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Reconstructing cancer evolution at the single-cell level in patient-derived organoids
No full textGenetic mutations in cancer driver genes are heritable and can confer aggressive and therapy-resistant properties to cells. However genetic driver events do not fully explain the malignant phenotype, and it is becoming increasingly clear that epigenetic drivers can also confer these qualities. Furthermore, phenotypic switching and plasticity can also enable malignant cells to adapt to a changing environment. The degree to which these properties are inherited and related to genetic state is unclear. Smart-RRBS is a dual-omic single-cell technique capable of profiling methylation of cytosines and the transcriptome from the same cell. Methylation of cytosines can be used to both define the epigenetic state and to assemble the genealogy of somatic cells through phylogenetic inference. Smart-RRBS, therefore, allows phylogenetic reconstruction at the single-cell level, enabling resolution of cell-to-cell ancestral relations, coupled with a read out of their epigenetic and phenotypic state. Here I optimise and validate Smart-RRBS, then assess its performance in our hands compared to published data. Following establishment of this methodology in our lab, I apply Smart-RRBS to a patient-derived organoid model system of colorectal cancer, comparing a treatment naïve line to the same line following exposure to a MEK inhibitor. Leveraging an expressible lineage tracing barcoding system to validate the phylogenetic tree, I find the tree accurately captures phylogenetic architecture. I explore transcriptional and epigenetic phenotypic differences in treated and untreated cells and investigate to what degree these are present prior to drug treatment. I identify concordant copy number alterations from both the methylation data and the transcription data, defining a clone with a unique copy number profile that emerges after selection by treatment. I also apply a new single-cell whole genome amplification assay to treatment-naive and Akt-inhibitor treated colorectal patient derived organoids and construct a phylogeny using single-cell SNVs
Developments in predictive biomarker testing and targeted therapy in advanced stage non-small cell lung cancer and their application across European countries.
No full textIn the past two decades, the treatment of metastatic non-small cell lung cancer (NSCLC), has undergone significant changes due to the introduction of targeted therapies and immunotherapy. These advancements have led to the need for predictive molecular tests to identify patients eligible for targeted therapy. This review provides an overview of the development and current application of targeted therapies and predictive biomarker testing in European patients with advanced stage NSCLC. Using data from eleven European countries, we conclude that recommendations for predictive testing are incorporated in national guidelines across Europe, although there are differences in their comprehensiveness. Moreover, the availability of recently EMA-approved targeted therapies varies between European countries. Unfortunately, routine assessment of national/regional molecular testing rates is limited. As a result, it remains uncertain which proportion of patients with metastatic NSCLC in Europe receive adequate predictive biomarker testing. Lastly, Molecular Tumor Boards (MTBs) for discussion of molecular test results are widely implemented, but national guidelines for their composition and functioning are lacking. The establishment of MTB guidelines can provide a framework for interpreting rare or complex mutations, facilitating appropriate treatment decision-making, and ensuring quality control
Nuclear rupture induced by capillary constriction forces promotes differential effects on metastatic and normal breast cells.
Get PDFDuring metastatic dissemination, circulating tumour cells (CTCs) enter capillary beds, where they experience mechanical constriction forces. The transient and persistent effects of these forces on CTCs behaviour remain poorly understood. Here, we developed a high-throughput microfluidic platform mimicking human capillaries to investigate the impact of mechanical constriction forces on malignant and normal breast cell lines. We observed that capillary constrictions induced nuclear envelope rupture in both cancer and normal cells, leading to transient changes in nuclear and cytoplasmic area. Constriction forces transiently activated cGAS/STING and pathways involved in inflammation (NF-κB, STAT and IRF3), especially in the non-malignant cell line. Furthermore, the non-malignant cell line experienced transcriptional changes, particularly downregulation of epithelial markers, while the metastatic cell lines showed minimal alterations. These findings suggest that mechanical constriction forces within capillaries may promote differential effects in malignant and normal cell lines
27-hydroxycholesterol and DNA damage repair: implication in prostate cancer.
No full textINTRODUCTION: We previously reported that cholesterol homeostasis in prostate cancer (PC) is regulated by 27-hydroxycholesterol (27HC) and that CYP27A1, the enzyme that converts cholesterol to 27HC, is frequently lost in PCs. We observed that restoring the CYP27A1/27HC axis inhibited PC growth. In this study, we investigated the mechanism of 27HC-mediated anti-PC effects. METHODS: We employed in vitro models and human transcriptomics data to investigate 27HC mechanism of action in PC. LNCaP (AR+) and DU145 (AR-) cells were treated with 27HC or vehicle. Transcriptome profiling was performed using the Affymetrix GeneChip™ microarray system. Differential expression was determined, and gene set enrichment analysis was done using the GSEA software with hallmark gene sets from MSigDB. Key changes were validated at mRNA and protein levels. Human PC transcriptomes from six datasets were analyzed to determine the correlation between CYP27A1 and DNA repair gene expression signatures. DNA damage was assessed via comet assays. RESULTS: Transcriptome analysis revealed 27HC treatment downregulated Hallmark pathways related to DNA damage repair, decreased expression of FEN1 and RAD51, and induced "BRCAness" by downregulating genes involved in homologous recombination regulation in LNCaP cells. Consistently, we found a correlation between higher CYP27A1 expression (i.e., higher intracellular 27HC) and decreased expression of DNA repair gene signatures in castration-sensitive PC (CSPC) in human PC datasets. However, such correlation was less clear in metastatic castration-resistant PC (mCRPC). 27HC increased expression of DNA damage repair markers in PC cells, notably in AR+ cells, but no consistent effects in AR- cells and decreased expression in non-neoplastic prostate epithelial cells. While testing the clinical implications of this, we noted that 27HC treatment increased DNA damage in LNCaP cells via comet assays. Effects were reversible by adding back cholesterol, but not androgens. Finally, in combination with olaparib, a PARP inhibitor, we showed additive DNA damage effects. DISCUSSION: These results suggest 27HC induces "BRCAness", a functional state thought to increase sensitivity to PARP inhibitors, and leads to increased DNA damage, especially in CSPC. Given the emerging appreciation that defective DNA damage repair can drive PC growth, future studies are needed to test whether 27HC creates a synthetic lethality to PARP inhibitors and DNA damaging agents in CSPC
Clinical practice guidelines for esophagogastric junction cancer: Upper GI Oncology Summit 2023.
No full textFUME-TCRseq Enables Sensitive and Accurate Sequencing of the T-cell Receptor from Limited Input of Degraded RNA.
No full textUNLABELLED: Genomic analysis of the T-cell receptor (TCR) reveals the strength, breadth, and clonal dynamics of the adaptive immune response to pathogens or cancer. The diversity of the TCR repertoire, however, means that sequencing is technically challenging, particularly for samples with low-quality, degraded nucleic acids. Here, we developed and validated FUME-TCRseq, a robust and sensitive RNA-based TCR sequencing methodology that is suitable for formalin-fixed paraffin-embedded samples and low amounts of input material. FUME-TCRseq incorporates unique molecular identifiers into each molecule of cDNA, allowing correction for sequencing errors and PCR bias. Using RNA extracted from colorectal and head and neck cancers to benchmark the accuracy and sensitivity of FUME-TCRseq against existing methods demonstrated excellent concordance between the datasets. Furthermore, FUME-TCRseq detected more clonotypes than a commercial RNA-based alternative, with shorter library preparation time and significantly lower cost. The high sensitivity and the ability to sequence RNA of poor quality and limited amount enabled quantitative analysis of small numbers of cells from archival tissue sections, which is not possible with other methods. Spatially resolved FUME-TCRseq analysis of colorectal cancers using macrodissected archival samples revealed the shifting T-cell landscapes at the transition to an invasive phenotype and between tumor subclones containing distinct driver alterations. In summary, FUME-TCRseq represents an accurate, sensitive, and low-cost tool for the characterization of T-cell repertoires, particularly in samples with low-quality RNA that have not been accessible using existing methodology. SIGNIFICANCE: FUME-TCRseq is a TCR sequencing methodology that supports sensitive and spatially resolved detection of TCR clones in archival clinical specimens, which can facilitate longitudinal tracking of immune responses through disease course and treatment
Roles of H3K4 methylation in biology and disease.
No full textEpigenetic modifications, including posttranslational modifications of histones, are closely linked to transcriptional regulation. Trimethylated H3 lysine 4 (H3K4me3) is one of the most studied histone modifications owing to its enrichment at the start sites of transcription and its association with gene expression and processes determining cell fate, development, and disease. In this review, we focus on recent studies that have yielded insights into how levels and patterns of H3K4me3 are regulated, how H3K4me3 contributes to the regulation of specific phases of transcription such as RNA polymerase II initiation, pause-release, heterogeneity, and consistency. The conclusion from these studies is that H3K4me3 by itself regulates gene expression and its precise regulation is essential for normal development and preventing disease
Acceleration-Based Kalman Tracking for Super-Resolution Ultrasound Imaging In Vivo.
No full textSuper-resolution ultrasound (SRUS) can image microvascular structure and flow at subwave-diffraction resolution based on localizing and tracking microbubbles (MBs). Currently, tracking MBs accurately under limited imaging frame rates and high MB concentrations remains a challenge, especially under the effect of cardiac pulsatility and in highly curved vessels. In this study, an acceleration-incorporated MB motion model is introduced into a Kalman tracking framework. The tracking performance was evaluated using simulated microvasculature with different MB motion parameters, concentrations, and acquisition frame rates, and in vivo human breast tumor US datasets. The simulation results show that the acceleration-based method outperformed the nonacceleration-based method at different levels of acceleration and acquisition frame rates and achieved significant improvement in true positive rate (TPR; up to 11.3%) and false negative rate (FNR; up to 13.2%). The proposed method can also reduce errors in vasculature reconstruction via the acceleration-based nonlinear interpolation, compared with linear interpolation (up to [Formula: see text]). The tracking results from temporally downsampled low frame rate in vivo datasets from human breast tumors show that the proposed method has better MB tracking performance than the baseline method, if using results from the initial high frame data as a reference. Finally, the acceleration estimated from tracking results also provides a spatial speed gradient map that may contain extra valuable diagnostic information
Fine-mapping analysis including over 254,000 East Asian and European descendants identifies 136 putative colorectal cancer susceptibility genes.
No full textGenome-wide association studies (GWAS) have identified more than 200 common genetic variants independently associated with colorectal cancer (CRC) risk, but the causal variants and target genes are mostly unknown. We sought to fine-map all known CRC risk loci using GWAS data from 100,204 cases and 154,587 controls of East Asian and European ancestry. Our stepwise conditional analyses revealed 238 independent association signals of CRC risk, each with a set of credible causal variants (CCVs), of which 28 signals had a single CCV. Our cis-eQTL/mQTL and colocalization analyses using colorectal tissue-specific transcriptome and methylome data separately from 1299 and 321 individuals, along with functional genomic investigation, uncovered 136 putative CRC susceptibility genes, including 56 genes not previously reported. Analyses of single-cell RNA-seq data from colorectal tissues revealed 17 putative CRC susceptibility genes with distinct expression patterns in specific cell types. Analyses of whole exome sequencing data provided additional support for several target genes identified in this study as CRC susceptibility genes. Enrichment analyses of the 136 genes uncover pathways not previously linked to CRC risk. Our study substantially expanded association signals for CRC and provided additional insight into the biological mechanisms underlying CRC development
Results and lessons learnt from the WISTERIA phase I trial combining AZD1775 with cisplatin pre- or post-operatively in head and neck cancer.
No full textBACKGROUND: Pre-clinical studies suggest AZD1775, a WEE1 kinase inhibitor, potentiates the activity of various chemotherapeutic agents. METHODS: WISTERIA was a prospective, parallel two-group, open-label, dose-finding, phase I clinical trial. Eligible patients had histologically confirmed oral, laryngeal, or hypopharyngeal squamous cell carcinoma, ECOG performance status 0/1, and aged ≥18-to-≤70 years. Primary outcomes were adverse events and defining recommended dose and schedule of AZD1775 in combination with cisplatin in pre-operative (Group A), or with cisplatin/radiotherapy in post-operative (Group B) patients. Dose determination was guided by a modified time-to-event continual reassessment method (mTITE-CRM). RESULTS: Between 30-Oct-2017 and 15-Jul-2019, nine patients were registered: Three into Group A and six into Group B. WISTERIA was closed early due to poor recruitment. Five dose-limiting toxicities (DLTs) were reported in four Group B patients. Seven serious adverse events were reported in four patients: One in Group A, and three in Group B. Three were related to treatment. No treatment-related deaths were reported. CONCLUSIONS: WISTERIA did not complete its primary objectives due to poor recruitment and toxicities reported in Group B. However, use of the novel mTITE-CRM improved flexibility in reducing accrual suspension periods and should be considered for future trials in complex patient populations. CLINICAL TRIAL REGISTRATION: ISRCTN76291951