Journal articles on the topic 'Tumour sequencing'
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Oey, Harald, Marissa Daniels, Vandana Relan, Tian Mun Chee, Morgan R. Davidson, Ian A. Yang, Jonathan J. Ellis, Kwun M. Fong, Lutz Krause, and Rayleen V. Bowman. "Whole-genome sequencing of human malignant mesothelioma tumours and cell lines." Carcinogenesis 40, no. 6 (April 25, 2019): 724–34. http://dx.doi.org/10.1093/carcin/bgz066.
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Abstract Pleural mesothelioma is a cancer of serosal surfaces caused by environmental exposure to asbestos. Clinical outcome remains poor and while trials of new treatments are ongoing it remains an understudied cancer. Mesothelioma cell lines can readily be grown from primary tumour and from tumour cells shed into pleural effusion with the latter representing a particularly valuable source of DNA in clinical settings, procurable without the need for additional invasive procedures. However, it is not well understood how accurately patient-derived cultured tumour cells represent the molecular characteristics of their primary tumour. We used whole-genome sequencing of primary tumour and matched cultured cells to comprehensively characterize mutations and structural alterations. Most cases had complex rearranged genomes with evidence of chromoanagenesis and rearrangements reminiscent of chromoplexy. Many of the identified driver mutations were structural, indicating that mesothelioma is often caused by structural alterations and catastrophic genomic events, rather than point mutations. Because the majority of genomic changes detected in tumours were also displayed by the genomes of cultured tumour cells, we conclude that low-passage cultured tumour cells are generally suitable for molecular characterization of mesothelioma and may be particularly useful where tissue samples with high tumour cell content are not available. However, the subclonal compositions of the cell lines did not fully recapitulate the subclonal diversity of the primary tumours. Furthermore, longitudinal acquisition of major alterations in subclonal cell populations was observed after long-term passaging. These two factors define limitations of tumour-derived cell lines as genomic substrate for clinical purposes.
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Lolkema, M. P. "Platforms for Tumour Sequencing: Pertinence and Practicability." Annals of Oncology 25 (September 2014): iv9. http://dx.doi.org/10.1093/annonc/mdu294.2.
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Navin, Nicholas, Jude Kendall, Jennifer Troge, Peter Andrews, Linda Rodgers, Jeanne McIndoo, Kerry Cook, et al. "Tumour evolution inferred by single-cell sequencing." Nature 472, no. 7341 (March 13, 2011): 90–94. http://dx.doi.org/10.1038/nature09807.
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Muers, Mary. "Sequencing to detect tumour DNA in circulation." Nature Reviews Genetics 14, no. 1 (December 18, 2012): 4. http://dx.doi.org/10.1038/nrg3397.
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Tang, Ka-Wei, and Erik Larsson. "Tumour virology in the era of high-throughput genomics." Philosophical Transactions of the Royal Society B: Biological Sciences 372, no. 1732 (September 11, 2017): 20160265. http://dx.doi.org/10.1098/rstb.2016.0265.
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With the advent of massively parallel sequencing, oncogenic viruses in tumours can now be detected in an unbiased and comprehensive manner. Additionally, new viruses or strains can be discovered based on sequence similarity with known viruses. Using this approach, the causative agent for Merkel cell carcinoma was identified. Subsequent studies using data from large collections of tumours have confirmed models built during decades of hypothesis-driven and low-throughput research, and a more detailed and comprehensive description of virus–tumour associations have emerged. Notably, large cohorts and high sequencing depth, in combination with newly developed bioinformatical techniques, have made it possible to rule out several suggested virus–tumour associations with a high degree of confidence. In this review we discuss possibilities, limitations and insights gained from using massively parallel sequencing to characterize tumours with viral content, with emphasis on detection of viral sequences and genomic integration events. This article is part of the themed issue ‘Human oncogenic viruses’.
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de Ruiter, J. R., L. F. A. Wessels, and J. Jonkers. "Mouse models in the era of large human tumour sequencing studies." Open Biology 8, no. 8 (August 2018): 180080. http://dx.doi.org/10.1098/rsob.180080.
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Cancer is a complex disease in which cells progressively accumulate mutations disrupting their cellular processes. A fraction of these mutations drive tumourigenesis by affecting oncogenes or tumour suppressor genes, but many mutations are passengers with no clear contribution to tumour development. The advancement of DNA and RNA sequencing technologies has enabled in-depth analysis of thousands of human tumours from various tissues to perform systematic characterization of their (epi)genomes and transcriptomes in order to identify (epi)genetic changes associated with cancer. Combined with considerable progress in algorithmic development, this expansion in scale has resulted in the identification of many cancer-associated mutations, genes and pathways that are considered to be potential drivers of tumour development. However, it remains challenging to systematically identify drivers affected by complex genomic rearrangements and drivers residing in non-coding regions of the genome or in complex amplicons or deletions of copy-number driven tumours. Furthermore, functional characterization is challenging in the human context due to the lack of genetically tractable experimental model systems in which the effects of mutations can be studied in the context of their tumour microenvironment. In this respect, mouse models of human cancer provide unique opportunities for pinpointing novel driver genes and their detailed characterization. In this review, we provide an overview of approaches for complementing human studies with data from mouse models. We also discuss state-of-the-art technological developments for cancer gene discovery and validation in mice.
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Patel, Areeba, Helin Dogan, Alexander Jung, Zaira Seferbekova, Alexander Payne, Michael Ritter, Daniel Schrimpf, et al. "PATH-46. COMPUTATIONAL HISTOPATHOLOGY INFORMED RAPID TARGETED NANOPORE SEQUENCING ENABLES AFFORDABLE NEXT DAY REPORTING OF COMPREHENSIVE MOLECULAR MARKERS FOR CNS TUMOUR DIAGNOSTICS." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii161. http://dx.doi.org/10.1093/neuonc/noac209.619.
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Abstract BACKGROUND Integrative brain tumour diagnostics indisputably requires comprehensive reporting of molecular markers. The 2021 WHO classification of central nervous system (CNS) tumours substantially increased the set of markers for routine evaluation, with greater significance to DNA methylation analysis in diagnostics. Limited by investment and batching, smaller labs and clinics might suffer major delays in delivering clinical decisions. To make precision diagnostics accessible, we introduce an integrated computational histopathology and adaptive nanopore sequencing workflow for next day CNS tumour diagnostics. METHODS We used CNS-CHiP- a multitask deep transfer learning model to predict key molecular alterations and methylation classification from H&E stained CNS tumour slides. For further characterisation and subtyping, we used the predictions to formulate a custom panel for each patient. Targeted sequencing and analyses were performed using Rapid-CNS2- a custom neurooncology nanopore sequencing pipeline for parallel copy-number, mutational and methylation analysis that is flexible in target selection with no additional library preparation and can be initiated upon receipt of frozen sections. Sequencing was performed on a portable MinION or GridION. RESULTS We demonstrate our workflow on diagnostic samples received by the Department of Neuropathology, University Hospital Heidelberg. CNS-CHiP predicted multiple pathognomonic alterations (eg. IDH mutation, 7 gain/10 loss) with reasonable accuracy. This provided basic information regarding the tumor type instantly. Personalised panels enabled small target sizes, resulting in low sequencing time (up to 24h) and competitive costs. The GPU-accelerated bioinformatics pipeline reduced analysis time from > 24h to < 3h. CONCLUSIONS Our workflow harnessing histology-based molecular predictions to instruct targeted nanopore sequencing can be set up with low initial investment and has the potential to facilitate reporting of molecular results on the next day of sample collection. CNS-CHiP combined with Rapid-CNS2 thus aims to make CNS tumour diagnostics affordable and accessible to smaller hospitals and labs especially in low- and middle-income countries.
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Knudsen, Erik S., Uthra Balaji, Brian Mannakee, Paris Vail, Cody Eslinger, Christopher Moxom, John Mansour, and Agnieszka K. Witkiewicz. "Pancreatic cancer cell lines as patient-derived avatars: genetic characterisation and functional utility." Gut 67, no. 3 (January 10, 2017): 508–20. http://dx.doi.org/10.1136/gutjnl-2016-313133.
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ObjectivePancreatic ductal adenocarcinoma (PDAC) is a therapy recalcitrant disease with the worst survival rate of common solid tumours. Preclinical models that accurately reflect the genetic and biological diversity of PDAC will be important for delineating features of tumour biology and therapeutic vulnerabilities.Design27 primary PDAC tumours were employed for genetic analysis and development of tumour models. Tumour tissue was used for derivation of xenografts and cell lines. Exome sequencing was performed on the originating tumour and developed models. RNA sequencing, histological and functional analyses were employed to determine the relationship of the patient-derived models to clinical presentation of PDAC.ResultsThe cohort employed captured the genetic diversity of PDAC. From most cases, both cell lines and xenograft models were developed. Exome sequencing confirmed preservation of the primary tumour mutations in developed cell lines, which remained stable with extended passaging. The level of genetic conservation in the cell lines was comparable to that observed with patient-derived xenograft (PDX) models. Unlike historically established PDAC cancer cell lines, patient-derived models recapitulated the histological architecture of the primary tumour and exhibited metastatic spread similar to that observed clinically. Detailed genetic analyses of tumours and derived models revealed features of ex vivo evolution and the clonal architecture of PDAC. Functional analysis was used to elucidate therapeutic vulnerabilities of relevance to treatment of PDAC.ConclusionsThese data illustrate that with the appropriate methods it is possible to develop cell lines that maintain genetic features of PDAC. Such models serve as important substrates for analysing the significance of genetic variants and create a unique biorepository of annotated cell lines and xenografts that were established simultaneously from same primary tumour. These models can be used to infer genetic and empirically determined therapeutic sensitivities that would be germane to the patient.
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Stankunaite, Reda, Sally L. George, Lewis Gallagher, Sabri Jamal, Ridwan Shaikh, Lina Yuan, Debbie Hughes, et al. "Circulating tumour DNA sequencing to determine therapeutic response and identify tumour heterogeneity in patients with paediatric solid tumours." European Journal of Cancer 162 (February 2022): 209–20. http://dx.doi.org/10.1016/j.ejca.2021.09.042.
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Tanner, Georgette, David R. Westhead, Alastair Droop, and Lucy F. Stead. "Simulation of heterogeneous tumour genomes with HeteroGenesis and in silico whole exome sequencing." Bioinformatics 35, no. 16 (January 4, 2019): 2850–52. http://dx.doi.org/10.1093/bioinformatics/bty1063.
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Abstract Summary Tumour evolution results in progressive cancer phenotypes such as metastatic spread and treatment resistance. To better treat cancers, we must characterize tumour evolution and the genetic events that confer progressive phenotypes. This is facilitated by high coverage genome or exome sequencing. However, the best approach by which, or indeed whether, these data can be used to accurately model and interpret underlying evolutionary dynamics is yet to be confirmed. Establishing this requires sequencing data from appropriately heterogeneous tumours in which the exact trajectory and combination of events occurring throughout its evolution are known. We therefore developed HeteroGenesis: a tool to generate realistically evolved tumour genomes, which can be sequenced using weighted-Wessim (w-Wessim), an in silico exome sequencing tool that we have adapted from previous methods. HeteroGenesis simulates more complex and realistic heterogeneous tumour genomes than existing methods, can model different evolutionary dynamics, and enables the creation of multi-region and longitudinal data. Availability and implementation HeteroGenesis and w-Wessim are freely available under the GNU General Public Licence from https://github.com/GeorgetteTanner, implemented in Python and supported on linux and MS Windows. Supplementary information Supplementary data are available at Bioinformatics online.
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Rico, Karen, Suzann Duan, Ritu L. Pandey, Yuliang Chen, Jayati T. Chakrabarti, Julie Starr, Yana Zavros, et al. "Genome analysis identifies differences in the transcriptional targets of duodenal versus pancreatic neuroendocrine tumours." BMJ Open Gastroenterology 8, no. 1 (November 2021): e000765. http://dx.doi.org/10.1136/bmjgast-2021-000765.
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ObjectiveGastroenteropancreatic neuroendocrine tumours (GEP-NETs) encompass a diverse group of neoplasms that vary in their secretory products and in their location within the gastrointestinal tract. Their prevalence in the USA is increasing among all adult age groups.AimTo identify the possible derivation of GEP-NETs using genome-wide analyses to distinguish small intestinal neuroendocrine tumours, specifically duodenal gastrinomas (DGASTs), from pancreatic neuroendocrine tumours.DesignWhole exome sequencing and RNA-sequencing were performed on surgically resected GEP-NETs (discovery cohort). RNA transcript profiles available in the Gene Expression Omnibus were analysed using R integrated software (validation cohort). Digital spatial profiling (DSP) was used to analyse paraffin-embedded GEP-NETs. Human duodenal organoids were treated with 5 or 10 ng/mL of tumor necrosis factor alpha (TNFα) prior to qPCR and western blot analysis of neuroendocrine cell specification genes.ResultsBoth the discovery and validation cohorts of small intestinal neuroendocrine tumours induced expression of mesenchymal and calcium signalling pathways coincident with a decrease in intestine-specific genes. In particular, calcium-related, smooth muscle and cytoskeletal genes increased in DGASTs, but did not correlate with MEN1 mutation status. Interleukin 17 (IL-17) and tumor necrosis factor alpha (TNFα) signalling pathways were elevated in the DGAST RNA-sequencing. However, DSP analysis confirmed a paucity of immune cells in DGASTs compared with the adjacent tumour-associated Brunner’s glands. Immunofluorescent analysis showed production of these proinflammatory cytokines and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) by the tumours and stroma. Human duodenal organoids treated with TNFα induced neuroendocrine tumour genes, SYP, CHGA and NKX6.3.ConclusionsStromal–epithelial interactions induce proinflammatory cytokines that promote Brunner’s gland reprogramming.
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Chong, Irene Y., Naureen Starling, Alistair Rust, John Alexander, Lauren Aronson, Marta Llorca-Cardenosa, Ritika Chauhan, et al. "The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing." Journal of Clinical Medicine 10, no. 2 (January 9, 2021): 215. http://dx.doi.org/10.3390/jcm10020215.
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1. Background: The application of massively parallel sequencing has led to the identification of aberrant druggable pathways and somatic mutations within therapeutically relevant genes in gastro-oesophageal cancer. Given the widespread use of formalin-fixed paraffin-embedded (FFPE) samples in the study of this disease, it would be beneficial, especially for the purposes of biomarker evaluation, to assess the concordance between comprehensive exome-wide sequencing data from archival FFPE samples originating from a prospective clinical study and those derived from fresh-frozen material. 2. Methods: We analysed whole-exome sequencing data to define the mutational concordance of 16 matched fresh-frozen and FFPE gastro-oesophageal tumours (N = 32) from a prospective clinical study. We assessed DNA integrity prior to sequencing and then identified coding mutations in genes that have previously been implicated in other cancers. In addition, we calculated the mutant-allele heterogeneity (MATH) for these samples. 3. Results: Although there was increased degradation of DNA in FFPE samples compared with frozen samples, sequencing data from only two FFPE samples failed to reach an adequate mapping quality threshold. Using a filtering threshold of mutant read counts of at least ten and a minimum of 5% variant allele frequency (VAF) we found that there was a high median mutational concordance of 97% (range 80.1–98.68%) between fresh-frozen and FFPE gastro-oesophageal tumour-derived exomes. However, the majority of FFPE tumours had higher mutant-allele heterogeneity (MATH) scores when compared with corresponding frozen tumours (p < 0.001), suggesting that FFPE-based exome sequencing is likely to over-represent tumour heterogeneity in FFPE samples compared to fresh-frozen samples. Furthermore, we identified coding mutations in 120 cancer-related genes, including those associated with chromatin remodelling and Wnt/β-catenin and Receptor Tyrosine Kinase signalling. 4. Conclusions: These data suggest that comprehensive genomic data can be generated from exome sequencing of selected DNA samples extracted from archival FFPE gastro-oesophageal tumour tissues within the context of prospective clinical trials.
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Chong, Irene Y., Naureen Starling, Alistair Rust, John Alexander, Lauren Aronson, Marta Llorca-Cardenosa, Ritika Chauhan, et al. "The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing." Journal of Clinical Medicine 10, no. 2 (January 9, 2021): 215. http://dx.doi.org/10.3390/jcm10020215.
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1. Background: The application of massively parallel sequencing has led to the identification of aberrant druggable pathways and somatic mutations within therapeutically relevant genes in gastro-oesophageal cancer. Given the widespread use of formalin-fixed paraffin-embedded (FFPE) samples in the study of this disease, it would be beneficial, especially for the purposes of biomarker evaluation, to assess the concordance between comprehensive exome-wide sequencing data from archival FFPE samples originating from a prospective clinical study and those derived from fresh-frozen material. 2. Methods: We analysed whole-exome sequencing data to define the mutational concordance of 16 matched fresh-frozen and FFPE gastro-oesophageal tumours (N = 32) from a prospective clinical study. We assessed DNA integrity prior to sequencing and then identified coding mutations in genes that have previously been implicated in other cancers. In addition, we calculated the mutant-allele heterogeneity (MATH) for these samples. 3. Results: Although there was increased degradation of DNA in FFPE samples compared with frozen samples, sequencing data from only two FFPE samples failed to reach an adequate mapping quality threshold. Using a filtering threshold of mutant read counts of at least ten and a minimum of 5% variant allele frequency (VAF) we found that there was a high median mutational concordance of 97% (range 80.1–98.68%) between fresh-frozen and FFPE gastro-oesophageal tumour-derived exomes. However, the majority of FFPE tumours had higher mutant-allele heterogeneity (MATH) scores when compared with corresponding frozen tumours (p < 0.001), suggesting that FFPE-based exome sequencing is likely to over-represent tumour heterogeneity in FFPE samples compared to fresh-frozen samples. Furthermore, we identified coding mutations in 120 cancer-related genes, including those associated with chromatin remodelling and Wnt/β-catenin and Receptor Tyrosine Kinase signalling. 4. Conclusions: These data suggest that comprehensive genomic data can be generated from exome sequencing of selected DNA samples extracted from archival FFPE gastro-oesophageal tumour tissues within the context of prospective clinical trials.
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Marziali, Andre. "Sensitive detection of tumor nucleic acids in plasma by mutation-enriched next generation sequencing." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): e22041-e22041. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e22041.
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e22041 Background: Next Generation DNA Sequencing (NGS) is becoming the new standard for mutational profiling of tumour tissue, due to its flexibility, speed, and decreasing cost. While generally exceptional in performance, NGS suffers from a sequencing error rate of 0.1% – 1%, largely due to amplification-induced artifacts in its workflow. While this does not constitute a significant problem in application of NGS to sequencing of tumour tissue, it makes NGS impractical as a method to search for low abundance mutation signatures in plasma samples. Numerous publications have shown the presence of tumour signatures in the cell-free DNA (cfDNA) circulating in plasma, but concordance between the tumour signature and the plasma signature has been limited. This is likely due to limitations in the detection technologies used to search for cfDNA in plasma. To maximize concordance between plasma and tissue, it will be essential that sensitivities reaching 0.01% and below (as little as a single tumour mutant allele per sample) be achieved, and ideally that multiple mutational hot spots be analysed to maximize the chance of detection. Current technologies are incapable of such sensitivity over a large number of mutation loci. Methods: We have developed a novel electrophoretic method that can enrich nucleic acid samples over 1,000,000-fold for up to 100 somatic mutations, enabling reliable profiling of samples containing as little as 0.01% mutant. By enriching nucleic acid samples for specific targets prior to amplification and sequencing, we enable the use of NGS in plasma-based mutation detection and profiling. Results: We present technical and clinical data demonstrating highly sensitive multiplexed mutation detection in plasma and tissue samples, demonstrating 0.01% sensitivity over 45 somatic mutations per sample. Conclusions: We have demonstrated a novel somatic mutation enrichment methodology that allows DNA sequencing to work beyond its usual limit of detection to accurately profile solid tumours by detecting their mutation signature in plasma, even when the tumour DNA is present in plasma at abundances below 0.01%.
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Nagaraju, Santhosh, Ion Boiangiu, Ian Brown, Hussien El-Maghraby, and U. Pohl. "Intracranial myxoid mesenchymal tumour with EWSR1-ATF1 fusion mimicking high grade glioma." Neuro-Oncology 23, Supplement_4 (October 1, 2021): iv23—iv24. http://dx.doi.org/10.1093/neuonc/noab195.059.
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Abstract Aims Molecular profiling is increasingly used in the diagnosis of CNS and non-CNS neoplasms. More than a quarter of all soft tissue tumours are characterized by specific recurrent chromosomal translocations which can be used as molecular signatures. With increasing frequency, EWSR1 rearrangements are found on both mesenchymal tumours and primary glial/neuronal tumours. Here we present a case of intracranial myxoid mesenchymal tumour (IMMT), a rare tumour which is becoming more recognised in recent years, affecting mainly children and young adults, and rarely older adults. It can be found in intraaxial and extraaxial location, with frequent dural connection. The tumour is defined by the genetic hallmark of EWSR1-CREB family gene fusion. Including our case, 16 intracranial tumours with this gene fusion have been reported to date. Our goal is to contribute further to the characterisation of the morphological spectrum, fusion partners and biological behaviour of rare EWSR1-CREB (non-ETS)-rearranged tumours of the CNS. Method Case: The patient is a 27 year old woman with a frontal lobe lesion, radiologically described as a tumour with dural attachment. She underwent surgical debulking, and tumour tissue was histologically examined with conventional immunohistochemistry. Additional genetic testing included targeted mutation screening, FISH, EPIC (Illumina BeadChip) methylation array and next generation sequencing. Histology showed a mitotically active neoplasm with relatively uniform cells, round nuclei and oligodendroglioma-like clear cell change, but no myxoid change. Glomeruloid microvascular proliferation and large areas of tumour necrosis were present. Immunohistochemistry was focally positive for GFAP, and negative or normal for synaptophysin, IDH1 R132H mutation, ATRX and p53. The ki-67 index reached ~20%. Sequencing of IDH1 and IDH2 did not reveal rare IDH mutations, and FISH did not show 1p19q codeletion. Testing for BRAF V600 mutation was negative. Results Although the histology initially suggested a diagnosis of oligodendroglioma, the integrated diagnosis was compatible with glioblastoma, IDH wildtype. Methylation array analysis by EPIC array did not result in classification of currently known entities, neither confirming glioblastoma, nor providing a new diagnosis, when analysed on both brain tumour and sarcoma classifier. This suggested a novel tumour entity not yet represented in the classifier algorithm. Additional testing including next generation sequencing revealed EWSR1 gene rearrangement with fusion partner ATF1 (EWSR1-ATF1 fusion). Based on this, the diagnosis was revised to the emerging new entity of ‘intracranial myxoid mesenchymal tumor’ (IMMT) characterised by EWSR1 fusion with members of the cAMP response element binding protein (CREB) family (ATF1, CREB1 and CREM). Subsequent immunohistochemistry demonstrated positive staining for CD99 and EMA but not desmin. The patient underwent various oncological treatments and is recurrence-free 3 years after initial diagnosis. Conclusion Histologically, IMMT demonstrates a spectrum of features that overlaps with other tumours, but often displays circumscribed growth, uniform cellularity, cytoplasmic clearing and variable myxoid change. The clinical behaviour of these tumours is not fully understood, however provisionally considered intermediate grade. EWSR1-CREB family fusion is not specific but shared with a diverse group of extracranial tumours including soft tissue, salivary gland, odontogenic and myoepithelial tumours. Therefore, clinico-radiologico-pathological correlation is essential to achieve the final diagnosis, and ensure the absence of a primary tumour elsewhere. Familiarisation with IMMT, its characteristic genetic profile and its as yet underreported natural course is crucial, as it can clinically mimic other intracranial tumours such as malignant meningioma or glioma but appears to behave less aggressively than high grade glioma. It is also important to further our understanding of its optimal treatment through review of larger case series and global comparison of patient management.
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Smyth, Robert J., Valentina Thomas, Joanna Fay, Ronan Ryan, Siobhan Nicholson, Ross K. Morgan, Liam Grogan, et al. "Tumour Genome Characterization of a Rare Case of Pulmonary Enteric Adenocarcinoma and Prior Colon Adenocarcinoma." Journal of Personalized Medicine 11, no. 8 (August 4, 2021): 768. http://dx.doi.org/10.3390/jpm11080768.
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Pulmonary enteric adenocarcinoma (PEAC) is a rare variant of lung adenocarcinoma first described in the early 1990s in a lung tumour with overlapping lung and small intestine features. It is a rare tumour with fewer than 300 cases described in the published literature and was only formally classified in 2011. Given these characteristics the diagnosis is challenging, but even more so in a patient with prior gastrointestinal malignancy. A 68-year-old Caucasian female presented with a cough and was found to have a right upper lobe mass. Her history was significant for a pT3N1 colon adenocarcinoma. The resected lung tumour showed invasive lung adenocarcinoma but also features of colorectal origin. Immuno-stains were strongly and diffusely positive for lung and enteric markers. Multi-region, whole-exome sequencing of the mass and archival tissue from the prior colorectal cancer showed distinct genomic signatures with higher mutational burden in the PEAC and very minimal overlap in mutations between the two tumours. This case highlights the challenge of diagnosing rare lung tumours, but more specifically PEAC in a patient with prior gastro-intestinal cancer. Our use of multi-region, next-generation sequencing revealed distinct genomic signatures between the two tumours further supporting our diagnosis, and evidence of PEAC intra-tumour heterogeneity.
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Castignani, Carla, Jonas Demeulemeester, Elizabeth Larose Cadieux, Robert E. Hynds, David R. Pearce, Stefan C. Dentro, Peter Van Loo, Charles Swanton, and TRACERx Consortium. "Abstract 1211: Allele-specific copy-number based deconvolution of bulk tumour RNA sequencing data from the TRACERx study." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1211. http://dx.doi.org/10.1158/1538-7445.am2022-1211.
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Abstract Introduction: Analyses of bulk RNA sequencing (RNA-seq) data are central to most large-scale tumour sequencing studies. Solid tumors often present a dynamic, heterogeneous environment consisting of both cancer (sub)clones and normal cells. Bulk expression data represent population averages and its interpretation is confounded by both normal cell contamination and somatic copy number alterations. Several computational methods to deconvolve tumour and normal expression profiles from bulk RNA-seq have been developed recently. However, these methods often rely on a set of cell-type-specific reference signatures and ignore the effect of copy number changes. Methods: To address these issues, we have developed a method that formalizes the relationship between allele-specific copy number, expression and sample purity to deconvolve the expression profiles of tumor and normal cells from bulk RNA-seq data in an unbiased manner. Our method was applied to sequencing data produced by the TRACERx consortium, a longitudinal study with multi-region whole-exome and RNA-seq of non-small-cell lung cancers. A total of 414 primary tumor regions and 140 adjacent normal tissue samples from 140 TRACERx patients with matched DNA and RNA sequencing data were processed. Results: Here, we were able to directly deconvolve a median of ~2,000 genes per sample and indirectly infer tumor and normal expression profiles of ~10,000 genes. The accuracy of the deconvolution was validated using in-silico mixtures of patient-derived tumour and normal cells and in regions with loss of heterogeneity (LOH) directly on the bulk sequencing data, where the total fraction of expression attributed to tumor cells can be computed directly using somatic mutations. Our method revealed a strong and constitutive genome-wide overexpression in cancer cells compared to admixed normal cells, this overexpression was more pronounced in lung squamous cell carcinoma than lung adenocarcinoma (p<0.001). Multidimensional projection of the purified tumor and normal expression profiles together with the adjacent normal tissue showed clear separation between the purified expression profiles and notably, the deconvolved normal expression was more similar to the normal adjacent profiles than the purified tumor profiles. Conclusion: Overall, these results suggest that our method is able to accurately disentangle the expression of tumour and normal cells from bulk RNA-seq without any previous knowledge. It has potential applications in many studies that include matched RNA-seq and copy number data and can provide new insights functional characterization, the taxonomy of cancer, and tumor evolution. Citation Format: Carla Castignani, Jonas Demeulemeester, Elizabeth Larose Cadieux, Robert E. Hynds, David R. Pearce, Stefan C. Dentro, Peter Van Loo, Charles Swanton, TRACERx Consortium. Allele-specific copy-number based deconvolution of bulk tumour RNA sequencing data from the TRACERx study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1211.
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Abuhusain, Hazem, and Veejay Bagga. "Redefining a Rare CNS Tumour Through Targeted Genetic Sequencing." Neuro-Oncology 24, Supplement_4 (October 1, 2022): iv15—iv16. http://dx.doi.org/10.1093/neuonc/noac200.068.
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Abstract AIMS Isolated cerebellar vermis lesions in adults is a rare location and entity with few literature case reports detailing gangliogliomas and gangliocytomas. Using targeted genetic sequencing, traditional histological diagnosis can be refined further, with a clear impact on prognosis as well as possible genetic counselling implications. METHOD A single case of cerebellar vermis lesion, identified incidentally through imaging of a neck nodule, with subtle features of ataxia and mild impairment of initiation of speech. Sub-total resection of lesion underwent standard histopathological work-up, followed by targeted genetic sequencing to obtain an integrated diagnosis. RESULTS Histological assessment identified atypical ganglion cells labelled with Synaptophysin on immunohistochemistry. Features closely resembled dysplastic cerebellar gangliocytoma (Lhermitte-Duclos Disease). Further targeted sequencing showed no evidence of IDH1, IDH2, TERT promoter, or histone mutation, however, BRAF mutation was present, supporting an alternate diagnosis of ganglioglioma. CONCLUSION Precision medicine is facilitated with advanced diagnostic techniques. This redefines categorisation of some CNS tumours, particularly rare entities. Techniques are especially valuable to individual patient management as they can have a direct impact on aspects of clinical work-up, prognosis, follow-up, and in some cases, genetic counselling.
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Hanna, Tom, Nick Bryan, Damian Bond, John Hunt, Chris Stanley, Paula Ghaneh, and William Greenhalf. "Next generation sequencing of Tp53 in circulating tumour cells." Pancreatology 14, no. 3 (June 2014): S83—S84. http://dx.doi.org/10.1016/j.pan.2014.05.663.
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Davis, John, Matthew Petterson, James Newell, Gregory Y. Lauwers, Thomas Royce, and Michael J. Demeure. "Micrometastatic gastric glomus tumour confirmed by next-generation sequencing." Histopathology 72, no. 2 (October 24, 2017): 351–54. http://dx.doi.org/10.1111/his.13303.
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Kuipers, Jack, Katharina Jahn, and Niko Beerenwinkel. "Advances in understanding tumour evolution through single-cell sequencing." Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1867, no. 2 (April 2017): 127–38. http://dx.doi.org/10.1016/j.bbcan.2017.02.001.
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de Sousa, Sílvia Ferreira, Marina Gonçalves Diniz, Josiane Alves França, Thaís dos Santos Fontes Pereira, Rennan Garcias Moreira, Jean Nunes dos Santos, Ricardo Santiago Gomez, and Carolina Cavalieri Gomes. "Cancer genes mutation profiling in calcifying epithelial odontogenic tumour." Journal of Clinical Pathology 71, no. 3 (November 10, 2017): 279–83. http://dx.doi.org/10.1136/jclinpath-2017-204813.
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AimsTo identify calcifying epithelial odontogenic tumour (CEOT) mutations in oncogenes and tumour suppressor genes.MethodsA panel of 50 genes commonly mutated in cancer was sequenced in CEOT by next-generation sequencing. Sanger sequencing was used to cover the region of the frameshift deletion identified in one sample.ResultsMissense single nucleotide variants (SNVs) with minor allele frequency (MAF) <1% were detected in PTEN, MET and JAK3. A frameshift deletion in CDKN2A occurred in association with a missense mutation in the same gene region, suggesting a second hit in the inactivation of this gene. APC, KDR, KIT, PIK3CA and TP53 missense SNVs were identified; however, these are common SNVs, showing MAF >1%.ConclusionCEOT harbours mutations in the tumour suppressor PTEN and CDKN2A and in the oncogenes JAK3 and MET. As these mutations occurred in only one case each, they are probably not driver mutations for these tumours.
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Macgeoch, Catriona, Diana M. Barnes, Julia A. Newton, Shehla Mohammed, Shirley V. Hodgson, Mun Ng, D. Timothy Bishop, and Nigel K. Spurr. "p53 Protein Detected By Immunohistochemical Staining is Not Always Mutant." Disease Markers 11, no. 5-6 (1993): 239–50. http://dx.doi.org/10.1155/1993/480686.
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The expression of the tumour suppressor gene p53 was analyzed in a variety of human solid tumours by immunohistochemistry and direct DNA sequencing. Positive nuclear staining using a panel of anti-p53 antibodies was used to select tumours for further genetic analysis. Using PCR amplification followed by immobilization onto magnetic beads and direct sequencing, we sequenced exons 5-9 of the p53 gene fro m 9 melanomas, 8 nasopharyngeal carcinomas, 16 sporadic breast carcinomas and 11 patients from familial breast cancer families. No sequence alterations of the p53 gene were detected in either the melanoma or nasopharyngeal tumours and only 19% of the primary breast carcinomas showed a variant band indicative of a mutation. Our results indicate firstly that p53 mutations are not generally involved in the tumour types studied and secondly the data emphasize the disparity encountered when attempting to correlate p53 immunohistochemical positivity with mutations within the p53 gene.
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Burghel, George J., Carolyn D. Hurst, Christopher M. Watson, Phillip A. Chambers, Helen Dickinson, Paul Roberts, and Margaret A. Knowles. "Towards a Next-Generation Sequencing Diagnostic Service for Tumour Genotyping: A Comparison of Panels and Platforms." BioMed Research International 2015 (2015): 1–6. http://dx.doi.org/10.1155/2015/478017.
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Detection of clinically actionable mutations in diagnostic tumour specimens aids in the selection of targeted therapeutics. With an ever increasing number of clinically significant mutations identified, tumour genetic diagnostics is moving from single to multigene analysis. As it is still not feasible for routine diagnostic laboratories to perform sequencing of the entire cancer genome, our approach was to undertake targeted mutation detection. To optimise our diagnostic workflow, we evaluated three target enrichment strategies using two next-generation sequencing (NGS) platforms (Illumina MiSeq and Ion PGM). The target enrichment strategies were Fluidigm Access Array custom amplicon panel including 13 genes (MiSeq sequencing), the Oxford Gene Technologies (OGT) SureSeq Solid Tumour hybridisation panel including 60 genes (MiSeq sequencing), and an Ion AmpliSeq Cancer Hotspot Panel including 50 genes (Ion PGM sequencing). DNA extracted from formalin-fixed paraffin-embedded (FFPE) blocks of eight previously characterised cancer cell lines was tested using the three panels. Matching genomic DNA from fresh cultures of these cell lines was also tested using the custom Fluidigm panel and the OGT SureSeq Solid Tumour panel. Each panel allowed mutation detection of core cancer genes includingKRAS,BRAF, andEGFR. Our results indicate that the panels enable accurate variant detection despite sequencing from FFPE DNA.
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Taylor, William S., John Pearson, Allison Miller, Sebastian Schmeier, Frank A. Frizelle, and Rachel V. Purcell. "MinION Sequencing of colorectal cancer tumour microbiomes—A comparison with amplicon-based and RNA-Sequencing." PLOS ONE 15, no. 5 (May 20, 2020): e0233170. http://dx.doi.org/10.1371/journal.pone.0233170.
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Moore, Ariane L., Aashil A. Batavia, Jack Kuipers, Jochen Singer, Elodie Burcklen, Peter Schraml, Christian Beisel, Holger Moch, and Niko Beerenwinkel. "Spatial Distribution of Private Gene Mutations in Clear Cell Renal Cell Carcinoma." Cancers 13, no. 9 (April 30, 2021): 2163. http://dx.doi.org/10.3390/cancers13092163.
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Intra-tumour heterogeneity is the molecular hallmark of renal cancer, and the molecular tumour composition determines the treatment outcome of renal cancer patients. In renal cancer tumourigenesis, in general, different tumour clones evolve over time. We analysed intra-tumour heterogeneity and subclonal mutation patterns in 178 tumour samples obtained from 89 clear cell renal cell carcinoma patients. In an initial discovery phase, whole-exome and transcriptome sequencing data from paired tumour biopsies from 16 ccRCC patients were used to design a gene panel for follow-up analysis. In this second phase, 826 selected genes were targeted at deep coverage in an extended cohort of 89 patients for a detailed analysis of tumour heterogeneity. On average, we found 22 mutations per patient. Pairwise comparison of the two biopsies from the same tumour revealed that on average, 62% of the mutations in a patient were detected in one of the two samples. In addition to commonly mutated genes (VHL, PBRM1, SETD2 and BAP1), frequent subclonal mutations with low variant allele frequency (<10%) were observed in TP53 and in mucin coding genes MUC6, MUC16, and MUC3A. Of the 89 ccRCC tumours, 87 (~98%) harboured private mutations, occurring in only one of the paired tumour samples. Clonally exclusive pathway pairs were identified using the WES data set from 16 ccRCC patients. Our findings imply that shared and private mutations significantly contribute to the complexity of differential gene expression and pathway interaction and might explain the clonal evolution of different molecular renal cancer subgroups. Multi-regional sequencing is central for the identification of subclones within ccRCC.
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Moorcraft, Sing Yu, David Gonzalez de Castro, David Cunningham, Brian A. Walker, Sanna Hulkki Wilson, Javier Diez Perez, Clare Peckitt, et al. "FOrMAT: Feasibility of a molecular characterization approach to treatment of patients (pts) with advanced gastrointestinal (GI) tumors." Journal of Clinical Oncology 33, no. 3_suppl (January 20, 2015): TPS227. http://dx.doi.org/10.1200/jco.2015.33.3_suppl.tps227.
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TPS227 Background: The molecular characteristics of a pt’s tumour can determine their suitability for treatment with targeted drugs. Several molecular alterations are only seen in a small percentage of pts and so it is often necessary to screen many pts to identify those who may benefit from targeted therapies. Testing each pt for multiple biomarkers is also increasingly important. Targeted next generation sequencing (NGS) can rapidly interrogate tumours for actionable genetic aberrations and therefore facilitate a personalised treatment approach. FOrMAT aims to assess the feasibility of delivering validated NGS results (to accredited standards) in a clinically meaningful timeframe and how this could be adopted into routine clinical practice in the United Kingdom’s National Health Service. Methods: FOrMAT (ClinicalTrials.gov identifier NCT02112357) is a single-centre translational study in pts with locally advanced/metastatic GI tumours (including gastroesophageal, pancreatic, biliary tract and colorectal). Targeted capture sequencing is performed on archival or fresh biopsy samples to detect mutations, copy number variations and translocations in 45 genes which have prognostic, predictive or pharmacogenomic significance or are targets in current/upcoming clinical trials. Results are discussed by a Sequencing Tumour Board to establish if a pt is potentially suitable for a targeted therapy (e.g. within another clinical trial, as part of standard of care or via a compassionate access program). Changes to pts’ treatment are not mandated. The primary endpoint is the percentage of pts in whom a currently actionable molecular alteration was detected. Secondary endpoints include evaluation of the time required to obtain sequencing results. Blood samples will be collected at baseline and at the time of each tumour response assessment for analysis of molecular markers, including ctDNA and microRNA. Pts may also consent to optional biopsies for exploratory research into tumour heterogeneity and development of pt-derived organoids. The study aims to recruit 200 pts over 2 years. Recruitment started in February 2014 and to date 56 pts have been recruited. Clinical trial information: NCT02112357.
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Winter, Helen, P. Kaisaki, Rebecca Carter, Anthony Cutts, Tessa Greenhalgh, Anna Schuh, Ricky A. Sharma, and Jenny C. Taylor. "Sequencing cell free DNA in patients receiving selective internal radiation therapy for colorectal liver metastases." Journal of Clinical Oncology 35, no. 4_suppl (February 1, 2017): 646. http://dx.doi.org/10.1200/jco.2017.35.4_suppl.646.
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646 Background: Tumour heterogeneity is a key determinants of cancer resistance. Serial sampling of cell free (cf)DNA may detect evolving somatic mutations. Monitoring cancers after therapies e.g. selective internal radiation therapy (SIRT) require new biomarkers as RECIST imaging was developed to assess response to chemotherapy and may not reflect tumour changes due to new therapeutic strategies. The utility of cfDNA to detect recurrence and predict response is emerging. The objective of this study was to sequence serial cfDNA samples from patients with liver predominant metastatic disease receiving SIRT, and explore the feasibility of using this method to detect evolution of somatic mutations after high dose radiation. Methods: A prospective imaging biomarker study was performed in patients with colorectal liver metastases (CRLM) receiving SIRT. Plasma was extracted and frozen within 4 hours at 3 time points: baseline, 4 and 10 weeks after SIRT. Ion Torrent Amplicon sequencing was performed using cancer hotspot panel v.2. Sequencing of primary tumours was obtained by pyrosequencing. Results: Twenty-four patients with CRLM were recruited from March – Dec 2015, and 18 had cfDNA extracted for sequencing at a minimum of two time points. Ion Torrent amplicon sequencing of baseline cfDNA showed high concordance with formalin-fixed paraffin-embedded (FFPE) tumour samples. Serial cfDNA sequencing in a patient with partial response by imaging, detected 22% KRAS G12C mutation at baseline, which decreased to 1.5% by 4 weeks, then was undetectable 10 weeks after SIRT. Sequencing of another patient’s cfDNA revealed persistence of KRAS G13D and a truncating APCmutation at both 4 and 10 weeks after SIRT, consistent with the patient’s progressive disease. Conclusions: Cell-free DNA is emerging as a biomarker in colorectal cancer. Our measurements of mutational status in baseline cfDNA and FFPE samples show high concordance. This study reports that serial cfDNA sequencing detects changes in mutational status and specific mutations following high dose radiation to the liver. This adds to the evidence of cfDNA as a tool to detect evolution of somatic mutations following therapy.
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Loddo, Marco, Keeda-Marie Hardisty, Alexander Llewelyn, Tiffany Haddow, Robert Thatcher, and Gareth Williams. "Utilisation of semiconductor sequencing for detection of actionable fusions in solid tumours." PLOS ONE 17, no. 8 (August 19, 2022): e0246778. http://dx.doi.org/10.1371/journal.pone.0246778.
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Oncogenic fusions represent compelling druggable targets in solid tumours highlighted by the recent site agnostic FDA approval of larotrectinib for NTRK rearrangements. However screening for fusions in routinely processed tissue samples is constrained due to degradation of nucleic acid as a result of formalin fixation., To investigate the clinical utility of semiconductor sequencing optimised for detection of actionable fusion transcripts in formalin fixed samples, we have undertaken an analysis of test trending data generated by a clinically validated next generation sequencing platform designed to capture 867 of the most clinically relevant druggable driver-partner oncogenic fusions. Here we show across a real-life cohort of 1112 patients with solid tumours that actionable fusions occur at high frequency (7.4%) with linkage to a wide range of targeted therapy protocols including seven fusion-drug matches with FDA/EMA approval and/or NCCN/ESMO recommendations and 80 clinical trials. The more prevalent actionable fusions identified were independent of tumour type in keeping with signalling via evolutionary conserved RAS/RAF/MEK/ERK, PI3K/AKT/MTOR, PLCy/PKC and JAK/STAT pathways. Taken together our data indicates that semiconductor sequencing for detection of actionable fusions can be integrated into routine diagnostic pathology workflows enabling the identification of personalised treatment options that have potential to improve clinical cancer management across many tumour types.
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Wang, Pin, Yunshan Wang, Sasha A. Langley, Yan-Xia Zhou, Kuang-Yu Jen, Qi Sun, Colin Brislawn, et al. "Diverse tumour susceptibility in Collaborative Cross mice: identification of a new mouse model for human gastric tumourigenesis." Gut 68, no. 11 (March 6, 2019): 1942–52. http://dx.doi.org/10.1136/gutjnl-2018-316691.
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ObjectiveThe Collaborative Cross (CC) is a mouse population model with diverse and reproducible genetic backgrounds used to identify novel disease models and genes that contribute to human disease. Since spontaneous tumour susceptibility in CC mice remains unexplored, we assessed tumour incidence and spectrum.DesignWe monitored 293 mice from 18 CC strains for tumour development. Genetic association analysis and RNA sequencing were used to identify susceptibility loci and candidate genes. We analysed genomes of patients with gastric cancer to evaluate the relevance of genes identified in the CC mouse model and measured the expression levels of ISG15 by immunohistochemical staining using a gastric adenocarcinoma tissue microarray. Association of gene expression with overall survival (OS) was assessed by Kaplan-Meier analysis.ResultsCC mice displayed a wide range in the incidence and types of spontaneous tumours. More than 40% of CC036 mice developed gastric tumours within 1 year. Genetic association analysis identified Nfκb1 as a candidate susceptibility gene, while RNA sequencing analysis of non-tumour gastric tissues from CC036 mice showed significantly higher expression of inflammatory response genes. In human gastric cancers, the majority of human orthologues of the 166 mouse genes were preferentially altered by amplification or deletion and were significantly associated with OS. Higher expression of the CC036 inflammatory response gene signature is associated with poor OS. Finally, ISG15 protein is elevated in gastric adenocarcinomas and correlated with shortened patient OS.ConclusionsCC strains exhibit tremendous variation in tumour susceptibility, and we present CC036 as a spontaneous laboratory mouse model for studying human gastric tumourigenesis.
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Pan, Xiuyi, Mengni Zhang, Jin Yao, Hao Zeng, Ling Nie, Jing Gong, Xueqin Chen, Miao Xu, Qiao Zhou, and Ni Chen. "Fumaratehydratase-deficient renal cell carcinoma: a clinicopathological and molecular study of 13 cases." Journal of Clinical Pathology 72, no. 11 (July 1, 2019): 748–54. http://dx.doi.org/10.1136/jclinpath-2019-205924.
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AimsHereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a newly recognised entity in the WHO 2016 classification defined as the germline mutation of FH gene. Fumaratehydratase-deficient renal cell carcinoma (FH-deficient RCC) is recommended for tumours with FH deficiency but lacking of genetic evidences of FH germline mutation. In this study, we described the clinicopathological and molecular changes of 13 FH-deficient RCCs.Methods and resultsHistology features, clinicopathological data, radiology performance and outcomes were collected for each patient. Next-generation sequencing and DNA sequencing of FH gene were performed to examine FH mutations. The patient group included five females and eight males. Different morphological patterns of papillary, nested, adenoid, foam adenoid, cribriform, tubular, tubulocystic, cystic and loose oedema stroma were observed. Except typical big nuclei with or without eosinophilic nucleoli and perinucleolar halos, raisin-like, hobnail-like and even low-grade nuclei were also observed in these tumours. Eleven cases with high-grade nuclei showed disease progression or death, but no disease progression was detected in two cases with low-grade nuclei and eosinophilic cytoplasm. FH expression was absent in tumour cells except for case 11. Next-generation sequencing and DNA sequencing verified seven FH germline mutations and four somatic mutations out of 13 cases.ConclusionsFH-deficient RCC is a rare renal tumour and has a wide morphological spectrum. Most of the tumours had high-grade nuclei and were aggressive. However, we observed a morphological subtype of FH-deficient RCC with low-grade nuclei and eosinophilic cytoplasm, which might mainly occur in young women and show a relatively good prognosis.
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Chen, Hsin-Pai, Jeng-Kai Jiang, Chia-Hao Chan, Wan-Huai Teo, Chih-Yung Yang, Yen-Chung Chen, Teh-Ying Chou, Chi-Hung Lin, and Yu-Jiun Chan. "Genetic polymorphisms of the human cytomegalovirus UL144 gene in colorectal cancer and its association with clinical outcome." Journal of General Virology 96, no. 12 (December 1, 2015): 3613–23. http://dx.doi.org/10.1099/jgv.0.000308.
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Human cytomegalovirus (HCMV) has been increasingly detected in colorectal cancer (CRC), and genetic polymorphisms in HCMV affect its pathogenesis. This study aimed to investigate HCMV genetic polymorphisms in CRC and its correlation with the clinical outcomes. We performed PCR and sequencing of a viral immunomodulatory gene, UL144, in clinical isolates and CRC specimens. The nucleotide and amino acid sequences were aligned, and a phylogenetic tree was constructed. The clinical, pathological and survival data were compared among tumours with different UL144 genotypes. HCMV was detected in 49 (47.8 %) of the tumour specimens. Genotype A predominated in 43 samples (22/43; 51.2 %) with successful sequencing, followed by genotype B (13/43; 30.2 %) and genotype C (8/43; 18.6 %). The genotypic distribution was similar to that of the clinical isolates and those reported in other Asian populations. The amino acid sequence of genotype B was the most conserved. For stage II and III CRC patients with HCMV-positive tumours, disease-free survival (DFS) varied among the three major genotypes (P = 0.0046). The presence of genotype B virus in the tumours was associated with a shorter DFS and independently predicted tumour recurrence in a multivariate Cox proportional hazards model (hazard ratio, 5.79; 95 % confidence interval, 1.30–25.81; P = 0.021). By reverse transcription PCR, tumour samples with genotype B viruses had the highest rate of UL144 expression. Our results suggest that genetic polymorphisms of HCMV UL144 are associated with clinical outcome in CRC and that HCMV may play an immunomodulatory role in the tumour microenvironment of CRC.
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Huang, Dachuan, Wan Lu Pang, Daryl Ming Zhe Cheah, Yurike Laurensia, Jing Quan Lim, Soo Yong Tan, Tiffany Tang, Soon Thye Lim, and Choon Kiat Ong. "A Patient Derived Xenograft As a Preclinical Model for Monomorphic Epitheliotropic Intestinal T-Cell Lymphoma." Blood 132, Supplement 1 (November 29, 2018): 2949. http://dx.doi.org/10.1182/blood-2018-99-110899.
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Abstract Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) is a type of rare aggressive lymphoma accounting for 5.4% of primary intestinal peripheral T-cell lymphoma and 10-25% of all primary intestinal lymphoma. MEITL also has an extremely poor prognosis with the median overall survival of only seven months. No effective treatment or targeted therapies are currently available to manage this disease. Therefore, there is an urgent need to devise and establish an appropriate preclinical model for a better understanding of this disease and for new therapeutic strategies to be developed on it. Here, we present the first MEITL patient derived xenograft (PDX) as both orthotropic intestinal and as a subcutaneous model. The histological analysis demonstrated high similarity in the overall immunomorphologic features between the primary tumour and PDX tumours. Importantly, all the PDX tumors carried the distinctive MEITL immunophenotype; CD3+CD4-CD8+CD56+ and extensive nuclear expression of MATK. We also had Sanger sequenced 83 somatic mutations in the original tumour and rediscovered them in the 6th passage of our xenografts, which suggest that the generated MEITL PDX tumours were genetically stable. Moreover, whole exome sequencing results showed that the clonal architecture in the primary tumour was well preserved in the PDX tumours. The clonal architecture was further analysed with single cell whole genome amplification following by Sanger sequencing. We found that the most of heterozygous mutations in the primary tumour were a mixture of wild type alleles and heterozygous/homozygous mutations from separated clones, including DUPS14 heterozygous mutation (p.R300W) which was known to activate MEK pathway, and SETD2 mutations, (g.chr3:47061285_ 47061286insA and g.chr3:47127805C>A) which were the synthetic lethal partner of Wee1. Interestingly, we found that the two SETD2 mutations alone were able to differentiate three subclones. This result indicates that the tumour was highly subclonal, which could have a strong impact on the selection of drug resistant clones in the drug treatment. In order to verify this hypothesis, we proceeded to test the efficacy of MEK inhibitor, Trametinib and Wee1 inhibitor, AZD1775 both separately and combined treatment, in our PDX subcutaneous model. The results showed that both 1mg/kg/day of Trametinib and 60mg/kg/day of AZD1775 treatments significantly delayed 60-70% of tumour growth as compared with the vehicle group. The tumour growth was completely arrested in the combined treatment group. In addition, 20% and 40% of the mice exhibited Trametinib and AZD1775 resistance, respectively. From our genomic analysis, all DUPS14 and SETD2 mutations from the pretreated tumours were preserved in the treatment-resistant tumours but absent in the sensitive post-treated residual tumours. The clonality analysis from the sequencing data of the pretreated tumour supports and suggests that single-agent regimes will often not be able to induce a complete response in MEITL patients. Altogether, the current results demonstrated that our MEITL PDX model preserved the principal histological and genetic characteristics of its original tumour and is genomic stable across passages. The model also showed that MEITL could be highly subclonal and combined targeted therapy might achieve a higher efficacy for MEITL treatment. Disclosures No relevant conflicts of interest to declare.
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Zhang, Qi, Yu Lou, Jiaqi Yang, Junli Wang, Jie Feng, Yali Zhao, Lin Wang, et al. "Integrated multiomic analysis reveals comprehensive tumour heterogeneity and novel immunophenotypic classification in hepatocellular carcinomas." Gut 68, no. 11 (June 21, 2019): 2019–31. http://dx.doi.org/10.1136/gutjnl-2019-318912.
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ObjectiveHepatocellular carcinoma (HCC) is heterogeneous, especially in multifocal tumours, which decreases the efficacy of clinical treatments. Understanding tumour heterogeneity is critical when developing novel treatment strategies. However, a comprehensive investigation of tumour heterogeneity in HCC is lacking, and the available evidence regarding tumour heterogeneity has not led to improvements in clinical practice.DesignWe harvested 42 samples from eight HCC patients and evaluated tumour heterogeneity using whole-exome sequencing, RNA sequencing, mass spectrometry-based proteomics and metabolomics, cytometry by time-of-flight, and single-cell analysis. Immunohistochemistry and quantitative polymerase chain reactions were performed to confirm the expression levels of genes. Three independent cohorts were further used to validate the findings.ResultsTumour heterogeneity is considerable with regard to the genomes, transcriptomes, proteomes, and metabolomes of lesions and tumours. The immune status of the HCC microenvironment was relatively less heterogenous. Targeting local immunity could be a suitable intervention with balanced precision and practicability. By clustering immune cells in the HCC microenvironment, we identified three distinctive HCC subtypes with immunocompetent, immunodeficient, and immunosuppressive features. We further revealed the specific metabolic features and cytokine/chemokine expression levels of the different subtypes. Determining the expression levels of CD45 and Foxp3 using immunohistochemistry facilitated the correct classification of HCC patients and the prediction of their prognosis.ConclusionThere is comprehensive intratumoral and intertumoral heterogeneity in all dimensions of HCC. Based on the results, we propose a novel immunophenotypic classification of HCCs that facilitates prognostic prediction and may support decision making with regard to the choice of therapy.
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Gullapalli, Veena, Hannah Hsu, Vanita Bhargava, and Peter Presgrave. "Synchronous Development of Acute Megakaryoblastic Leukaemia and Disseminated Melanoma following Treatment of a Germ Cell Tumour: A Case Report." Case Reports in Oncology 14, no. 3 (November 17, 2021): 1638–44. http://dx.doi.org/10.1159/000519663.
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Somatic malignant transformation of germ cell tumours is a well-described but poorly understood phenomenon. It is characterized by differentiation of pluripotent teratoma cells into somatic tumour cells. Following malignant transformation, the most common histologies are sarcomas and primitive neuroectodermal tumours; however, other subtypes have been recognized including melanoma, leukaemia, and renal cell carcinoma. We report a case of a 38-year-old male who had recently completed treatment for a mediastinal germ cell tumour with teratomatous components. He presented several months after completion of chemotherapy with metastatic lesions in his spine and liver accompanied with severe pancytopenia. He was subsequently diagnosed with acute megakaryoblastic leukaemia (AMKL), and a biopsy of a liver lesion was consistent with metastatic melanoma. This case illustrates the simultaneous development of 2 rare malignant entities: mediastinal germ cell tumour-associated AMKL and somatic malignant transformation to melanoma. It also highlights the importance of close surveillance to detect these metastatic sequelae and the emerging role of tumour sequencing to establish targetable pathways.
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Ooi, Wen Fong, Amrita M. Nargund, Kevin Junliang Lim, Shenli Zhang, Manjie Xing, Amit Mandoli, Jing Quan Lim, et al. "Integrated paired-end enhancer profiling and whole-genome sequencing reveals recurrent CCNE1 and IGF2 enhancer hijacking in primary gastric adenocarcinoma." Gut 69, no. 6 (September 21, 2019): 1039–52. http://dx.doi.org/10.1136/gutjnl-2018-317612.
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ObjectiveGenomic structural variations (SVs) causing rewiring of cis-regulatory elements remain largely unexplored in gastric cancer (GC). To identify SVs affecting enhancer elements in GC (enhancer-based SVs), we integrated epigenomic enhancer profiles revealed by paired-end H3K27ac ChIP-sequencing from primary GCs with tumour whole-genome sequencing (WGS) data (PeNChIP-seq/WGS).DesignWe applied PeNChIP-seq to 11 primary GCs and matched normal tissues combined with WGS profiles of >200 GCs. Epigenome profiles were analysed alongside matched RNA-seq data to identify tumour-associated enhancer-based SVs with altered cancer transcription. Functional validation of candidate enhancer-based SVs was performed using CRISPR/Cas9 genome editing, chromosome conformation capture assays (4C-seq, Capture-C) and Hi-C analysis of primary GCs.ResultsPeNChIP-seq/WGS revealed ~150 enhancer-based SVs in GC. The majority (63%) of SVs linked to target gene deregulation were associated with increased tumour expression. Enhancer-based SVs targeting CCNE1, a key driver of therapy resistance, occurred in 8% of patients frequently juxtaposing diverse distal enhancers to CCNE1 proximal regions. CCNE1-rearranged GCs were associated with high CCNE1 expression, disrupted CCNE1 topologically associating domain (TAD) boundaries, and novel TAD interactions in CCNE1-rearranged primary tumours. We also observed IGF2 enhancer-based SVs, previously noted in colorectal cancer, highlighting a common non-coding genetic driver alteration in gastric and colorectal malignancies.ConclusionIntegrated paired-end NanoChIP-seq and WGS of gastric tumours reveals tumour-associated regulatory SV in regions associated with both simple and complex genomic rearrangements. Genomic rearrangements may thus exploit enhancer-hijacking as a common mechanism to drive oncogene expression in GC.
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Ślaska, Brygida, Magdalena Surdyka, Adam Brodzki, Sylwia Nisztuk, Artur Gurgul, Monika Bugno-Poniewierska, Anna Śmiech, Dorota Różańska, and Maciej Orzelski. "Mitochondrial D-loop mutations can be detected in sporadic malignant tumours in dogs." Bulletin of the Veterinary Institute in Pulawy 58, no. 4 (December 1, 2014): 631–37. http://dx.doi.org/10.2478/bvip-2014-0096.
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Abstract The aim of this study was to identify mutations in the D-loop region of mtDNA in head, neck, and limb tumours in dogs, and determination of their relationship with the process of neoplastic transformation. Blood and tumour tissue samples from 19 dogs with diagnosed sporadic malignant tumours were analysed. DNA extraction, amplification, and sequencing of the mtDNA D-loop, and bioinformatic analyses were performed. Five mutations and 19 polymorphisms were observed in 68.42% of all tumours. Polymorphic variants were noted in 42.86% of the head and neck tumours and in 58.33% of the limb tumours. Mutations were observed in 21.05% of dogs. The mutations were found in 28.57% of the head and neck tumours and in 16.66% of the limb tumours. The mutations were identified in 50% of the studied epithelial cancers. In the mesenchymal tumours, no mutations in the D-loop region were observed. Mitochondrial haplotype A17 was found in over 40% cases of limb tumours. No association between the age, breed, sex, type of tumour, and detected polymorphic variants were observed. Different mutational changes in the D-loop sequences of mtDNA identified in the blood and tumour tissues may indicate a relationship between the type of tumour and individual changes in the D-loop nucleotide sequences of mtDNA.
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O'Byrne, Kenneth John, Joanna Kapeleris, Arutha Kulasinghe, Majid E. Warkiani, Connor Gerard O'Leary, Rahul Ladwa, Ian Vela, Paul Leo, Peter Sternes, and Chamindie Punyadeera. "Culture of circulating tumour cells derived from non-small cell lung cancer." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e21692-e21692. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e21692.
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e21692 Background: Tumour tissue-based information is limited. Liquid biopsy can provide valuable real-time information through circulating tumour cells (CTCs). Profiling and expanding CTCs may provide avenues to study transient metastatic disease. Methods: 70 NSCLC patients were recruited. CTCs were enriched using the spiral microfluidic chip and a RosetteSep™ using bloods from NSCLC patients. CTC cultures were carried out using the Clevers media under hypoxic conditions. CTCs were characterized using immunofluorescence and mutation-specific antibodies for samples with known mutation profiles. Exome sequencing was used to characterized CTC cultures. Results: CTCs ( > 2 cells) were detected in 38/70 (54.3%) of patients ranging from 0-385 CTCs per 7.5ml blood. In 4/5 patients where primary tumours harboured an EGFR exon 19 deletion, this EGFR mutation was also captured in CTCs. ALK translocation was confirmed on CTCs from a patient harbouring an ALK-rearrangement in the primary tumour. Short term CTC cultures were successfully generated in 9/70 NSCLC patients. Whole exome sequencing confirmed the presence of somatic mutations in the CTC cultures with mutational signatures consistent with NSCLC. Conclusions: This study demonstrates a workflow for ex vivo culture of CTCs from NSCLC blood samples.
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Li, Rong, Shuangxiu Wu, Yuanyuan Chang, Yin Wang, and Boyi Li. "Whole-exome sequencing on circulating tumor cells explores platinum-based chemotherapy resistance in advanced non-small cell lung cancer." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e21021-e21021. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e21021.
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e21021 Background: Circulating tumour cells (CTCs) have important applications in clinical practice on early tumour diagnosis, prognostic prediction and treatment evaluation. Platinum-based chemotherapy is the first-line treatment for non-small cell lung cancer (NSCLC) patients without targeted drugs. But most patients progressed after a period of treatment. Therefore, to dissect the genetic information contributing to drug resistance and tumour progression in CTCs is valuable for guidance of treatment adjustment. Methods: In this study, we enrolled 9 NSCLC patients treated with platinum-based chemotherapy. For each patient, 10 CTCs were isolated after progression to perform a single cell-level whole-exome sequencing (WES). Meanwhile the patients’ paired primary-diagnosed FFPE sample and progressive biopsy specimens were also performed WES. Results: Comparisons of WES data between primary and progressive specimens, as well as CTCs revealed that more than half patients’ tumour mutation burden (TMB) increased after progression. Dozes to hundreds of single-nucleotide variants (SNVs) and insertions or deletions (Indels) were detected in the CTCs. Slightly more proportion of SNVs/Indels in CTCs shared with paired primary tumours (1.2%-23.1%) than with progressive samples (0.6%-11.7%). Conclusions: Functional annotation on SNVs/Indels showed that CTCs not only harboured cancer driver gene mutations, such as EGFR and TP53, shared with primary and/or progressive tumours, but also harboured chemotherapy-resistance and stem cell-related gene mutations, including SHKBP1, NUMA1, ZNF143, MUC16, ORC1, PON1, PELP1, etc. which have crucial roles in drug resistance and poor prognosis for NSCLCs. Thus, detection of genetic information in CTCs was necessary for guidance of individual therapy and drug resistance study.
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Ma, E. S. K., C. L. P. Wong, F. B. F. Law, W.-K. Chan, and D. Siu. "Detection of KRAS mutations in colorectal cancer by high-resolution melting analysis." Journal of Clinical Pathology 62, no. 10 (September 25, 2009): 886–91. http://dx.doi.org/10.1136/jcp.2008.063677.
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Aims:Mutation of the KRAS gene predicts the clinical response to the monoclonal antibody cetuximab in patients with advanced colorectal cancer (CRC). This study aimed to perform KRAS mutation detection on formalin-fixed paraffin-embedded (FFPE) tumour tissue by two different methods for comparison.Methods:The FFPE sample was microdissected to enrich for tumour cells. KRAS exon 2 mutations were performed on 100 Chinese patients with CRC by direct nucleotide sequencing and high-resolution melting (HRM) analysis.Results:KRAS exon 2 mutations were detected in a total of 62 patients with the two methods combined, comprising 11 different mutant alleles. Three common mutations p.Gly12Asp, p.Gly12Val and p.Gly13Asp accounted for approximately 70% of all cases. The concordant rate between the two methods was 95%. Four mutations not initially detected by direct sequencing were identified by HRM and confirmed by sequencing of the HRM amplicons. One mutation detected by direct sequencing was inadvertently grouped as a wild-type allele by HRM software, but this was readily rectified through manual review.Conclusion:HRM analysis is a sensitive method of detecting KRAS mutation on FFPE tumour tissue to guide cetuximab treatment and is applicable to routine molecular diagnostic service. Utilisation of HRM to screen for mutations upfront economises the resource used in the sequencing reaction.
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Hiley, Crispin T., Kevin Litchfield, Oriol Pich, David Moore, Cristina Naceur-Lombardelli, Selvaraju Veeriah, Maise Al Bakir, et al. "Abstract 645: Heterogeneity of immunotherapy biomarkers in the TRACERx non-small cell lung cancer multi-region lung cancer cohort study." Cancer Research 82, no. 12_Supplement (June 15, 2022): 645. http://dx.doi.org/10.1158/1538-7445.am2022-645.
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Abstract Immunotherapy containing regimens have become the standard of care first-line therapy for non-small cell lung cancer (NSCLC) in the metastatic setting and are being explored in the adjuvant and neo-adjuvant setting. However, as intratumor heterogeneity (ITH) is pervasive in cancer we investigated the impact of ITH on established and putative biomarkers predictive of immunotherapy response. We have used whole exome sequencing, RNA sequencing and PDL1 immunohistochemistry (IHC) derived from the multiregion treatment naïve TRACERx cohort to investigate this. Whole exome sequencing data was available for 432 primary tumours from 421 patients with greater than 1500 unique tumour regions. Tumour mutational burden (TMB) was calculated using best practice guidelines from the TMB harmonisation project. Using the FDA approved threshold of ≥ 10 mutations per megabase to define high TMB we found that 41% of patients with high TMB had at least one region with <10 mutation per megabase. This could result in misclassification of patients so we modelled increased sampling in this cohort and found that at least 5 spatially separated samples would be required to ensure no patients are misclassified. Contrary to the published literature we did not find TMB was confounded by tumour purity. Multi-region PDL1 IHC data using the FDA approved companion diagnostic assay (22c3) was available for 132 spatially separated tumour regions from 36 patients. Tumour regions were scored according to the established tumour proportion scoring system (≥50%, 49-1%, <1%). Using only a single sample 31% of patients would have been misclassified. Finally, we investigated several putative transcriptomic biomarkers including 10 single genes (e.g. CXCL9), 15 expression signatures (e.g cytolytic score, TIDE) and 2 tumour microenvironment classification systems. We found that 20-45% of these putative biomarkers were discordant when applied to multi-region data and this increased with the number of tumour regions per patients but was not impacted by tumour purity or sequencing depth. To investigate this further we compared the mean geneset RNA-ITH score between a geneset of all the immune biomarker genes with 100 randomly selected non-immune, but expressed, genesets. The immune biomarker gene set was subject to significantly more intra tumour heterogeneity of expression (Mean RNA-ITH Score 0.50 vs 0.31 p=6.4e-12). In conclusion we demonstrate significant heterogeneity of immune biomarkers in NSCLC and in particular a lack of robustness of expression-based predictors of response to immunotherapy. This highlights the need for biomarkers that are robust to intra-tumour heterogeneity and sampling bias or the need for more representative tumour sampling techniques. Citation Format: Crispin T. Hiley, Kevin Litchfield, Oriol Pich, David Moore, Cristina Naceur-Lombardelli, Selvaraju Veeriah, Maise Al Bakir, Simranpreet Summan, Kristiana Grigoriadis, Carlos Martinez Ruiz, Clare Puttick, Katey Enfield, Sophia Ward, Alexander Frankell, Dhruva Biswas, Rachel Rosenthal, Nicolai J. Birkbak, Mariam Jamal-Hanjani, Nicholas McGranahan, Charles Swanton, TRACERx Consortium. Heterogeneity of immunotherapy biomarkers in the TRACERx non-small cell lung cancer multi-region lung cancer cohort study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 645.
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LeBlanc, VG, D. Trinh, M. Hughes, I. Luthra, D. Livingstone, MD Blough, JG Cairncross, JJ Kelly, and MA Marra. "1450-1545 Young Investigator Awards & Presentations Basic/Translational Exploring cellular subpopulations in glioblastoma and matched organoids using single-cell RNA-seq 52." Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques 45, S3 (June 2018): S13—S14. http://dx.doi.org/10.1017/cjn.2018.297.
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Glioblastomas (GBMs) account for nearly half of all primary malignant brain tumours, and current therapies are often only marginally effective. Our understanding of the underlying biology of these tumours and the development of new therapies have been complicated in part by widespread inter- and intratumoural heterogeneity. To characterize this heterogeneity, we performed regional subsampling of primary glioblastomas and derived organoids from these tissue samples. We then performed single-cell RNA-sequencing (scRNA-seq) on these primary regional subsamples and 1-3 matched organoids per sample. We have profiled samples from six tumour sets to date and have obtained sequencing data for 21,234 primary tissue cells and 14,742 organoid cells. While the most apparent differences in gene expression appear to be between individual tumours, we were also able to identify similar cellular subpopulations across tissue samples and across organoids. Importantly, organoids derived from the same tissue sample appeared to be composed of similar cellular subpopulations and were highly comparable to each other, indicating that replicate organoids faithfully represent the original tumour tissue. Overall, our scRNA-seq approach will help evaluate the utility of tumour-derived organoids as model systems for GBM and will aid in identifying cellular subpopulations defined by gene expression patterns, both in primary GBM regional subsamples and their associated organoids. These analyses will allow for the characterization of clonal or subclonal populations that are likely to respond to different therapeutic approaches and may also uncover novel therapeutic targets previously unrevealed through bulk analyses.
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Caushi, Justina X., Jiajia Zhang, Zhicheng Ji, Ajay Vaghasia, Boyang Zhang, Emily Han-Chung Hsiue, Brian J. Mog, et al. "Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers." Nature 596, no. 7870 (July 21, 2021): 126–32. http://dx.doi.org/10.1038/s41586-021-03752-4.
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AbstractPD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a ‘barcode’ to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein–Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.
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Ghorani, Ehsan, James L. Reading, Jake Y. Henry, Marc Robert De Massy, Rachel Rosenthal, Andrew J. S. Furness, Assma Ben Aissa, et al. "Association of the imbalance between early and late differentiated intra-tumor CD4 T cells with mutational burden in non-small cell lung cancer." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): 2590. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.2590.
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2590 Background: CD4 T helper cells are key orchestrators of immunity in states of persistent antigen exposure. In chronic viral infection, loss of immune control is associated with CD4 differentiation skewing (CD4ds) resulting from decline of early progenitors and gain in abundance of exhausted and terminally differentiated subsets. Here, we set out to identify whether a similar process occurs within the tumour microenvironment, contributing to immune dysfunction. Methods: Multiregional samples of tumour and non-tumour lung tissue from patients with untreated, surgically resected non-small cell lung cancer (NSCLC) within the first 100 recruited to the prospective lung TRACERx study were analysed by high dimensional flow cytometry of tumour infiltrating lymphocytes (TILs) and paired bulk tumour exome and RNA sequencing. We additionally reanalysed publically available single T cell and bulk tumour RNA sequencing from patients with NSCLC. Results: Unsupervised clustering and dimension reduction revealed a heterogenous landscape of CD4 TILs, with evidence of CD4ds in association with tumour mutational burden. Loss of PD1-CCR7+ T central memory enriched early differentiated cells was accompanied by gain in abundance of PD1+ populations with exhausted (CD57-ICOShiCTLA4hi) and terminally differentiated effector (CD57+Eomes+) phenotypes. Further characterisation of these populations by single cell RNA sequencing revealed differential expression of key genes involved in transcriptional regulation, co-inhibition and co-stimulatory pathways. A validated gene signature of CD4ds was associated with worse outcomes in TRACERx and TCGA cohorts. Conclusions: Our findings reveal remodelling of the CD4 differentiation landscape in association with tumour genomic characteristics, underscoring how mutational burden in the context of chronic stimulation may lead to loss of immune fitness and elucidating key regulatory pathways in potentially clinically relevant CD4 subsets.
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Crisafulli, Giovanni, Benedetta Mussolin, Andrea Cassingena, Monica Montone, Alice Bartolini, Ludovic Barault, Antonia Martinetti, et al. "Whole exome sequencing analysis of urine trans-renal tumour DNA in metastatic colorectal cancer patients." ESMO Open 4, no. 6 (November 2019): e000572. http://dx.doi.org/10.1136/esmoopen-2019-000572.
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BackgroundThe analysis of circulating free tumour DNA (ctDNA) in blood, commonly referred as liquid biopsy, is being used to characterise patients with solid cancers. Tumour-specific genetic variants can also be present in DNA isolated from other body fluids, such as urine. Unlike blood, urine sampling is non-invasive, can be self-performed, and allows recurrent longitudinal monitoring. The features of tumour DNA that clears from the glomerular filtration barrier, named trans-renal tumour DNA (trtDNA), are largely unexplored.Patients and methodsSpecimens were collected from 24 patients with KRAS or BRAF mutant metastatic colorectal cancer (mCRC). Driver mutations were assessed by droplet digital PCR (ddPCR) in ctDNA from plasma and trtDNA from urine. Whole exome sequencing (WES) was performed in DNA isolated from tissue, plasma and urine.ResultsOut of the 24 CRC cases, only four had sufficient DNA to allow WES analyses in urine and plasma. We found that tumour alterations primarily reside in low molecular weight fragments (less than 112 bp). In patients whose trtDNA was more than 2.69% of the urine derived DNA, cancer-specific molecular alterations, mutational signatures and copy number profiles identified in urine DNA are comparable with those detected in plasma ctDNA.ConclusionsWith current technologies, WES analysis of trtDNA is feasible in a small fraction of mCRC patients. Tumour-related genetic information is mainly present in low molecular weight DNA fragments. Although the limited amounts of trtDNA poses analytical challenges, enrichment of low molecular weight DNAs and optimised computational tools can improve the detection of tumour-specific genetic information in urine.
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Yaegashi, Mizunori, Takeshi Iwaya, Noriyuki Sasaki, Masashi Fujita, Zhenlin Ju, Doris Siwak, Tsuyoshi Hachiya, et al. "Frequent post-operative monitoring of colorectal cancer using individualised ctDNA validated by multiregional molecular profiling." British Journal of Cancer 124, no. 9 (March 3, 2021): 1556–65. http://dx.doi.org/10.1038/s41416-021-01266-4.
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Abstract Background Circulating tumour DNA (ctDNA) is known as a tumour-specific personalised biomarker, but the mutation-selection criteria from heterogeneous tumours remain a challenge. Methods We conducted multiregional sequencing of 42 specimens from 14 colorectal tumours of 12 patients, including two double-cancer cases, to identify mutational heterogeneity to develop personalised ctDNA assays using 175 plasma samples. Results “Founder” mutations, defined as a mutation that is present in all regions of the tumour in a binary manner (i.e., present or absent), were identified in 12/14 tumours. In contrast, “truncal” mutations, which are the first mutation that occurs prior to the divergence of branches in the phylogenetic tree using variant allele frequency (VAF) as continuous variables, were identified in 12/14 tumours. Two tumours without founder and truncal mutations were hypermutators. Most founder and truncal mutations exhibited higher VAFs than “non-founder” and “branch” mutations, resulting in a high chance to be detected in ctDNA. In post-operative long-term observation for 10/12 patients, early relapse prediction, treatment efficacy and non-relapse corroboration were achievable from frequent ctDNA monitoring. Conclusions A single biopsy is sufficient to develop custom dPCR probes for monitoring tumour burden in most CRC patients. However, it may not be effective for those with hypermutated tumours.
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Asmann, Y., K. Parikh, M. Borad, L. Bergsagel, and A. S. Mansfield. "84MO Tumour-only sequencing led to inflated tumour mutational burden estimation especially in under-represented ethnic groups." Annals of Oncology 31 (September 2020): S274—S275. http://dx.doi.org/10.1016/j.annonc.2020.08.205.
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Cheng, Michael L., Michael F. Berger, David M. Hyman, and David B. Solit. "Clinical tumour sequencing for precision oncology: time for a universal strategy." Nature Reviews Cancer 18, no. 9 (July 20, 2018): 527–28. http://dx.doi.org/10.1038/s41568-018-0043-2.
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Yadav, Mahesh, Suchit Jhunjhunwala, Qui T. Phung, Patrick Lupardus, Joshua Tanguay, Stephanie Bumbaca, Christian Franci, et al. "Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing." Nature 515, no. 7528 (November 26, 2014): 572–76. http://dx.doi.org/10.1038/nature14001.
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Mahamdallie, Shazia, Shawn Yost, Emma Poyastro-Pearson, Esty Holt, Anna Zachariou, Sheila Seal, Anna Elliott, et al. "Identification of new Wilms tumour predisposition genes: an exome sequencing study." Lancet Child & Adolescent Health 3, no. 5 (May 2019): 322–31. http://dx.doi.org/10.1016/s2352-4642(19)30018-5.
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