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ObjectiveTo determine the rates of asymptomatic viral carriage and seroprevalence of SARS-CoV-2 antibodies in healthcare workers.DesignA cross-sectional study of asymptomatic healthcare workers undertaken on 24/25 April 2020.SettingUniversity Hospitals Birmingham NHS Foundation Trust (UHBFT), UK.Participants545 asymptomatic healthcare workers were recruited while at work. Participants were invited to participate via the UHBFT social media. Exclusion criteria included current symptoms consistent with COVID-19. No potential participants were excluded.InterventionParticipants volunteered a nasopharyngeal swab and a venous blood sample that were tested for SARS-CoV-2 RNA and anti-SARS-CoV-2 spike glycoprotein antibodies, respectively. Results were interpreted in the context of prior illnesses and the hospital departments in which participants worked.Main outcome measureProportion of participants demonstrating infection and positive SARS-CoV-2 serology.ResultsThe point prevalence of SARS-CoV-2 viral carriage was 2.4% (n=13/545). The overall seroprevalence of SARS-CoV-2 antibodies was 24.4% (n=126/516). Participants who reported prior symptomatic illness had higher seroprevalence (37.5% vs 17.1%, χ2=21.1034, pConclusions and relevanceWe identify differences in the occupational risk of exposure to SARS-CoV-2 between hospital departments and confirm asymptomatic seroconversion occurs in healthcare workers. Further investigation of these observations is required to inform future infection control and occupational health practices.

The BEACON CRC trial demonstrated a survival advantage over chemotherapy for a combination of targeted agents comprising the potent BRAF inhibitor encorafenib together with cetuximab and binimetinib. Resistance to BRAF inhibition in CRC arises in part through the generation and activation of RAF dimers resulting in MEK-ERK pathway reactivation. Paradox breaker BRAF inhibitors, such as PLX8394, are designed to inhibit RAF dimer formation. We analyzed whether paradox breakers reduce pathway reactivation and so have enhanced potency compared with encorafenib in BRAF mutant CRC. The potency of encorafenib and PLX8394 was greater than vemurafenib and the degree of pathway reactivation somewhat less. However, dose response curves for encorafenib and PLX8394 were similar and there was no significant differences in degree of pathway reactivation. To our knowledge these data represent the first comparative data of encorafenib and paradox breaker inhibitors in BRAF mutant CRC. Whilst these results support further investigation of PLX8394, all three agents tested reactivated the pathway in melanoma cells, a disease in which monotherapy is effective. Strategies focused on restricting RAF dimerization fail to address the impact that specific context of BRAF mutation in CRC has on targeted therapy outcomes.

Introduction: Tumor specific MHC class II (tsMHC-II) expression impacts tumor microenvironmental immunity. tsMHC-II positive cancer cells act as surrogate antigen presenting cells and targets for CD4+ T cell-mediated lysis. In colorectal cancer (CRC), tsMHC-II negativity is common and in cell lines is attributable to CIITA promoter methylation. To clarify mechanisms of tsMHC-II repression in CRC we analyzed CRC organoids which are epigenetically faithful to tissue of origin. Methods: 15 primary CRC organoids were treated with IFNγ +/- epigenetic modifiers. Flow cytometry was used to assess for tsMHC-II inducibility. RT-qPCR, total RNAseq, nanopore sequencing, bisulfite conversion/pyrosequencing and western blotting were used to quantitate CIITA, STAT1, IRF1 and JAK1 expression, gene structure and promoter methylation and ChIP-PCR to quantitate H3K9ac, H3K9Me2 and EZH2 occupancy at CIITA. Results: We define three types of tsMHC-II response to IFNγ in CRC: strong- (≥50%), delayed/weak- (11-49%) and non-inducibility (≤10%). 8/15 demonstrated strong expression, 4/15 delayed/weak: although it was possible to overcome this weak expression with prolonged IFNγ exposure in 3/15 without additional pharmacological treatment. In 1/15 organoid, expression was restricted even with prolonged exposure due to IFNγ-mediated EZH2 occupancy at CIITA: tsMHC-II expression was enhanced by EZH2 and HDAC inhibition. Non-inducibility is seen in 3 CMS1 organoids due to JAK1 mutation. These demonstrated low level (11%) JAK1 promoter methylation, but tsMHC-II inducibility was not rescued with DNMT inhibition. No organoid demonstrated CIITA promoter methylation, despite confirming the presence in previously described 2D cell lines, suggesting this is acquired through culture methods and not reflective of disease biology. Conclusion: We demonstrate using epigenetically appropriate models the individual variation in pharmacologic tractability of epigenetic modification in CRC. Providing IFNγ signaling is intact, most CRC organoids are class II inducible. Up-regulation of restricted tsMHC-II through targeted epigenetic therapy is seen in 1/15 organoid. These data may explain some of the disappointing results of immuno-epigenetic approaches in the clinic. Citation Format: Oliver J. Pickles, Kasun Wanigasooriya, Anetta Ptasinska, Akshay J. Patel, Helen L. Robbins, Claire Bryer, Celina M. Whalley, Louise Tee, Neeraj Lal, Maria Pinna, Nahla Elzefzafy, Phillipe Taniere, Andrew D. Beggs, Gary W. Middleton. Colorectal cancer displays three patterns of tumor-specific MHC-II inducibility and dynamics, with implications for combining epigenetic therapy with immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2989.

Partial or total resistance to preoperative chemoradiotherapy occurs in more than half of locally advanced rectal cancer patients. Several novel or repurposed drugs have been trialled to improve cancer cell sensitivity to radiotherapy, with limited success. To understand the mechanisms underlying this resistance and target them effectively, we initially compared treatment-naive transcriptomes of radiation-resistant and radiation-sensitive patient-derived organoids (PDO) to identify biological pathways involved in radiation resistance. Pathway analysis revealed that PI3K/AKT/mTOR and epithelial mesenchymal transition pathway genes were upregulated in radioresistant PDOs. Moreover, single-cell sequencing of pre & post-irradiation PDOs showed mTORC1 upregulation, which was confirmed by a genome-wide CRSIPR-Cas9 knockout screen using irradiated colorectal cancer (CRC) cell lines. Based on these findings, we evaluated cancer cell viability in vitro when treated with radiation in combination with dual PI3K/mTOR inhibitors apitolisib or dactolisib. Significant AKT phosphorylation was detected in HCT116 cells two hours post-irradiation (p=0.027). Dual PI3K/mTOR inhibitors radiosensitised HCT116 and radiation-resistant PDO lines. The PI3K/AKT/mTOR pathway upregulation contributes to radioresistance and its pharmacological inhibition leads to significant radiosensitisation in an organoid model of CRC and is a target for clinical trials.

SummaryThe rapid emergence of COVID-19 has sent shockwaves through healthcare systems globally, with cancer patients at increased risk. The interplay of the virus and host immune system has been implicated in the development of ARDS. Immunotherapy agents have the potential to adversely potentiate this phenomenon, requiring careful real-world observation.

Background In January 2020 reports of unidentified severe respiratory illness were described in Wuhan, China. A rapid expansion in cases affecting most countries around the globe led to major changes in the way people live their daily lives. In the United Kingdom, the Department of Health and Social Care directed healthcare providers to establish additional resources to manage the anticipated surge in cases that could overwhelm the health services. A priority area was testing for SARS-CoV-2 RNA and its detection by qualitative RT-PCR. Design A laboratory workflow twinning research environment with clinical laboratory capabilities was implemented and validated in the University of Birmingham within 4 days of the project initiation. The diagnostic capability was centred on an IVD CE-marked RT-PCR kit and designed to provide surge capacity to the nearby Queen Elizabeth Hospital. The service was initially tasked with testing healthcare workers (HCW) using throat swabs, and subsequently the process investigated the utility of using saliva as an alternative sample type. Results Between the 8th April 2020 and the 30th April 2020, the laboratory tested a total of 1282 HCW for SARS-CoV-2 RNA in throat swabs. RNA was detected in 54 % of those who reported symptoms compatible with COVID-19, but in only 4% who were asymptomatic. Conclusion This capability was established rapidly and utilised a cold-chain free methodology, applicable to a wide range of settings, and which can provide surge capacity and support to clinical laboratories facing increasing pressure during periods of national crisis.

Purpose: KRAS mutation is a common canonical mutation in colorectal cancer, found at differing frequencies in all consensus molecular subtypes (CMS). The independent immunobiological impacts of RAS mutation and CMS are unknown. Thus, we explored the immunobiological effects of KRAS mutation across the CMS spectrum. Experimental Design: Expression analysis of immune genes/signatures was performed using The Cancer Genome Atlas (TCGA) RNA-seq and the KFSYSCC microarray datasets. Multivariate analysis included KRAS status, CMS, tumor location, MSI status, and neoantigen load. Protein expression of STAT1, HLA-class II, and CXCL10 was analyzed by digital IHC. Results: The Th1-centric co-ordinate immune response cluster (CIRC) was significantly, albeit modestly, reduced in KRAS-mutant colorectal cancer in both datasets. Cytotoxic T cells, neutrophils, and the IFNγ pathway were suppressed in KRAS-mutant samples. The expressions of STAT1 and CXCL10 were reduced at the mRNA and protein levels. In multivariate analysis, KRAS mutation, CMS2, and CMS3 were independently predictive of reduced CIRC expression. Immune response was heterogeneous across KRAS-mutant colorectal cancer: KRAS-mutant CMS2 samples have the lowest CIRC expression, reduced expression of the IFNγ pathway, STAT1 and CXCL10, and reduced infiltration of cytotoxic cells and neutrophils relative to CMS1 and CMS4 and to KRAS wild-type CMS2 samples in the TCGA. These trends held in the KFSYSCC dataset. Conclusions: KRAS mutation is associated with suppressed Th1/cytotoxic immunity in colorectal cancer, the extent of the effect being modulated by CMS subtype. These results add a novel immunobiological dimension to the biological heterogeneity of colorectal cancer. Clin Cancer Res; 24(1); 224–33. ©2017 AACR.

Background More than 70% of patients diagnosed with colorectal cancer with liver metastasis have unresectable disease, so the available treatment is systemic chemotherapy that has limited efficacy and causes harmful side-effects. Thus novel and more effective drugs are needed to improve patients’ survival and quality of life. Recent studies have identified several genomic alterations in CRLM that can theoretically be targeted by novel cancer-specific drugs. Among those is the gain-of-function mutations in PIK3CA – the gene encoding the p110a catalytic subunit of PI3K – that are present in 18% of metastatic colorectal tumours. Following the recent FDA-approval of Alpelisib (a PI3Ka-specific inhibitor) HR-positive breast cancer, there is increasing interest to test this drug efficacy to treat CRLM patients with PIK3CA mutations. Although widely used to test cancer cell response, immortalized cell lines fail to represent tumour genomic alterations and heterogeneity. Our goal is to develop patient-derived primary cell models that can be used to reliably test patients’ drug response. Methods Fresh colorectal cancer liver metastasis surgical specimens were dissociated and digested into single cells and seeded in matrigel with appropriate culture media. Originated organoid cultures were then submitted to a minimum of 5 passages followed by a freeze-thaw cycle before deemed established culture. To validate organoid tumour origin, we then performed whole-genome sequencing of paired organoid and respective fresh tumour. Results The established patient-derived organoids from colorectal liver metastasis add an overall mutation match of > 80% when compared to with the original tumour. We have found so far two CRLM organoid lines with mutations in the helical domain of PIK3CA (E545A and E542K). Conclusions We have demonstrated patient-derived organoids recapitulate the genome of their tumour of origin. Libraries of these primary cell models should in the near future be used to more accurately determine drug effectiveness prior to clinical trials. Ongoing testing of Alpelisib alone or in combination with other PI3K pathway target genes will determine whether these patients could benefit from these drugs. Legal entity responsible for the study Barros-Silva, J.D. Funding Cancer Research UK. Disclosure All authors have declared no conflicts of interest.

A recent association scan using a genome-wide set of nonsynonymous coding single-nucleotide polymorphisms (nsSNPs) conducted in four diseases including Graves' disease (GD), identified nine novel possible regions of association with GD. We used a case-control approach in an attempt to replicate association of these nine regions in an independent collection of 1578 British GD patients and 1946 matched Caucasian controls. Although none of these loci showed evidence of association with GD in the independent data set, when combined with the original Wellcome Trust Case-Control Consortium study group, minor differences in allele frequencies (P>or=10(-3)) remained in the combined collection of 5924 subjects for four of the nsSNPs, present within HDLBP, TEKT1, JSRP1 and UTX. An additional 29 Tag SNPs were screened within these four gene regions to determine if further associations could be detected. Similarly, minor differences only (P=0.042-0.002) were detected in two HDLBP and two TEKT1 Tag SNPs in the combined UK GD collection. In conclusion, it is unlikely that the SNPs selected in this replication study have a significant effect on the risk of GD in the United Kingdom. Our study confirms the need for large data sets and stringent analysis criteria when searching for susceptibility loci in common diseases.