenced dabrafenib therapy including six individuals who had progressed on a prior BRAF-inhibitor treatment. The majority of patients had cerebral metastases (n=17) and/or a poor performance status [Eastern Cooperative Oncology Group (ECOG)≥2, n=11]. Median overall survival was 5.6 months (range 0.1-22 months). Median PFS was 3.3 months (range 0.1-21) and was similar despite performance status. One patient had a complete response and eight showed partial responses to treatment. Patients with cerebral metastases (n=17) had a median PFS of 4.6 months. Five patients (16%) had dose-limiting toxicities. Despite several poor prognostic features, dabrafenib is a safe and effective treatment in the community setting, with occasional impressive outcomes.

Background Patients prefer oral to intravenous (IV) palliative chemotherapy, provided that oral therapy is not less effective. We compared the efficacy and safety of oral and IV fluoropyrimidines for treatment of colorectal cancer (CRC). Objectives To compare the effects of oral and IV fluoropyrimidine chemotherapy in patients treated with curative or palliative intent for CRC. Search methods We searched the Cochrane Central Register of Controlled Trials (CENTRAL; 2016, Issue 5), along with OVID MEDLINE, OVID Embase, and Web of Science databases, in June 2016. We also searched five clinical trials registers, several conference proceedings, and reference lists from study reports and systematic reviews. We contacted pharmaceutical companies to identify additional studies. Selection criteria We included randomised controlled trials (RCTs) comparing oral and IV fluoropyrimidine chemotherapy in patients treated with curative or palliative intent for CRC. Data collection and analysis Three review authors extracted data and assessed risk of bias independently. We assessed the seven domains in the Cochrane 'Risk of bias' tool and three additional domains: schedules of outcome assessment and/or follow-up; use of intention-to-treat analysis; and baseline comparability of treatment arms. Main results We included nine RCTs (total of 10,918 participants) that examined treatment with curative intent for CRC with neoadjuvant and/or adjuvant chemotherapy. We included 35 RCTs (total of 12,592 participants) that examined treatment with palliative intent for inoperable advanced or metastatic CRC with chemotherapy (31 first-line studies, two second-line studies, and two studies of first- or second-line chemotherapy). All studies included male and female participants, and no studies included participants younger than 18 years of age. Patients treated with curative intent for CRC with neoadjuvant and/or adjuvant chemotherapy • Disease-free survival (DFS): DFS did not differ between participants treated with oral versus IV fluoropyrimidines (hazard ratio (HR) 0.93, 95% confidence interval (CI) 0.87 to 1.00; seven studies, 8903 participants; moderate-quality evidence). • Overall survival (OS): OS did not differ between participants treated with oral versus IV fluoropyrimidines (HR 0.92, 95% CI 0.84 to 1.00; seven studies, 8902 participants analysed; high-quality evidence). • Grade ≥ 3 adverse events (AEs): Participants treated with oral fluoropyrimidines experienced less grade ≥ 3 neutropenia/granulocytopenia (odds ratio (OR) 0.14, 95% CI 0.11 to 0.16; seven studies, 8087 participants; moderate-quality evidence), stomatitis (OR 0.21, 95% CI 0.14 to 0.30; five studies, 4212 participants; low-quality evidence), and any grade ≥ 3 AEs (OR 0.82, 95% CI 0.74 to 0.90; five studies, 7741 participants; low-quality evidence). There was more grade ≥ 3 hand foot syndrome (OR 4.59, 95% CI 2.97 to 7.10; five studies, 5731 participants; low-quality evidence) in patients treated with oral fluoropyrimidines. There were no differences between participants treated with oral versus IV fluoropyrimidines in occurrence of grade ≥ 3 diarrhoea (OR 1.12, 95% CI 0.99 to 1.25; nine studies, 9551 participants; very low-quality evidence), febrile neutropenia (OR 0.59, 95% CI 0.18 to 1.90; four studies, 2925 participants; low-quality evidence), vomiting (OR 1.05, 95% CI 0.83 to 1.34; eight studies, 9385 participants; low-quality evidence), nausea (OR 1.21, 95% CI 0.97 to 1.51; seven studies, 9233 participants; low-quality evidence), mucositis (OR 0.64, 95% CI 0.25 to 1.62; four studies, 2233 participants; very low-quality evidence), and hyperbilirubinaemia (OR 1.67, 95% CI 0.52 to 5.38; three studies, 2757 participants; very low-quality evidence). Patients treated with palliative intent for inoperable advanced or metastatic CRC with chemotherapy • Progression-free survival (PFS): Overall, PFS was inferior in participants treated with oral versus IV fluoropyrimidines (HR 1.06, 95% CI 1.02 to 1.11; 23 studies, 9927 participants; moderate-quality evidence). Whilst PFS was worse in participants treated with oral compared with IV fluoropyrimidines when UFT/Ftorafur or eniluracil with oral 5-fluorouracil (5-FU) was used, PFS did not differ between individuals treated with oral versus IV fluoropyrimidines when capecitabine, doxifluridine, or S-1 was used. • OS: Overall, OS did not differ between participants treated with oral versus IV fluoropyrimidines (HR 1.02, 95% CI 0.99 to 1.05; 29 studies, 12,079 participants; high-quality evidence). OS was inferior in participants treated with oral versus IV fluoropyrimidines when eniluracil with oral 5-fluorouracil (5-FU) was used. • Time to progression (TTP): TTP was inferior in participants treated with oral versus IV fluoropyrimidines (HR 1.07, 95% CI 1.01 to 1.14; six studies, 1970 participants; moderate-quality evidence). • Objective response rate (ORR): ORR did not differ between participants treated with oral versus IV fluoropyrimidines (OR 0.98, 95% CI 0.90 to 1.06; 32 studies, 11,115 participants; moderate-quality evidence). • Grade ≥ 3 AEs: Participants treated with oral fluoropyrimidines experienced less grade ≥ 3 neutropenia/granulocytopenia (OR 0.17, 95% CI 0.15 to 0.18; 29 studies, 11,794 participants; low-quality evidence), febrile neutropenia (OR 0.27, 95% CI 0.21 to 0.36; 19 studies, 9407 participants; moderate-quality evidence), stomatitis (OR 0.26, 95% CI 0.20 to 0.33; 21 studies, 8718 participants; low-quality evidence), mucositis (OR 0.17, 95% CI 0.12 to 0.24; 12 studies, 4962 participants; low-quality evidence), and any grade ≥ 3 AEs (OR 0.83, 95% CI 0.74 to 0.94; 14 studies, 5436 participants; low-quality evidence). There was more grade ≥ 3 diarrhoea (OR 1.66, 95% CI 1.50 to 1.84; 30 studies, 11,997 participants; low-quality evidence) and hand foot syndrome (OR 3.92, 95% CI 2.84 to 5.43; 18 studies, 6481 participants; moderate-quality evidence) in the oral fluoropyrimidine arm. There were no differences between oral and IV fluoropyrimidine arms in terms of grade ≥ 3 vomiting (OR 1.18, 95% CI 1.00 to 1.40; 23 studies, 9528 participants; low-quality evidence), nausea (OR 1.16, 95% CI 0.99 to 1.36; 25 studies, 9796 participants; low-quality evidence), and hyperbilirubinaemia (OR 1.62, 95% CI 0.99 to 2.64; nine studies, 2699 participants; low-quality evidence). Authors' conclusions Results of this review should provide confidence that treatment for CRC with most of the oral fluoropyrimidines commonly used in current clinical practice is similarly efficacious to treatment with IV fluoropyrimidines. Treatment with eniluracil with oral 5-FU was associated with inferior PFS and OS among participants treated with palliative intent for CRC, and eniluracil is no longer being developed. Oral and IV fluoropyrimidines have different patterns of side effects; future research may focus on determining the basis for these differences.

Purpose: Although there have been reports describing the use of 1% voriconazole eye drops in the treatment of fungal infections, little is known about the penetration of voriconazole eye drops into the vitreous humour. The aim of this study was to elucidate if topical application of 1% voriconazole eye drops could reach therapeutic levels in the vitreous humour. Methods: A prospective, open-label trial involving 10 participants selected from patients scheduled to undergo elective, posterior segment surgery. Participants received 1% voriconazole solution, preserved with 0.01% benzalkonium chloride, hourly for four doses or four times a day for 3 days. Vitreous humour was obtained at the start of surgery and analysed using a validated high-performance liquid chromatography assay. Results: The minimum and maximum voriconazole concentration after hourly dosing (n = 5) was 0.1 and 1.1 µg/mL, respectively, whereas the median was 0.3 µg/mL. Samples were taken between 0.8 and 5.4 h after the last dose, with the median at 1.2 h. Almost all voriconazole concentrations from the four times a day group (n = 5) were below the limit of quantification. Conclusions: One per cent voriconazole eye drops are able to penetrate into human vitreous humour. Adequate concentration for treatment of sensitive Candida species can be achieved upon hourly administration.

We investigated involvement of feral swine in contamination of agricultural fields and surface waterways with Escherichia coli O157:H7 after a nationwide outbreak traced to bagged spinach from California. Isolates from feral swine, cattle, surface water, sediment, and soil at 1 ranch were matched to the outbreak strain.

Objective To determine the therapeutic efficacy of adjuvant use of voriconazole, 1 %, eyedrops in the treatment of refractory fungal keratitis. Methods A prospective open-label trial was conducted to determine voriconazole levels obtained in human aqueous humor after administration of a 1% solution, preserved with 0.01% benzalkonium chloride, every 6 hours for 3 days, or hourly for 4 doses. Ten participants were selected among patients scheduled to undergo elective anterior segment surgery, and samples were tested using validated high-performance liquid chromatography. Results The mean (SD) voriconazole concentrations after hourly dosing (n = 5) was 1.90 (1.12) μg/mL and after a single dosing every 6 hours (n = 5) was 0.94 (1.21) μg/mL, respectively. The mean (SD) sampling times after the last administration of eyedrops were 1.1 (0.5) hours after hourly dosing and 2.1 (0.6) hours after a single dosing every 6 hours. Conclusions Voriconazole, 1%, eyedrops are well tolerated and penetrate into human aqueous humor when administered at hourly or 6-hourly intervals. They are effective in treatingCandidaandAspergilluskeratitis, are substantially more affordable than oral therapy, and have less potential to cause systemic adverse effects.