Biology subjects

Hamdy, F. C.

Publications and source records attributed to Hamdy, F. C..

13 recordsLinked to original sources

bioRxiv2026

Novel Methylation Markers in a Prostate Cancer Cohort are Associated with Disease Development and Relapse

Prostate cancer remains one of the most common cancers among men globally. While significant strides have been made in diagnosis and treatment, understanding the complex genetic and epigenetic underpinnings of the disease remains crucial for guiding intervention and developing more personalized and effective therapies. The importance of DNA methylation in prostate cancer has been known for some time, but important facets of the modulation of the epigenome during carcinogenesis remain obscure, partly because the bulk of cancer methylation data have been produced using microarray technologies. Here we utilise the TruSeq methyl capture method (EPICseq) to profile the, previously defined, UK Prostate ICGC cohort of well-annotated primary prostate cancers. To this we add methylation sequencing of benign tissue from the same men. These data allow us to identify differentially methylated regions distinguishing cancerous and non-cancerous prostate tissue, while identifying numerous genes whose methylation profiles can perform that task as well as distinguishing between classes of prostate cancer. We describe a describe a methylation-based control mechanism for prostate-cancer-associated SNPs, and show that this seems a likely mechanism of action for a SNP near the MMP7 gene. We describe three novel molecular signatures that arise from different aspects of the biology of prostate cancer revealed by sequencing. Each is shown to be an independent classifier of cancers into groups with different expected times to relapse. These consist of patterns in driver gene methylation, strand-specific methylation, and signal arising in mitochondrial reads. We show that these signatures, combined with existing molecular tools, provide a powerful predictor of time to recurrence. By substantially enhancing understanding of prostate cancer risk, detection, and prognosis, we pave the way for the development of clinical practices that will benefit patients and improve outcomes.

cancer biology↗

bioRxiv2025

3D pathology-guided microdissection

Traditional micro- and macro-dissection techniques enable the extraction of localized regions in thin tissue sections for molecular analysis. Despite the growing use of 3D microscopy, analogous methods for volumetric microdissection are lacking. We have developed a 3D microdissection method based on computer numerical controlled (CNC) milling integrated with open-top light-sheet microscopy. We demonstrate the ability to study tumor evolution along convoluted 3D branching architectures, which is inaccessible to 2D methods.

bioengineering↗

bioRxiv2024

IGFs regulate cancer cell immune evasion in prostate cancer

Insulin-like growth factor-1 (IGF-1) promotes prostate cancer (PCa) development and lethality, with immunosuppressive properties in other disease models. These studies investigated the tumor-intrinsic immune effects of IGF-1 in PCa to understand mechanisms underlying its poor immunotherapy response. Transcriptional profiling of human (DU145, 22Rv1) and murine (Myc-CaP) PCa cells revealed, through pathway enrichment, that cytokine signalling, antigen processing and presentation, and other immune regulatory pathways, were most suppressed by IGF-1. We went on to investigate changes in the expression of components of two key pathways responsible for cancer cell recognition by immune cells and immune evasion: antigen processing and presentation, and PD-L1 checkpoint expression. These pathways are crucial for determining immunotherapy response. IGF-1 downregulated transporters associated with antigen processing (TAPs), endoplasmic reticulum aminopeptidase-1 (ERAP-1), and Class I component {beta}2-microglobulin, without major changes in Class I allele expression. These effects were associated with reduced presentation of Class I complexes on the Myc-CaP cell surface suggesting altered peptide transport, processing, and/or presentation. In contrast, IGF-1 upregulated immune checkpoint CD274 (PD-L1) via IGF receptor/AKT/ERK-dependent signaling. Analysis of public data (TCGA Firehose Legacy PCa) revealed increased CD274 expression in PCa with high endogenous IGF1 and IGFBP5, markers of high IGF axis activity. Additionally, in primary PCa (n=32), multiplex immunofluorescence revealed higher PD-L1 expression in the central tumor of men with high serum IGF-1 promoting cancer cell immune evasion in PCa. These findings indicate a novel mechanism by which IGF-1 mediates immunosuppressive effects in malignant prostate epithelium, potentially explaining the poor responsiveness of PCa to immunotherapy and highlighting the complex interplay between IGF signaling and immune evasion.

cancer biology↗

bioRxiv2023

Stress-induced Rab11a-exosomes induce AREG-mediated cetuximab resistance in colorectal cancer

Exosomes are secreted vesicles made intracellularly in the endosomal system. We have previously shown that exosomes are not only made in late endosomes, but also in recycling endosomes marked by the monomeric G-protein Rab11a. These vesicles, termed Rab11a-exosomes, are preferentially secreted under nutrient stress from several cancer cell types, including HCT116 colorectal cancer (CRC) cells. HCT116 Rab11a-exosomes have particularly potent signalling activities, some mediated by the Epidermal Growth Factor Receptor (EGFR) ligand, Amphiregulin (AREG). Mutant activating forms of KRAS, a downstream target of EGFR, are often found in advanced CRC. When absent, monoclonal antibodies, such as cetuximab, which target the EGFR and block the effects of EGFR ligands, such as AREG, can be administered. Patients, however, inevitably develop resistance to cetuximab, either by acquiring KRAS mutations or via non-genetic microenvironmental changes. Here we show that nutrient stress in several CRC cell lines causes the release of AREG-carrying Rab11a-exosomes. We demonstrate that while soluble AREG has no effect, much lower levels of AREG bound to Rab11a-exosomes from cetuximab-resistant KRAS-mutant HCT116 cells, can suppress the effects of cetuximab on KRAS-wild type Caco-2 CRC cells. Using neutralising anti-AREG antibodies and an intracellular EGFR kinase inhibitor, we show that this effect is mediated via AREG activation of EGFR, and not transfer of activated KRAS. Therefore, presentation of AREG on Rab11a-exosomes affects its ability to compete with cetuximab. We propose that this Rab11a-exosome-mediated mechanism contributes to the establishment of resistance in cetuximab-sensitive cells and may explain why in cetuximab-resistant tumours only some cells carry mutant KRAS. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=157 HEIGHT=200 SRC="FIGDIR/small/572341v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@18f47bborg.highwire.dtl.DTLVardef@1ae627corg.highwire.dtl.DTLVardef@1c3cb23org.highwire.dtl.DTLVardef@14da12e_HPS_FORMAT_FIGEXP M_FIG C_FIG This study highlights a clinically relevant mechanism in which stress-induced Rab11a-exosomes carrying the EGFR ligand, Amphiregulin (AREG) transfer drug resistance between genetically distinct colorectal cancer cells. Resistance to cetuximab, an anti-EGFR therapy, can be passed via Rab11a-exosomes from drug-resistant KRAS-mutant cells to previously drug-responsive KRAS-wild type cells. Unlike soluble AREG, Rab11a-exosome-associated AREG competes with cetuximab to activate EGFR signalling and promote EGFR-dependent outcomes, such as growth. This mechanism may support the co-operative evolution of clonal heterogeneity during tumour progression.

cancer biology↗

bioRxiv2023

Molecular analysis of archival diagnostic prostate cancer biopsies identifies genomic similarities in cases with progression post-radiotherapy, and those with de novo metastatic disease.

PurposeIt is important to identify molecular features that improve prostate cancer (PCa) risk stratification before radical treatment with curative intent. Molecular analysis of historical diagnostic formalin-fixed paraffin-embedded (FFPE) prostate biopsies from cohorts with post-radiotherapy (RT) long-term clinical follow-up has been limited. Utilizing parallel sequencing modalities, we performed a proof-of-principle sequencing analysis of historical diagnostic FFPE prostate biopsies. We compared patients with i) stable PCa post-primary or salvage RT (sPCa), ii) progressing PCa post-RT (pPCa), and iii) de novo metastatic PCa (mPCa). Experimental DesignA cohort of 19 patients with diagnostic prostate biopsies (n=6 sPCa, n=5 pPCa, n=8 mPCa) and mean 4 years 10 months follow-up (diagnosed 2009-2016) underwent nucleic acid extraction from demarcated malignancy. Samples underwent 3RNA sequencing (3RNAseq) (n=19), nanoString analysis (n=12) and Illumina 850k methylation (n=8) sequencing. Bioinformatic analysis was performed to coherently identify differentially expressed genes (DEGs) and methylated genomic regions (MGRs). Results18 of 19 samples provided useable 3RNAseq data. Principal Component Analysis (PCA) demonstrated similar expression profiles between pPCa and mPCa cases, versus sPCa. Coherently differentially methylated probes between these groups identified [~]600 differentially MGRs. The top 50 genes with increased expression in pPCa patients were associated with reduced progression-free survival post-RT (p<0.0001) in an external cohort. Conclusions3RNAseq, nanoString and 850K-methylation analyses are each achievable from historical FFPE diagnostic pre-treatment prostate biopsies, unlocking the potential to utilize large cohorts of historic clinical samples. Profiling similarities between individuals with pPCa and mPCa suggests biological similarities and historical radiological staging limitations, which warrant further investigation.

genomics↗

bioRxiv2023

Spatial transcriptomic analysis of virtual prostate biopsy reveals confounding effect of heterogeneity on genomic signature scoring

Genetic signatures have added a molecular dimension to prognostics and therapeutic decision-making. However, tumour heterogeneity in prostate cancer and current sampling methods could confound accurate assessment. Based on previously published spatial transcriptomic data from multifocal prostate cancer, we created virtual biopsy models that mimic conventional biopsy placement and core size. We then analysed the gene expression of different prognostic signatures (OncotypeDx(R), Decipher(R), Prostadiag(R)) using a step-wise approach increasing resolution from pseudo-bulk analysis of the whole biopsy, to differentiation by tissue subtype (benign, stroma, tumour), followed by distinct tumour grade and finally clonal resolution. The gene expression profile of virtual tumour biopsies revealed clear differences between grade groups and tumour clones, compared to a benign control, which were not reflected in bulk analyses. This suggests that bulk analyses of whole biopsies or tumour-only areas, as used in clinical practice, may provide an inaccurate assessment of gene profiles. The type of tissue, the grade of the tumour and the clonal composition all influence the gene expression in a biopsy. Clinical decision making based on biopsy genomics should be made with caution while we await more precise targeting and cost-effective spatial analyses. Patient summaryProstate cancers are very variable, including within a single tumour. Current genetic scoring systems, which are sometimes used to make decisions for how to treat patients with prostate cancer, are based on sampling methods which do not reflect these variations. We found, using state-of-the-art spatial genetic technology to simulate accurate assessment of variation in biopsies, that the current approaches miss important details which could negatively impact clinical decisions. Take home messageVirtual biopsies from spatial transcriptomic analysis of a whole prostate reveal that current genomic risk scores potentially deliver misleading results as they are based on bulk analysis of prostate biopsies and ignore tumour heterogeneity.

genomics↗

bioRxiv2023

Clonal phylogenies inferred from bulk, single cell, and spatial transcriptomic analysis of cancer

Epithelial cancers are typically heterogeneous with primary prostate cancer being a typical example of histological and genomic variation. Prostate cancer is the second most common male cancer in western industrialized countries. Prior studies of primary prostate cancer tumor genetics revealed extensive inter and intra-patient tumor heterogeneity. Recent advances have enabled extensive single-cell and spatial transcriptomic profiling of tissue specimens. The ability to resolve accurate prostate cancer tumor phylogenies at high spatial resolution would provide tools to address questions in tumorigenesis, disease progression, and metastasis. Recent advances in machine learning have enabled the inference of ground-truth genomic single-nucleotide and copy number variant status from transcript data. The inferred SNV and CNV states can be used to resolve clonal phylogenies, however, it is still unknown how faithfully transcript-based tumor phylogenies reconstruct ground truth DNA-based tumor phylogenies. We sought to study the accuracy of inferred-transcript to recapitulate DNA-based tumor phylogenies. We first performed in-silico comparisons of inferred and directly resolved SNV and CNV status, from single cancer cells, from three different cell lines. We found that inferred SNV phylogenies accurately recapitulate DNA phylogenies (entanglement = 0.097). We observed similar results in iCNV and CNV based phylogenies (entanglement = 0.11). Analysis of published prostate cancer DNA phylogenies and inferred CNV, SNV and transcript based phylogenies demonstrated phylogenetic concordance. Finally, a comparison of pseudo-bulked spatial transcriptomic data to adjacent sections with WGS data also demonstrated recapitulation of ground truth (entanglement = 0.35). These results suggest that transcript-based inferred phylogenies recapitulate conventional genomic phylogenies. Further work will need to be done to increase accuracy, genomic, and spatial resolution.

bioinformatics↗

bioRxiv2023

Intra-prostatic tumour evolution, steps in metastatic spread and histogenomic associations revealed by integration of multi-region whole genome sequencing with histopathological features

Extension of prostate cancer beyond the primary site into the surrounding organs by local invasion or nodal metastasis is associated with poor prognosis. The emergence and evolution of cancer clones at this early stage of expansion and spread has not been studied in detail. We performed whole genome sequencing on 42 prostate cancer samples from the prostate, seminal vesicles and regional lymph nodes of five treatment-naive patients with locally advanced disease who underwent radical prostatectomy. Using cancer cell fractions computed from single nucleotide variants and copy number alterations, we reconstructed the tumour phylogenies, which in turn allowed us to infer key molecular steps in the progression of prostate cancer in these individuals. We mapped the clonal composition of cancer sampled across the prostate in each individual and inferred the routes of spread of cancer cells within the prostate and to seminal vesicles and lymph nodes. Based on these data, we delineated the route of tumour progression and metastasis following the transformation of adenocarcinoma to amphicrine morphology, the molecular events leading to whole genome duplication associated with a single clonal expansion and identified putative driver events associated with local invasion and lymph node metastasis. We also correlated genomic changes associated with differences in morphology and identified putative driver events associated with spread to seminal vesicle invasion and lymph node metastasis. Taken together, these findings have implications for diagnosis and risk stratification, in addition to providing a rationale for further studies to characterise the genetic changes associated with morphological transformation. Our results demonstrate the value of integrating multi-region sequencing with histopathological data to study tumour evolution and identify mechanisms of prostate cancer spread.

cancer biology↗

bioRxiv2021

The spatial landscape of clonal somatic mutations in benign and malignant tissue

Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. Here, we provide an unsupervised approach to study spatial genome integrity in situ to gain molecular insight into clonal relationships. We employed spatially resolved transcriptomics to infer spatial copy number variations in >120 000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of an unsupervised approach to capture the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.

genomics↗

bioRxiv2021

Rbf/E2F1 control growth and endoreplication via steroid-independent Ecdysone Receptor signalling in Drosophila prostate-like secondary cells

Dysregulation of cell cycle components results in the development and progression of several cancer types. Unusually, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 have been linked to late-stage tumour progression in prostate cancer, rather than early-stage events. This change is associated with an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. We have previously shown that binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG) share several functional and signalling similarities with human prostate epithelial cells. Upon mating, SC growth regulation switches from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control that induces genome endoreplication. Here, we demonstrate that the Drosophila Rb homologue, Rbf, and E2F1, as well as cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key mediators of SC growth and endoreplication both in virgin and mated males. Importantly, we show that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and associated growth in SCs after mating, mirroring changes in CRPC. We also demonstrate that excess Rbf activity reversibly suppresses binucleation in adult SCs. Overall, our work reveals mechanistic parallels between the physiological switch to hormone-independent EcR signalling in SCs, and the pathological switch seen in CRPC, and suggests that the latter may represent the dysregulation of a currently unidentified physiological process, which permits AR signalling when androgen levels are low.

cell biology↗

bioRxiv2020

Tumor irradiation combined with vascular-targeted photodynamic therapy enhances anti-tumor effects in preclinical prostate cancer.

RationaleThere is an important clinical need to improve the treatment of high risk localized and locally advanced prostate cancer (PCa), and to reduce the side effects of these treatments. We hypothesized that multi-modality therapy combining radiotherapy and vascular-targeted photodynamic therapy (VTP) could PCa tumour control compared against monotherapy with each of these treatments alone. This could provide proof-of-concept to take to the clinic. VTP is a focal therapy for localized PCa, which rapidly destroys targeted tumors through vascular disruption. Tumor vasculature is characterized by vessel immaturity, increased permeability, aberrant branching and inefficient flow. Fractionated radiotherapy (FRT) alters the tumor microenvironment and promotes transient vascular normalization. ObjectiveWe investigated whether sequential delivery of FRT followed by VTP 7 days later improves PCa tumor control compared to monotherapy with FRT or VTP alone. FindingsFRT induced vascular normalization changes in PCa flank tumor allografts, improving vascular function as demonstrated using dynamic contrast enhanced magnetic resonance imaging. FRT followed by VTP significantly delayed tumor growth in flank PCa allograft pre-clinical models, compared with monotherapy with FRT or VTP alone, and improved overall survival. ConclusionTaken together, these results suggest that combining FRT and VTP could become a promising multimodal clinical strategy in PCa therapy. This provides proof-of-concept for this multi-modality therapy approach to take forward to early phase clinical trials.

cancer biology↗

bioRxiv2019

A genetic hazard score to personalize prostate cancer screening, applied to population data

BackgroundGenetic risk stratification may inform decisions of whether--and when--a man should undergo prostate cancer (PCa) screening. We previously validated a polygenic hazard score (PHS), a weighted sum of 54 single-nucleotide polymorphism genotypes, for accurate prediction of age of onset of aggressive PCa and improved screening performance. We now assess the potential impact of PHS-informed screening.\n\nMethodsUnited Kingdom population data were fit to a continuous model of age-specific PCa incidence. Using hazard ratios estimated from ProtecT trial data, age-specific incidence rates were calculated for percentiles of genetic risk. Incidence of higher-grade PCa (Gleason[≥]7) was estimated from age-specific data from the linked CAP trial. PHS and incidence data were combined to give a risk-equivalent age, when a man with a given PHS percentile will have risk of higher-grade PCa equivalent to that of a typical man at age 50 (50-years standard). Positive predictive value (PPV) of PSA testing was calculated using PHS-adjusted (PCa-risk-equivalent age) groups identified from ProtecT.\n\nResultsExpected age of onset of higher-grade PCa is modulated by 19 years between the 1st and 99th PHS percentiles. A man with PHS in the 99th percentile reaches 50-years-standard risk at age 41; conversely, a man in the 1st percentile reaches this risk at age 60. PPV of PSA was higher for men with higher PHS-adjusted age.\n\nConclusionsPHS informs PCa screening strategies with individualized estimates of risk-equivalent age for higher-grade PCa. Screening initiation could be adjusted according to a mans genetic hazard score, improving PPV of PSA screening.

genetics↗

bioRxiv2016

A genetic risk score to guide age-specific, personalized prostate cancer screening

BackgroundProstate-specific-antigen (PSA) screening resulted in reduced prostate cancer (PCa) mortality in a large clinical trial, but due to a high false-positive rate, among other concerns, many guidelines do not endorse universal screening and instead recommend an individualized decision based on each patients risk. Genetic risk may provide key information to guide the decisions of whether and at what age to screen an individual man for PCa.\n\nMethodsGenotype, PCa status, and age from 34,444 men of European ancestry from the PRACTICAL consortium database were analyzed to select single-nucleotide polymorphisms (SNPs) associated with prostate cancer diagnosis. These SNPs were then incorporated into a survival analysis to estimate their effects on age at PCa diagnosis. The resulting polygenic hazard score (PHS) is an assessment of individual genetic risk. The final model was validated in an independent dataset comprised of 6,417 men with screening PSA and genotype data. PHS was calculated for these men to test for prediction of PCa-free survival. PHS was also combined with age-specific PCa incidence data from the U.S. population to generate a PCa-Risk (PCaR) age that relates a given mans risk to that of the population average. PHS and PCaR age were evaluated for prediction of positive predictive value (PPV) of PSA screening.\n\nFindingsPHS calculated from 54 SNPs was very highly predictive of age at PCa diagnosis for men in the validation set (p =10-53). PPV of PSA screening varied from 0.18 to 0.52 for men with low and high genetic risk, respectively. PHS modulates PCa-free survival curves by an estimated 20 years between men in the 1st or 99th percentiles of genetic risk.\n\nInterpretationPolygenic hazard scores give personalized genetic risk estimates and can inform the decisions of whether and at what age to screen a man for PCa.\n\nFundingDepartment of Defense #W81XWH-13-1-0391

genetics↗