Intra-promoter switch of transcription initiation sites in proliferation signaling-dependent RNA metabolism.

Global changes in transcriptional regulation and RNA metabolism are crucial features of cancer development. However, little is known about the role of the core promoter in defining transcript identity and post-transcriptional fates, a potentially crucial layer of transcriptional regulation in cancer. In this study, we use CAGE-seq analysis to uncover widespread use of dual-initiation promoters in which non-canonical, first-base-cytosine (C) transcription initiation occurs alongside first-base-purine initiation across 59 human cancers and healthy tissues. C-initiation is often followed by a 5' terminal oligopyrimidine (5'TOP) sequence, dramatically increasing the range of genes potentially subjected to 5'TOP-associated post-transcriptional regulation. We show selective, dynamic switching between purine and C-initiation site usage, indicating transcription initiation-level regulation in cancers. We additionally detail global metabolic changes in C-initiation transcripts that mark differentiation status, proliferative capacity, radiosensitivity, and response to irradiation and to PI3K-Akt-mTOR and DNA damage pathway-targeted radiosensitization therapies in colorectal cancer organoids and cancer cell lines and tissues.

MHC Class II is Induced by IFNγ and Follows Three Distinct Patterns of Expression in Colorectal Cancer Organoids.

Tumor-specific MHC class II (tsMHC-II) expression impacts tumor microenvironmental immunity. tsMHC-II positive cancer cells may act as surrogate antigen-presenting cells and targets for CD4+ T cell-mediated lysis. In colorectal cancer, tsMHC-II negativity is common, in cell lines due to CIITA promoter methylation. To clarify mechanisms of tsMHC-II repression in colorectal cancer, we analyzed colorectal cancer organoids which are epigenetically faithful to tissue of origin. 15 primary colorectal cancer organoids were treated with IFNγ ± epigenetic modifiers: flow cytometry was used for tsMHC-II expression. qRT-PCR, total RNA sequencing, nanopore sequencing, bisulfite conversion/pyrosequencing, and Western blotting was used to quantitate CIITA, STAT1, IRF1, and JAK1 expression, mutations and promoter methylation and chromatin immunoprecipitation to quantitate H3K9ac, H3K9Me2, and EZH2 occupancy at CIITA. We define three types of response to IFNγ in colorectal cancer: strong, weak, and noninducibility. Delayed and restricted expression even with prolonged IFNγ exposure was due to IFNγ-mediated EZH2 occupancy at CIITA. tsMHC-II expression was enhanced by EZH2 and histone deacetylase inhibition in the weakly inducible organoids. Noninducibility is seen in three consensus molecular subtype 1 (CMS1) organoids due to JAK1 mutation. No organoid demonstrates CIITA promoter methylation. Providing IFNγ signaling is intact, most colorectal cancer organoids are class II inducible. Upregulation of tsMHC-II through targeted epigenetic therapy is seen in one of fifteen organoids. Our approach can serve as a blueprint for investigating the heterogeneity of specific epigenetic mechanisms of immune suppression across individual patients in other cancers and how these might be targeted to inform the conduct of future trials of epigenetic therapies as immune adjuvants more strategically in cancer. Significance: Cancer cell expression of MHC class II significantly impacts tumor microenvironmental immunity. Previous studies investigating mechanisms of repression of IFNγ-inducible class II expression using cell lines demonstrate epigenetic silencing of IFN pathway genes as a frequent immune evasion strategy. Unlike cell lines, patient-derived organoids maintain epigenetic fidelity to tissue of origin. In the first such study, we analyze patterns, dynamics, and epigenetic control of IFNγ-induced class II expression in a series of colorectal cancer organoids.

The impact of the COVID-19 pandemic on elective laparoscopic cholecystectomy: A retrospective Cohort study.

The coronavirus disease 2019 (COVID-19) pandemic had a significant impact on elective surgery for benign disease. We examined the effects of COVID-19 related delays on the outcomes of patients undergoing elective laparoscopic cholecystectomy (LC) in an upper gastrointestinal surgery unit in the UK. We have analysed data retrospectively of patients undergoing elective LC between 01/03/2019 to 01/05/2019 and 01/04/2021 to 11/06/2021. Demographics, waiting time to surgery, intra-operative details and outcome data were compared between the two cohorts. Indications for surgery were grouped as inflammatory (acute cholecystitis, gallstone pancreatitis, CBD stone with cholangitis) or non-inflammatory (biliary colic, gallbladder polyps, CBD stone without cholangitis). A p value of <0.05 was used for statistical significance. Out of the 159 patients included, 106 were operated pre-pandemic and 53 during the pandemic recovery phase. Both groups had similar age, gender, ASA-grades and BMI. In the pre-pandemic group, 68 (64.2%) were operated for a non-inflammatory pathology compared to 19 (35.8%) from the recovery phase cohort (p < 0.001). The waiting time to surgery was significantly higher amongst patients operated during the recovery phase (p = 0000.1). Less patients had complete cholecystectomy during the pandemic recovery phase (p = 0.04). There were no differences in intraoperative times and patient outcomes. These results demonstrate the impact of COVID-19 related delays to our cohort, however due to the retrospective nature of this study, the current results need to be backed up by higher evidence in order for strong recommendations to be made.

Patient Derived Organoids Confirm That PI3K/AKT Signalling Is an Escape Pathway for Radioresistance and a Target for Therapy in Rectal Cancer.

Objectives: 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. We aimed to understand the mechanisms of resistance to chemoradiotherapy in rectal cancer using patient derived organoid models. Design: To understand the mechanisms underlying this resistance, we compared the pre-treatment transcriptomes of patient-derived organoids (PDO) with measured radiotherapy sensitivity to identify biological pathways involved in radiation resistance coupled with single cell sequencing, genome wide CRISPR-Cas9 and targeted drug screens. Results: RNA sequencing enrichment analysis revealed upregulation of PI3K/AKT/mTOR and epithelial mesenchymal transition pathway genes in radioresistant PDOs. Single-cell sequencing of pre & post-irradiation PDOs showed mTORC1 and PI3K/AKT upregulation, which was confirmed by a genome-wide CRSIPR-Cas9 knockout screen using irradiated colorectal cancer (CRC) cell lines. We then tested the efficiency of dual PI3K/mTOR inhibitors in improving cancer cell sensitivity to radiotherapy. After irradiation, significant AKT phosphorylation was detected (p=0.027) which was abrogated with dual PI3K/mTOR inhibitors and lead to significant radiosensitisation of the HCT116 cell line and radiation resistant PDO lines. Conclusions: The PI3K/AKT/mTOR pathway upregulation contributes to radioresistance and its targeted pharmacological inhibition leads to significant radiosensitisation in CRC organoids, making it a potential target for clinical trials.

The Effectiveness of a Foundation Year 1 Doctor Preparation Course for Final Year Medical Students.

BACKGROUND: Starting work as a junior doctor can be daunting for any medical student. There are numerous aspects of the hidden curriculum which many students fail to acquire during their training. OBJECTIVES: To evaluate the effectiveness of a novel foundation year one (FY1) doctor preparation course focusing on certain core topics, practical tips and components of the hidden curriculum. The primary objective was to improve the confidence level and knowledge of final year medical students transitioning to FY1 doctors. METHOD: A 2-day, practical course titled 'Preparation 2 Practice' delivering hands-on, small-group and lecture-based teaching, covering core medical student undergraduate curriculum topics in medicine and surgery. The course content spanned therapeutics, documentation skills and managing acute clinical tasks encountered by FY1 doctors during an on-call shift. A pre- and post-course survey and knowledge assessment were carried out to assess the effectiveness of the course. The assessment was MCQ-based, derived from topics covered within our course. The 20-question test and a short survey were administered electronically. RESULTS: Twenty students from a single UK medical school attended the course. 100% participation was observed in the pre- and post-course test and survey. The median post-course test result was 22 (IQR 20.25-23.75) which was higher than the median pre-course test score of 18.75 (IQR 17-21.75). A Wilcoxon sign rank test revealed a statistically significant difference between the pre- and post-course test results (P = .0003). The self-reported confidence score of delegates on starting work as a junior doctor was also significantly higher following the course (P = .004). CONCLUSION: The results show a significant improvement in perceived confidence and knowledge on core curriculum topics amongst final year medical students having attended our FY1 doctor preparation course. We conclude that there is scope for similar supplementary courses as an adjunct to the undergraduate medical curriculum.

Radiosensitising Cancer Using Phosphatidylinositol-3-Kinase (PI3K), Protein Kinase B (AKT) or Mammalian Target of Rapamycin (mTOR) Inhibitors.

Radiotherapy is routinely used as a neoadjuvant, adjuvant or palliative treatment in various cancers. There is significant variation in clinical response to radiotherapy with or without traditional chemotherapy. Patients with a good response to radiotherapy demonstrate better clinical outcomes universally across different cancers. The PI3K/AKT/mTOR pathway upregulation has been linked to radiotherapy resistance. We reviewed the current literature exploring the role of inhibiting targets along this pathway, in enhancing radiotherapy response. We identified several studies using in vitro cancer cell lines, in vivo tumour xenografts and a few Phase I/II clinical trials. Most of the current evidence in this area comes from glioblastoma multiforme, non-small cell lung cancer, head and neck cancer, colorectal cancer, and prostate cancer. The biological basis for radiosensitivity following pathway inhibition was through inhibited DNA double strand break repair, inhibited cell proliferation, enhanced apoptosis and autophagy as well as tumour microenvironment changes. Dual PI3K/mTOR inhibition consistently demonstrated radiosensitisation of all types of cancer cells. Single pathway component inhibitors and other inhibitor combinations yielded variable outcomes especially within early clinical trials. There is ample evidence from preclinical studies to suggest that direct pharmacological inhibition of the PI3K/AKT/mTOR pathway components can radiosensitise different types of cancer cells. We recommend that future in vitro and in vivo research in this field should focus on dual PI3K/mTOR inhibitors. Early clinical trials are needed to assess the feasibility and efficacy of these dual inhibitors in combination with radiotherapy in brain, lung, head and neck, breast, prostate and rectal cancer patients.

A review of retroperitoneal liposarcoma genomics.

Retroperitoneal liposarcomas are rare tumours that carry a poorer prognosis than their extremity counterparts. Within their subtypes - well differentiated (WDL), dedifferentiated (DDL), myxoid (MLS) and pleomorphic (PLS) - they exhibit a diverse genomic landscape. With recent advances in next generation sequencing, the number of studies exploring this have greatly increased. The recent literature has deepened our understanding of the hallmark MDM2/CDK4 amplification in WDL/DDL and addressed concerns about toxicity and resistance when targeting this. The FUS-DDIT3 fusion gene remains the primary focus of interest in MLS with additional potential targets described. Whole genome sequencing has driven identification of novel genes and pathways implicated in WDL/DDL outside of the classic 12q13-15 amplicon. Due to their rarity; anatomical location and histologic subtype are infrequently mentioned when reporting the results of these studies. Reports can include non-adipogenic or extremity tumours, making it difficult to draw specific retroperitoneal conclusions. This narrative review aims to provide a summary of retroperitoneal liposarcoma genomics and the implications for therapeutic targeting.

Malnutrition is associated with adverse postoperative outcome in patients undergoing elective colorectal cancer resections.

PURPOSE: Malnutrition results in a significant increase in postoperative morbidity and mortality after abdominal surgery. Apart from the anthropometric assessments, malnutrition can be also assessed using laboratory scores, with the most widely used being Onodera's Prognostic Nutritional Index (PNI). The purpose of our study was to assess if the presence of malnutrition as calculated by the Onodera's PNI was associated with higher postoperative morbidity after elective colorectal cancer resection. METHODS: We performed a retrospective analysis of our institutional database including the patients who underwent elective colorectal cancer resection over a 24-month period. PNI scores were calculated and correlated amongst other parameters, such as cancer stage, severity of postoperative complications, unplanned transfusion of blood products, need for unplanned level 2/3 care after surgery and overall length of hospitalization. RESULTS: A total of 213 patients were included in this analysis, with 22.5% being classified as malnourished based on the preoperative PNI. Of note, PNI values were inversely associated with advanced-stage disease, severity of postoperative complications and unplanned intensive care unit (ICU) admission postoperatively. Also, malnourished patients had a statistically significant prolonged length of in-hospital stay. No difference in PNI scores was identified between groups requiring unplanned blood products' transfusions. CONCLUSIONS: Preoperative malnutrition status as defined by PNI is associated with greater postoperative morbidity after elective surgery for colorectal cancer. Routine nutritional assessment and ad hoc nutritional support prior to surgery could contribute to an improvement of postoperative outcome after colorectal cancer resections.

Institutional variations in nutritional aspects of enhanced recovery pathways after elective surgery for colon cancer.

PURPOSE: Perioperative nutritional optimisation is one of the key aspects of the Enhanced Recovery after Surgery (ERAS) pathway after elective colorectal cancer resections. Despite the general acceptance of ERAS as a safe and cost-effective perioperative care bundle, significant variations in terms of nutritional support exist among colorectal units. METHODS: To assess these variations, we performed a cross-sectional online survey among colorectal surgical residents within a UK region. RESULTS: Our survey of practice demonstrated that despite the international recommendations, a considerable percentage of colorectal units would still advocate prolonged fasting prior to surgery without routine use of carbohydrate loading, as well as delaying resumption of oral intake postoperatively. In addition, in almost one in five colorectal units, the patients would not be assessed by the specialist nutrition team for potential support during their hospitalisation. CONCLUSION: Therefore, we believe that further education of the medical and allied health professional staff is required regarding the correct implementation of ERAS pathway guidelines after elective surgery for colorectal cancer.