43 research outputs found
Variable Responses of MYC Translocation Positive Lymphoma Cell Lines To Different Combinations of Novel Agents: Impact of BCL2 Family Protein Expression.
No full textSeveral newly developed drugs including JQ1 (BET inhibitor), ABT199 (BCL2 inhibitor), and bortezomib (proteasome inhibitor) may offer novel therapeutic strategies for aggressive diffuse large B-cell lymphoma (DLBCL). We tested these drugs together with doxorubicin in a series of combinations in 16 DLBCL cell lines including 4 ABC-DLBCL (OCI-Ly3, OCI-Ly10, SUDHL2, RIVA) and 12 GCB-DLBCL lines (OCI-Ly4, OCI-Ly18, BJAB, SUDHL4, SUDHL6, SUDHL10, DB, PR1, VAL, SC1, Karpas-231, Karpas-422). Among these cell lines, ABT199 and doxorubicin, and to a lesser extent JQ1 and bortezomib, showed high variations in their ED50 values. Of the six cell lines showing high ABT199 ED50 values, four (SUDHL10, OCI-Ly4, SUDHL2, and BJAB) had no or little BCL2 expression, and SUDHL6 also displayed a low BCL2 expression. There was no association between the ED50 value of doxorubicin, JQ1 and bortezomib, and TP53/MYC/BCL2 genetic abnormalities or cell of origin subtype. A synergistic effect in all or the majority of drug combinations was seen in 11 cell lines, while an antagonistic effect in a high proportion of drug combinations was observed in the remaining 5 cell lines including the 3 (SUDHL10, OCI-Ly4, and SUDHL2) with little BCL2 expression, and additionally OCI-Ly18 and RIVA. Extensive Western blot analyses revealed high MCL1 expression in SUDHL10 and OCI-Ly4 but no apparent alterations in other cell lines. The molecular mechanism underlying the antagonistic effect of drug combinations in DLBCL is heterogeneous with the altered BCL2 family protein expression (absent BCL2, but high MCL1) in some cell lines
CUPiD: A cfDNA methylation-based tissue-of-origin classifier for Cancers of Unknown Primary - figure generation data and code
No full text<p>This repository holds code behind the article "A cfDNA methylation-based tissue-of-origin classifier for Cancers of Unknown Primary" by Conway, Pearce, Clipson et al, <a href="https://www.nature.com/articles/s41467-024-47195-7">published in Nature Communications</a>. This contains the code and data required to regenerate all the figures in the paper.</p>
<p>The code is structured as follows:</p>
<ul>
<li><strong>figure_inputs</strong>: Inputs required to generate the figures. Some of these are generated as part of the classifier generation/application process, some are additional clinical data. </li>
<li><strong>figure_generation</strong>: RMarkdown reports to generate the figures in the paper, given all the inputs in figures_inputs. These are separate RMarkdown files, and should be self-contained, and are able to be ran directly based on the files within `figure_inputs`. They may be ran in any order, except that CUP_mutation_analysis should be ran after CUPiD_cfDNA_application.</li>
</ul>
<p>The code will generate outputs in two folders, one called <strong>output</strong> and one called <strong>source_data;</strong> these are provided here for completeness. The source_data folder contains data for Nature Communications Source Data files, including data behind the ROC curves that are too large to be included there, while the output folder generates supplementary data tables and copies of the plots.</p>
<p>Data and code to generate the classifier is available upon request from <a href="../records/10678015">https://zenodo.org/uploads/10678015</a>.</p>
Target-guided synthesis approach to the discovery of novel bivalent inhibitors of Glutathione Transferases
No full textTarget-guided synthesis is an approach to drug discovery that uses the
biological target as a template to direct synthesis of its own best inhibitors from
small molecule fragments. The process bridges the gap between chemical synthesis
of drug candidates and their biological binding assay, merging the two operations
into a single process whereby the active site or a binding pocket within the structure
of the biological target directly controls the assembly of the best inhibitor in situ.
Two different approaches to target-guided synthesis, the thermodynamic approach,
making use of reversible reactions, and the kinetic approach, which uses an
irreversible reaction, have been employed to discover novel, isoform selective
inhibitors of the glutathione transferase (GST) enzyme family – possible drug targets
in cancer and parasitic disease treatments.
The thermodynamic approach described in this thesis uses the
aniline-catalysed reversible acyl hydrazone formation reaction to create a dynamic
covalent library of bivalent ligands designed to bind the dimeric structure of GST. In
the presence of GST one of the bivalent ligands was selectively amplified at the
expense of the other library members. This ligand was shown, via biological assays,
to be a specific inhibitor for one isoform of GST, the mu isoform mGSTM1-1.
A kinetic approach has also been investigated as a way to identify novel
bivalent GST inhibitors utilising the Huisgen 1, 3 dipolar cycloaddition reaction. An
azide and alkyne fragment library was designed to bind across the dimeric GST
structure. The inhibitor structures are therefore bivalent, containing two anchoring
fragments known to bind to the GST active site, linked by a triazolopeptide spacer.
The triazole provides the click chemistry disconnection, enabling rapid in situ
screening of candidate alkyne and azide fragments for inhibitor discovery. Whilst the
in situ reaction with GST yielded inconclusive results, a number of the triazole
products were found to have low nanomolar inhibitory activity towards GST
Problem definition in a participatory design process.
No full textAlthough there exist substantial literatures on individual design processes and upon problem definition, there is little attention to how participatory design groups define the problems on which they work. This lack occurs because of the difficulty in learning about what goes on inside design groups. However, participatory and collaborative design is a widespread and growing mode of design practice that demands a better understanding. This dissertation consists of an in depth study of one participatory design process that occurred in a federal laboratory setting. It was a multi-disciplinary activity that initially involved representatives from five research and development departments. These representatives collaborated over seven months to create a project proposal that would incorporate technology and design contributions from each of their specialities. The final proposal comprised a design problem definition including specific design components, schedule, budget, and system integration requirements. The central thesis states that collaborative or participatory design is especially appropriate for defining a design problem, as opposed to systematic design methods that are more appropriate for seeking a solution to that design problem. Several propositions devolve from this thesis, notably the idea of alternative paths toward a common goal. The research approach was research-in-action. The author served as a facilitator of the participatory design process, recording their activities and the outcomes of their efforts. The facilitators developed and employed a number of participatory design methods to address specific issues and to encourage the participants to build a consensus upon a proposal. Most of these methods failed to produce the intended results but at least one succeeded for each issue, and eventually the participants took the initiative to forge the essential consensus. This consensus proved critical to the proposal's success. The analysis of the results relies upon direct observation and upon an extensive literature review of precedents in design participation. The attendance data reveal that most of the facilitator and participant interaction occurred outside the formal meetings. A meta-analysis of the observations shows they are largely consistent with precedent, plus a few important new insights. The findings indicate that the design problem definition moved through a sequence of states known in the literature: from "condition of irresolution," to, "wicked problem," to "ill-defined" problem, to a final "well-structured" problem state. The participatory design group's consensus upon the project content and internal relationships proved a crucial step in forging the final design problem definition. The conclusion presents a new model of design participation, in which the design group's consensus upon a problem definition acts as a gateway between problem-defining and solution-seeking.Doctor of Architecture (DArch)ArchitectureUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104731/1/9610061.pdfDescription of 9610061.pdf : Restricted to UM users only
CUPiD, A cfDNA methylation-based tissue-of-origin classifier for Cancers of Unknown Primary - classifier data and code
No full text<p>This repository holds code behind the article "A cfDNA methylation-based tissue-of-origin classifier for Cancers of Unknown Primary" by Conway, Pearce, Clipson et al, <a href="https://www.nature.com/articles/s41467-024-47195-7">published in Nature Communications</a>. This contains the code and data required to generate the CUPiD classifier itself.</p>
<p>Data and code to reproduce the figures in the paper are available from <a href="../records/10684337">https://zenodo.org/uploads/10684337</a> (unrestricted).</p>
<p>Methyl-Binding Domain protein sequencing (MBD-Seq) was applied to circulating cell-free DNA (cfDNA) samples derived from patients with a range of known cancer types (143 patients), as well as 106 non-cancerous controls (79 used in training). </p>
<p>The objects deposited here include R data files containing qseaSets from the R package qsea, which includes the read counts per sample per 300 base pair window across the genome, as well as information on copy number variation and metadata tables. These are provided in the <code>inputFiles/nextflowOutput</code> folder, and are some of the outputs of the nextflow pipeline.</p>
<p>The scripts folder contains numbered sub-folders, with numbered scripts within them, which should be ran in order. The scripts are setup to be run on a PBS-Torque system; files ending ".pbs" should be submitted via qsub, files ending ".sh" should be ran on a node and will submit individual jobs within a loop. R scripts without an associated .pbs or .sh file should just be ran directly. All files should be submitted from the base of the repository (e.g. <code>qsub scripts/01-downloadData/01-getRawData.pbs</code>) to set the paths appropriately via the environment variable <code>PBS_O_WORKDIR</code>. </p>
<ul>
<li><code>01-downloadData</code> contains scripts to download and preprocess all the required data.</li>
<li><code>02-qseaSetNextFlowPipeline</code> contains our custom in-house DSL2 Nextflow pipeline which takes fastq files to processed qseaSets, including QC checks. This requires the fastq files which will be deposited in EGA.</li>
<li><code>03-convertArrays</code> converts downloaded (pre-processed) arrays into estimated qseaSets (containing solely the array sample), and then mixes each array with each NCC cfDNA at varying proportions.</li>
<li><code>04-DMRs</code> calculates pairwise DMRs between each class.</li>
<li><code>05-prepForClassifier</code> selects up to 10000 mixture sets per class, and generates a large table suitable for input into the ML model.</li>
<li><code>06-fitClassifier</code> fits the ML model using <code>xgboost</code> within the <code>tidymodels</code> framework. This is repeated 100 times with different subsets of the mixture sets as input.</li>
<li><code>07-applyClassifier</code> applies these classifiers to the "independent test cohort" - the set of 143 known tumour types, 27 additional NCCs and the 41 patients with CUP. These have been ran through the Nextflow pipeline separately to the 79 NCCs used to derive CUPiD, and have not been used to derive the classifier.</li>
<li><code>08-UMAPs</code> generates some UMAPs on the array data.</li>
</ul>
<p>A subset of these output files are provided in <a href="../records/10684337">https://zenodo.org/uploads/10684337</a> , along with the code to reproduce the figures.</p>
Mutation screening using formalin-fixed paraffin-embedded tissues: a stratified approach according to DNA quality.
No full textDNA samples from formalin-fixed paraffin-embedded tissues are highly degraded with variable quality, and this imposes a big challenge for targeted sequencing due to false positives, largely caused by PCR errors and cytosine deamination. To eliminate false positives, a common practice is to validate the detected variants by Sanger sequencing or perform targeted sequencing in duplicate. Technically, PCR errors could be removed by molecular barcoding of template DNA prior to amplification as in the HaloPlexHS design. Nonetheless, it is uncertain to what extent variants detected using this approach should be further validated. Here, we addressed this question by correlating variant reproducibility with DNA quality using HaloPlexHS target enrichment and Illumina HiSeq4000, together with an in-house validated variant calling algorithm. The overall sequencing coverage, as shown by analyses of 70 genes in 266 cases of large B-cell lymphoma, was excellent (98%) in DNA samples amenable for PCR of ≥400 bp, but suboptimal (92%) and poor (80%) in those amenable for PCR of 300 bp and 200 bp respectively. By mutation analysis in duplicate in 93 cases, we demonstrated that 20 alternative allele depth (AAD) was an optimal cut-off value for separating reproducible from non-reproducible variants in DNA samples amenable for PCR of ≥300 bp, with 97% sensitivity and 100% specificity. By cross validation with a previously established targeted sequencing protocol by Fluidigm-PCR and Illumina MiSeq, the HaloPlexHS protocol was shown to be highly sensitive and specific in mutation screening. To conclude, we proposed a stratified approach for mutation screening by HaloplexHS and Illumina HiSeq4000 according to DNA quality. DNA samples with good quality (≥400 bp) are amenable for mutation analysis with a single replicate, with only variants at 15-20 AAD requiring for further validation, while those with suboptimal quality (300 bp) are better analysed in duplicate with reproducible variants at >15 AAD regarded as true genetic changes
Gene expression profiling in an open-label randomised phase 3 trial (REMoDL-B) of bortezomib added to standard chemoimmunotherapy for diffuse large B-cell lymphoma
No full textBackground: Biologically distinct sub-types of diffuse large B-cell lymphoma (DLBCL) can be identified using gene expression analysis to determine their cell of origin (COO), corresponding to germinal centre (GCB) or activated B‐cells (ABC). This study investigated whether adding bortezomib(B) to standard therapy could improve outcomes in these subtypes.Methods: The REMoDL-B trial is an open-label adaptive two-stage randomised controlled trial at 107 centres in the United Kingdom and Switzerland. Eligible patients (pts) had previously untreated, histologically confirmed DLBCL with sufficient diagnostic material for gene expression profiling and pathology review; age 18 years or older; Eastern Cooperative Oncology Group performance status of ≤2; bulky stage I or stage II-IV requiring full course chemotherapy; measurable disease, and cardiac, lung, renal and liver function sufficient to tolerate chemotherapy. Pts initially received 1 cycle of standard R‐CHOP. During this time, gene expression profiling by whole genome cDNA-mediated annealing, selection, extension and ligation assay was performed on routine diagnostic biopsy material. Patients were then centrally assigned (1:1) via a web-based system, with block randomisation stratified by international prognostic index and COO subtype to continue R‐CHOP +/‐ bortezomib (1.3 mg/m2 IV or 1.6 mg/m2 SC) days 1+8 for cycles 2‐6. The primary endpoint was 30 month progression‐free survival (PFS) for the GCB + ABC population. The primary analysis was intention-to-treat. The safety population consisted of all participants who received at least one dose of study drug. The study was registered at ClinicalTrials.gov: NCT01324596. We report the PFS and safety outcomes for patients in the follow-up phase after the required number of events occurred. Recruitment and treatment has completed for all participants, with long-term follow-up continuingFindings: Between June 2011 and June 2015, 1128 eligible pts were registered and a total of 918 randomised. There was no evidence for a difference in PFS in the combined GCB + ABC population between R‐CHOP (N=361)and RB‐CHOP (N=358) (30 month PFS: 70.6% vs 75.2% respectively) adjusted HR = 0.82, 95% CI 0.63 - 1.08; P=0.16. The most common Grade ≥3 adverse event experienced was haematological toxicity, with 178 (39.8%) and 187 (42.1%) of pts receiving R-CHOP and RB-CHOP experienced, respectively. However, RB‐CHOP was not associated with increased haematological toxicity and 87% of pts completed 6 cycles; Grade ≥3 neuropathy occurred in 3.8% RB‐CHOP vs 1.8% R‐CHOP pts. Serious adverse events occurred in 190 (42.5%) and 223 (50.2%) of pts, including 5 and 4 treatment-related deaths in pts receiving R-CHOP and RB-CHOP, respectively.Interpretation: This is the first large-scale study in DLBCL to use real-time molecular characterisation for prospective stratification and randomisation, and subsequent analysis of biologically distinct subgroups. The addition of bortezomib did not improve outcomes in the ABC subgroup as expected, but proteosome inhibition could be investigated as a possible means to improve the treatment of cytogenetic 'double-hit' DLBCL.Funding: Janssen-Cilag, Bloodwise and Cancer Research UK. <br/
Distinct genetic changes reveal evolutionary history and heterogeneous molecular grade of DLBCL with MYC/BCL2 double-hit.
No full textUsing a Burkitt lymphoma-like gene expression signature, we recently defined a high-risk molecular high-grade (MHG) group mainly within germinal centre B-cell like diffuse large B-cell lymphomas (GCB-DLBCL), which was enriched for MYC/BCL2 double-hit (MYC/BCL2-DH). The genetic basis underlying MHG-DLBCL and their aggressive clinical behaviour remain unknown. We investigated 697 cases of DLBCL, particularly those with MYC/BCL2-DH (n = 62) by targeted sequencing and gene expression profiling. We showed that DLBCL with MYC/BCL2-DH, and those with BCL2 translocation, harbour the characteristic mutation signatures that are associated with follicular lymphoma and its high-grade transformation. We identified frequent MYC hotspot mutations that affect the phosphorylation site (T58) and its adjacent amino acids, which are important for MYC protein degradation. These MYC mutations were seen in a subset of cases with MYC translocation, but predominantly in those of MHG. The mutations were more frequent in double-hit lymphomas with IG as the MYC translocation partner, and were associated with higher MYC protein expression and poor patient survival. DLBCL with MYC/BCL2-DH and those with BCL2 translocation alone are most likely derived from follicular lymphoma or its precursor lesion, and acquisition of MYC pathogenic mutations may augment MYC function, resulting in aggressive clinical behaviour
Abstract 2808: cfDNA multi-omics profiling and tissue of origin predictions in cancers of unknown primary
No full textCancers of Unknown Primary (CUP) comprise a heterogeneous group of metastatic cancers where the primary site is undetectable. It accounts for 2% of all new UK cancer diagnoses and is the 6th leading cause of cancer death. Favourable subsets of CUP exist, based on clinico-pathological features, and although these patients have improved clinical outcomes, they make up only 20% of patients with CUP. The evolution of molecular profiling and tissue-of-origin (TOO) classifiers has the potential to transform diagnosis and management of patients with CUP. Reliable molecular profiling requires good quality and quantity tumour tissue material, often scarce in patients with CUP. Liquid biopsies offer an alternative, minimally invasive approach. We developed a novel cell-free DNA (cfDNA)-based multi-omics assay combining methylation profiling with mutation and copy number alterations (CNA) detection, to enable TOO prediction and biomarker discovery to aid treatment stratification.Methods: Two novel library preparation methods were applied to 41 CUP cfDNA samples, 80 non-cancer controls and 80 cfDNA samples from known tumour types (breast, lung, colorectal cancer): 1) Targeted (641 gene panel) next generation sequencing (NGS); 2) A multiplex genome-wide methylation capture assay (T7-MBDSeq). Both sequencing outputs can estimate CNA. Cancer Genome Interpreter (CGI) was used to evaluate the potential actionability of molecular alterations detected. A novel TOO classifier, generated utilising in silico dilution mixture sets derived from The Cancer Genome Atlas (TCGA) methylation data, demonstrated an AUROC of 0.96 across 15 different cancer groups. This was applied to cfDNA methylation data from known tumour types to test performance accuracy and subsequently applied to CUP cfDNA samples.Results: Multi-omics profiling from cfDNA was completed for 34/41 patients with CUP. cfDNA mutation profiling and estimated CNA analysis so far revealed >350 alterations across 225 genes. 17/34 (50%) of patients harboured potentially actionable alterations by CGI and, where performed, 68% of tumour mutations were concordant in the cfDNA. cfDNA methylation profiling revealed tumour-specific methylation changes in breast, lung and colorectal cancers. Our preliminary TOO classifier performance in these tumour types had an overall sensitivity and specificity of 85% and 98%, respectively. In patients with CUP, a predicted TOO was made in 26/41 (63%) patients and for 6 patients, where a subsequent primary tumour diagnosis was made, the TOO prediction was correct.Conclusions: cfDNA multi-omics profiling revealed potential therapeutic benefit in 77% of this retrospective CUP cohort and illustrates the potential to re-classify patients to primary tumour types where alternative therapies may improve clinical outcomes. Further multi-omics cfDNA profiling of the CUP cohort and validation in known primary cancer types are ongoing.Citation Format: Alicia-Marie Conway, Alexandra Clipson, Francesca Chemi, Simon Pearce, Katarzyna Kamieniecka, Saba Ferdous, Sophie Richardson, Elaine Kilgour, Matthew G. Krebs, Alastair Kerr, Dominic Rothwell, Natalie Cook, Caroline Dive. cfDNA multi-omics profiling and tissue of origin predictions in cancers of unknown primary [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 2808
The prognosis of MYC translocation positive diffuse large B-cell lymphoma depends on the second hit.
No full textA proportion of MYC translocation positive diffuse large B-cell lymphomas (DLBCL) harbour a BCL2 and/or BCL6 translocation, known as double-hit DLBCL, and are clinically aggressive. It is unknown whether there are other genetic abnormalities that cooperate with MYC translocation and form double-hit DLBCL, and whether there is a difference in clinical outcome between the double-hit DLBCL and those with an isolated MYC translocation. We investigated TP53 gene mutations along with BCL2 and BCL6 translocations in a total of 234 cases of DLBCL, including 81 with MYC translocation. TP53 mutations were investigated by PCR and sequencing, while BCL2 and BCL6 translocation was studied by interphase fluorescence in situ hybridization. The majority of MYC translocation positive DLBCLs (60/81 = 74%) had at least one additional genetic hit. In MYC translocation positive DLBCL treated by R-CHOP (n = 67), TP53 mutation and BCL2, but not BCL6 translocation had an adverse effect on patient overall survival. In comparison with DLBCL with an isolated MYC translocation, cases with MYC/TP53 double-hits had the worst overall survival, followed by those with MYC/BCL2 double-hits. In MYC translocation negative DLBCL treated by R-CHOP (n = 101), TP53 mutation, BCL2 and BCL6 translocation had no impact on patient survival. The prognosis of MYC translocation positive DLBCL critically depends on the second hit, with TP53 mutations and BCL2 translocation contributing to an adverse prognosis. It is pivotal to investigate both TP53 mutations and BCL2 translocations in MYC translocation positive DLBCL, and to distinguish double-hit DLBCLs from those with an isolated MYC translocation
