Circulating tumour DNA to direct the management of patients with gastrointestinal malignancies

Background Management of patients with gastrointestinal (GI) malignancies requires a personalised approach to improve survival while minimising risk. Colorectal and oesophagogastric (OG) malignancies account for 20% of all cancer deaths, with significant global socioeconomic impact. Novel technologies are required to customise treatment. Circulating tumour DNA (ctDNA) is emerging as a prognostic biomarker and predictive biomarker of treatment response and can be used to guide management. Hypotheses ctDNA can be used to predict recurrence and guide adjuvant chemotherapy (ACT) treatment decisions in patients with resected early-stage colorectal cancer (CRC) and evaluate response to therapies in metastatic CRC and OGA. Aims and objectives 1. Determine the association between detectable ctDNA and recurrence free survival (RFS) in patients with resected CRC using a tissue-free approach to ctDNA detection. 2. Assess the feasibility of using a tissue-free (versus tumour-informed) ctDNA assay to detect minimal residual disease (MRD) and guide a de-escalation strategy of ACT in patients with curatively resected CRC who are ctDNA negative post-operatively. 3. Determine the use of serial ctDNA monitoring and immune profiling to detect biomarkers of response and resistance to immunotherapy combinations in metastatic GI cancers. Methodology 1. Clinical validity of ctDNA to detect MRD based on a tissue-free ctDNA assay from TRACC Part B UK-based multi-centre observational translational study data of curatively treated CRC patients. 2. Clinical utility of ctDNA to guide adjuvant management of CRC by means of development of the TRACC Part C interventional randomised MRD study, involving additional clinical validation and process implementation and evaluation. 3. ctDNA and immune profiling in immunotherapy response monitoring in an academic phase II study of anti-PD1 and an HDAC inhibitor (EMERGE study) in patients with metastatic OG and CRC Significance ctDNA has potential for wide ranging applications in early and metastatic GI malignancies to directly impact patient care

Elucidating Prostate Cancer Biology Through Integrative Computational Multi-omic Analyses

in this thesis, I aimed to elucidate the microbial landscape of castration-resistant prostate cancer (CRPC), focusing on novel biomarkers of therapeutic resistance. We employed metagenomic sequencing of over 200 CRPC patients, alongside in vitro cell Line and ex vivo patient-derived xenograft (POX), models, to investigate microbial biomarkers of progression from hormone-sensitive prostate cancer {HSPC) to CRPC. Metagenomic sequencing effectively characterised the gut microbiome ofCRPC patients. While microbiome alpha diversity between HSPC and CRPC I remained similar, species-level changes, including an increased abundance of Adlercreutzia equolifaciens, were observed in CRPC. Further, L- 1 citrulline biosynthesis by the microbiota was linked to improved AR -negative prostate cancer cell viability, highlighting its potential role in CRPC i progression. Analysis ofCRPC biopsy samples provided inconclusive evidence for intra-tumour micro biota presence, with contamination confounding microbial classifications. Single-nucleus sequencing revealed significant myeloid cell heterogeneity in CRPC, with varying degrees of oxidative stress-induced DNA damage I linked to myeloid-derived reactive oxygen species {ROS). This heterogeneity extended to tumour subclones, with genomic instability and replication I stress driving therapeutic sensitivity to ATR inhibition. Notably, the overexpression of the prognostic marker POLQ was associated with replication [ stress and ATR inhibition sensitivity, highlighting a potential therapeutic target for CRPC. These findings provide insights into the complex interplay: between the microbiome, inflammation, and cellular mechanisms driving CRPC progression and resistance and merit further functional validations

Mechanisms of replication stress: the role of RAD52 in replication and a new vulnerability in mismatch repair deficient cancers

Faithful duplication of chromosomes is critical for maintaining survival and preventing genomic instability. However, both endogenous and exogenous factors can disrupt the DNA synthesis process, leading to replication stress (RS). In turn, RS leads to the accumulation of genetic modifications causing genomic instability, a hallmark of tumorigenesis. Understanding the causes and consequences of replication stress is critical, not only for furthering the knowledge of the mechanistic landscape of tumorigenesis but also for the development of better anti-cancer strategies targeting cancer vulnerabilities. The first part of the thesis explores the consequences of RAD52 knockout in non-cancerous (RPE1) and cancerous cells (HeLa and HCT116) under conditions of low-dose aphidicolin (APH) treatment and translesion synthesis inhibition (TLSi). While TLSi alone reduces global DNA synthesis in cancer cells, it has minimal impact on untransformed cells. Notably, combined TLSi and APH treatment exacerbates the DNA synthesis defect in all cell lines, highlighting a requirement for TLS in mitigating RS. Interestingly, RAD52 loss is synergistic with these effects only in HCT116 cells. Treatment with APH leads to underreplicated regions that complete DNA synthesis in G2. Further, analysis of these G2 DNA synthesis events reveals that TLS plays a critical role in maintaining replication at these regions. The data also shows RAD52 to be necessary in HCT116, but dispensable in RPE1 cells. Investigation using chemical inhibitors and siRNA-mediated depletion of RAD52 indicates that the phenotypes observed in HCT116 are likely due to compensatory mechanisms rather than direct functional loss of RAD52. Additionally, a potential synthetic lethal interaction between MLH1 loss and TLSi was identified, warranting further exploration. The biological complexities in studying the functional role of RAD52 across various cell types and conditions led to a shift in focus towards exploring novel synthetic lethality relationship between mismatch repair (MMR) deficiency and TLSi in the second part of the thesis. This lethality was confirmed across multiple untransformed cell lines (RPE1, RPE1P53KO, 1BR3, MCF10A). Notably, MMR-deficient colon cancer cell lines exhibit increased sensitivity to TLSi compared to MMR-proficient colorectal cancers. A proof-of-concept analysis of the TCGA cohort further demonstrates that this interaction could be exploited in gastrointestinal and endometrial cancers. At a molecular level, it is demonstrated that combined loss of MMR and TLS leads to ssDNA accumulation, increased p21 levels, heterochromatinization and cell cycle delays. Furthermore, new insights reveal that loss of MMR genes alone modulates cellular homeostasis, significantly impacting chromatin compaction, replication speed, and cell cycle progression

Proteogenomic discovery of RB1-defective phenocopy in cancer predicts disease outcome, response to treatment, and therapeutic targets.

Genomic defects caused by truncating mutations or deletions in the Retinoblastoma tumor suppressor gene (RB1) are frequently observed in many cancer types leading to dysregulation of the RB pathway. Here, we propose an integrative proteogenomic approach that predicts cancers with dysregulation in the RB pathway. A subset of these cancers, which we term as "RBness," lack RB1 genomic defects and yet phenocopy the transcriptional profile of RB1-defective cancers. We report RBness as a pan-cancer phenomenon, associated with patient outcome and chemotherapy response in multiple cancer types, and predictive of CDK4/6 inhibitor response in estrogen-positive breast cancer. Using RNA interference and a CRISPR-Cas9 screen in isogenic models, we find that RBness cancers also phenocopy synthetic lethal vulnerabilities of cells with RB1 genomic defects. In summary, our findings suggest that dysregulation of the RB pathway in cancers lacking RB1 genomic defects provides a molecular rationale for how these cancers could be treated

Fitness and transcriptional plasticity of human breast cancer single-cell-derived clones.

Clonal fitness and plasticity drive cancer heterogeneity. We used expressed lentiviral-based cellular barcodes combined with single-cell RNA sequencing to associate single-cell profiles with in vivo clonal growth. This generated a significant resource of growth measurements from over 20,000 single-cell-derived clones in 110 xenografts from 26 patient-derived breast cancer xenograft models. 167,375 single-cell RNA profiles were obtained from 5 models and revealed that rare propagating clones display a highly conserved model-specific differentiation program with reproducible regeneration of the entire transcriptomic landscape of the original xenograft. In 2 models of basal breast cancer, propagating clones demonstrated remarkable transcriptional plasticity at single-cell resolution. Dichotomous cell populations with different clonal growth properties, signaling pathways, and metabolic programs were characterized. By directly linking clonal growth with single-cell transcriptomes, these findings provide a profound understanding of clonal fitness and plasticity with implications for cancer biology and therapy

Radiation-induced extracellular matrix remodelling drives prognosis and predicts radiotherapy response in muscle-invasive bladder cancer.

Muscle-invasive bladder cancer (MIBC) is a prevalent disease that can be treated with radiotherapy, but has a poor prognosis. Radiation-induced extracellular matrix (ECM) remodelling and fibrosis can induce tumour resistance and recurrence, but have not been studied in MIBC. Here, we aimed to characterise the impact of radiation on the ECM composition of MIBC. Three MIBC cell lines (T24, UMUC3, J82) were treated with fractionated radiation. We used proteomics to analyse the ECM composition produced by surviving cancer cells and immunofluorescence to investigate changes in the morphology and number of ECM fibres. We evaluated the RNA expression of identified ECM proteins (FN1, COL5A1, COL1A1, TNF6AIP6, FLG) in one cystectomy (TCGA-BLCA, n=397) and two radiotherapy (BC2001, n=313; BCON, n=151) cohorts. There were 613 proteins affected by radiation (padj2 or <-2), 68 of which were ECM-associated proteins. There was a general increase in proteases and protease regulators but heterogeneity across cell lines. Enrichment analysis showed ECM organisation was the primary pathway affected. Immunofluorescence confirmed radiation affected ECM structure, generally, reducing the number, length and width of fibres. Five ECM genes of interest were identified (COL1A1, COL5A2, FN1, FLG, TNFAIP6), constituting an ECM signature. High FN1, COL1A1, TNF6AIP6 mRNA levels and ECM signature scores were independent poor prognostic markers, while FLG mRNA expression independently predicted radiotherapy benefit in a meta-analysis (n=861). We found high COL1A1 expression levels predicted hypoxia-modifying treatment benefit. Prognostic significance of COL5A2, FN1 and the ECM signature was dependent on patients harbouring TP53-mutations. Radiation alters the composition and structure of the ECM produced by MIBC. As a proof-of-concept, we showed that radiation-affected ECM genes are independent prognostic and predictive markers of radiotherapy benefit in MIBC. Future studies should validate these radiation-induced ECM changes in clinical samples, and explore the role of FLG in radioresistance

Alcohol intake and pancreatic cancer risk: An analysis from 30 prospective studies across Asia, Australia, Europe, and North America.

BACKGROUND: Alcohol is a known carcinogen, yet the evidence for an association with pancreatic cancer risk is considered as limited or inconclusive by international expert panels. We examined the association between alcohol intake and pancreatic cancer risk in a large consortium of prospective studies. METHODS AND FINDINGS: Population-based individual-level data was pooled from 30 cohorts across four continents, including Asia, Australia, Europe, and North America. A total of 2,494,432 participants without cancer at baseline (62% women, 84% European ancestries, 70% alcohol drinkers [alcohol intake ≥ 0.1 g/day], 47% never smokers) were recruited between 1980 and 2013 at the median age of 57 years and 10,067 incident pancreatic cancer cases were recorded. In age- and sex-stratified Cox proportional hazards models adjusted for smoking history, diabetes status, body mass index, height, education, race and ethnicity, and physical activity, pancreatic cancer hazard ratios (HR) and 95% confidence intervals (CI) were estimated for categories of alcohol intake and in continuous for a 10 g/day increase. Potential heterogeneity by sex, smoking status, geographic regions, and type of alcoholic beverage was investigated. Alcohol intake was positively associated with pancreatic cancer risk, with HR30-to-<60 g/day and HR≥60 g/day equal to 1.12 (95% CI [1.03,1.21]) and 1.32 (95% CI [1.18,1.47]), respectively, compared to intake of 0.1 to <5 g/day. A 10 g/day increment of alcohol intake was associated with a 3% increased pancreatic cancer risk overall (HR: 1.03; 95% CI [1.02,1.04]; pvalue < 0.001) and among never smokers (HR: 1.03; 95% CI [1.01,1.06]; pvalue = 0.006), with no evidence of heterogeneity by sex (pheterogeneity = 0.274) or smoking status (pheterogeneity = 0.624). Associations were consistent in Europe-Australia (HR10 g/day = 1.03, 95% CI [1.00,1.05]; pvalue = 0.042) and North America (HR10 g/day = 1.03, 95% CI [1.02,1.05]; pvalue < 0.001), while no association was observed in cohorts from Asia (HR10 g/day = 1.00, 95% CI [0.96,1.03]; pvalue = 0.800; pheterogeneity = 0.003). Positive associations with pancreatic cancer risk were found for alcohol intake from beer (HR10 g/day = 1.02, 95% CI [1.00,1.04]; pvalue = 0.015) and spirits/liquor (HR10 g/day = 1.04, 95% CI [1.03,1.06]; pvalue < 0.001), but not wine (HR10 g/day = 1.00, 95% CI [0.98,1.03]; pvalue = 0.827). The differential associations across geographic regions and types of alcoholic beverages might reflect differences in drinking habits and deserve more investigations. CONCLUSIONS: Findings from this large-scale pooled analysis support a modest positive association between alcohol intake and pancreatic cancer risk, irrespective of sex and smoking status. Associations were particularly evident for baseline alcohol intake of at least 15 g/day in women and 30 g/day in men

Pembrolizumab plus Abiraterone Acetate and Prednisone in Patients with Chemotherapy-naïve Metastatic Castration-resistant Prostate Cancer: Results from KEYNOTE-365 Cohort D.

BACKGROUND AND OBJECTIVE: Abiraterone acetate (abiraterone) plus prednisone is approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Our aim was to evaluate the efficacy and safety of pembrolizumab plus abiraterone in mCRPC. METHODS: In cohort D of the phase 1b/2 KEYNOTE-365 study (NCT02861573), patients were chemotherapy-naïve, had disease progression ≤6 mo before screening, and had either not received prior next-generation hormonal agents for mCRPC or had received prior enzalutamide for mCRPC and had disease progression or became intolerant to enzalutamide. Patients received pembrolizumab 200 mg intravenously every 3 wk plus abiraterone 1000 mg orally once daily and prednisone 5 mg orally twice daily. The primary endpoints were safety, prostate-specific antigen (PSA) response rate, and objective response rate (ORR) according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) by blinded independent central review (BICR). Secondary endpoints included radiographic progression-free survival (rPFS) according to Prostate Cancer Clinical Trials Working Group 3-modified RECIST v1.1 by BICR and overall survival (OS). KEY FINDINGS AND LIMITATIONS: For the 103 patients who were treated, median follow-up was 28 mo (interquartile range 26-31). The confirmed PSA response rate was 56% (58/103 patients). The ORR for patients with RECIST v1.1-measurable disease was 16% (6/37 patients). Median rPFS was 15 mo (95% confidence interval 9.2-22) and median OS was 30 mo (95% confidence interval 23-not reached); the estimated 24-mo OS rate was 58%. In total, 91% of patients experienced treatment-related adverse events, and 39% experienced grade 3-5 events. Grade 3/4 elevation of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) was observed in 12% and 6.8% of patients, respectively. One patient died due to treatment-related myasthenic syndrome. Study limitations include the single-arm design. CONCLUSIONS: Pembrolizumab plus abiraterone and prednisone demonstrated antitumor activity and acceptable safety in patients with chemotherapy-naïve mCRPC. Higher incidence of grade 3/4 elevated ALT/AST occurred than was reported for the individual agents. PATIENT SUMMARY: For patients with metastatic castratation-resistant prostate cancer, the drug combination of pembrolizumab plus abiraterone and prednisone showed antitumor activity and acceptable safety

Sub-3 Å resolution protein structure determination by single-particle cryo-EM at 100 keV.

Cryoelectron microscopy (cryo-EM) has transformed structural biology by providing high-resolution insights into biological macromolecules. We report sub-3 Å resolution structures using the 100 keV Tundra cryo-TEM, equipped with the Falcon C direct electron detector (DED). This system combines advanced optics, extreme-brightness field emission gun (XFEG), and SP-TWIN lens to enhance coherence and resolution. The semi-automated loader reduced contamination and drift, enabling extended data collection, while the high detective quantum efficiency (DQE) of Falcon C improved signal-to-noise ratio. We validated performance by determining structures of biological samples, including apoferritin (2.1 Å), T20S proteasome (2.7 Å), GABAA receptor (2.8 Å), hemoglobin (5.0 Å), transthyretin (3.5 Å), and AAV9 capsid (2.8 Å), spanning 50 kDa-3.9 MDa. This work highlights the potential of 100 keV transmission electron microscopes (TEMs) to make cryo-EM more accessible. It sets a precedent for using lower voltage TEMs not only for screening, but also for high-resolution protein structure determination