International journal of laboratory hematology, 2019
Clinical genetic testing in the myeloid malignancies is undergoing a rapid transition from the era of cytogenetics and single-gene testing to an era dominated by next-generation sequencing (NGS). This transition promises to better reveal the genetic alterations underlying disease, but there are distinct risks and benefits associated with different NGS testing platforms. NGS offers the potential benefit of being able to survey alterations across a wider set of genes, but analytic and clinical challenges associated with incidental findings, germ line variation, turnaround time, and limits of detection must be addressed. Additionally, transcriptome-based testing may offer several distinct benefits beyond traditional DNA-based methods. In addition to testing at disease diagnosis, research indicates potential benefits of genetic testing both prior to disease onset and at remission. In this review, we discuss the transition from the era of cytogenetics and single-gene tests to the era of NGS panels and genome-wide sequencing-highlighting both the potential and drawbacks of these novel technologies.
JAMA network open, 2019
A molecular diagnostic method that incorporates information about the transcriptional status of all genes across multiple tissue types can strengthen confidence in cancer diagnosis.
To understand gene function, the cre/loxP conditional system is the most powerful available for temporal and spatial control of expression in mouse. However, the research community requires more cre recombinase expressing transgenic mouse strains (cre-drivers) that restrict expression to specific cell types. To address these problems, a high-throughput method for large-scale production that produces high-quality results is necessary. Further, endogenous promoters need to be chosen that drive cell type specific expression, or we need to further focus the expression by manipulating the promoter. Here we test the suitability of using knock-ins at the docking site 5' of for rapid development of numerous cre-driver strains focused on expression in adulthood, using an improved cre tamoxifen inducible allele (icre/ERT2), and testing a novel inducible-first, constitutive-ready allele (icre/f3/ERT2/f3). In addition, we test two types of promoters either to capture an endogenous expression pattern (MaxiPromoters), or to restrict expression further using minimal promoter element(s) designed for expression in restricted cell types (MiniPromoters). We provide new cre-driver mouse strains with applicability for brain and eye research. In addition, we demonstrate the feasibility and applicability of using the locus 5' of for the rapid generation of substantial numbers of cre-driver strains. We also provide a new inducible-first constitutive-ready allele to further speed cre-driver generation. Finally, all these strains are available to the research community through The Jackson Laboratory.
Transfusion medicine reviews, 2019
Promising efficacy results of chimeric antigen receptor (CAR) T-cell therapy have been tempered by safety considerations. Our objective was to comprehensively summarize the efficacy and safety of CAR-T cell therapy in patients with relapsed or refractory hematologic or solid malignancies. MEDLINE, Embase, and the Cochrane Register of Controlled Trials (inception - November 21, 2017). Interventional studies investigating CAR-T cell therapy in patients with malignancies were included. Our primary outcome of interest was complete response (defined as the absence of detectable cancer). Two independent reviewers extracted relevant data, assessed risk of bias, and graded the quality of evidence using established methods. A total of 42 hematological malignancy studies and 18 solid tumor studies met were included (913 participants). Of 486 evaluable hematologic patients, 54.4% [95% CI, 42.5%-65.9%] experienced complete response in 27 CD19 CAR-T cell therapy studies. Of 65 evaluable hematologic patients, 24.4% [95% CI, 9.4%-50.3%] experienced complete response in seven non-CD19 CAR-T cell therapy studies. Cytokine release syndrome was experienced by 55.3% [95% CI, 40.3%-69.4%] of patients and neurotoxicity 37.2% [95% CI, 28.6%-46.8%] of patients with hematologic malignancies. Of 86 evaluable solid tumor patients, 4.1% [95% CI, 1.6%-10.6%] experienced complete response in eight CAR-T cell therapy studies. Limitations include heterogeneity of study populations, as well as high risk of bias of included studies. There was a strong signal for efficacy of CAR-T cell therapy in patients with CD19+ hematologic malignancies and no overall signal in solid tumor trials published to date. These results will help inform patients, physicians, and other stakeholders of the benefits and risks associated with CAR-T cell therapy.
Although generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in more than 90% of cases in malaria-endemic regions, and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumor EBV status or geographic origin. Through an integrative analysis of whole-genome and transcriptome data, we show a striking genome-wide increase in aberrant somatic hypermutation in EBV-positive tumors, supporting a link between EBV and activation-induced cytidine deaminase (AICDA) activity. In addition to identifying novel candidate BL genes such as , , and , we demonstrate that EBV-positive tumors had significantly fewer driver mutations, especially among genes with roles in apoptosis. We also found immunoglobulin variable region genes that were disproportionally used to encode clonal B-cell receptors (BCRs) in the tumors. These include IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Our results suggest that tumor EBV status defines a specific BL phenotype irrespective of geographic origin, with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply rational use of DNA-damaging chemotherapy in some patients with BL and targeted agents such as the CDK4/6 inhibitor palbociclib in others, whereas the importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk, and Src family kinases among these patients.
The Journal of molecular diagnostics : JMD, 2019
Recurrent activating point mutations in KRAS are critical drivers in pancreatic cancer and have been attributed to resistance to anti-epidermal growth factor receptor therapy in colorectal cancer. Although KRAS genotyping provides limited clinical utility in the diagnosis and management of pancreatic cancer patients at present, inferences about the fractional abundance of KRAS mutations may inform on tumor purity in traditionally challenging clinical specimens and their potential use in precision medicine. KRAS genetic testing has indeed become an essential tool to guide treatment decisions in colorectal cancer, but an unmet need for methods standardization exists. Here, we present a unique droplet digital PCR method that enables the simultaneous detection and quantification of KRAS exon 2, 3, and 4 point mutations and copy number alterations. We have validated 13 mutations (G12S, G12R, G12D, G12A, G12V, G12C, G13D, G60V, Q61H, Q61L, A146V, A146T, and A146P) and focal KRAS amplifications by conducting this assay in a cohort of 100 DNA samples extracted from fresh frozen tumor biopsies, formaldehyde-fixed, paraffin-embedded tissue, and liquid biopsy specimens. Despite its modest lower limit of detection (approximately 1%), this assay will be a rapid cost-effective means to infer the purity of biopsy specimens carrying KRAS mutations and can be used in noninvasive serial monitoring of circulating tumor DNA to evaluate clinical response and/or detect early signs of relapse.
Journal of proteome research, 2019
Optimizing the quality of proteomics data collected from a mass spectrometer (MS) requires careful selection of acquisition parameters and proper assessment of instrument performance. Software tools capable of extracting a broad set of information from raw files, including meta, scan, quantification, and identification data, are needed to provide guidance for MS system management. In this work, direct extraction and utilization of these data is demonstrated using RawTools, a standalone tool for extracting meta and scan data directly from raw MS files generated on Thermo Orbitrap instruments. RawTools generates summarized and detailed plain text outputs after parsing individual raw files, including scan rates and durations, duty cycle characteristics, precursor and reporter ion quantification, and chromatography performance. RawTools also contains a diagnostic module that includes an optional "preview" database search for facilitating informed decision-making related to optimization of MS performance based on a variety of metrics. RawTools has been developed in C# and utilizes the Thermo RawFileReader library and thus can process raw MS files with high speed and high efficiency on all major operating systems (Windows, MacOS, Linux). To demonstrate the utility of RawTools, the extraction of meta and scan data from both individual and large collections of raw MS files was carried out to identify problematic characteristics of instrument performance. Taken together, the combined rich feature-set of RawTools with the capability for interrogation of MS and experiment performance makes this software a valuable tool for proteomics researchers.
The analysis of cell-free circulating tumor DNA (ctDNA) is potentially a less invasive, more dynamic assessment of cancer progression and treatment response than characterizing solid tumor biopsies. Standard isolation methods require separation of plasma by centrifugation, a time-consuming step that complicates automation. To address these limitations, we present an automatable magnetic bead-based ctDNA isolation method that eliminates centrifugation to purify ctDNA directly from peripheral blood (PB). To develop and test our method, ctDNA from cancer patients was purified from PB and plasma. We found that allelic fractions of somatic single-nucleotide variants from target gene capture libraries were comparable, indicating that the PB ctDNA purification method may be a suitable replacement for the plasma-based protocols currently in use.
In its third edition, the Vancouver Autophagy Symposium presented a platform for vibrant discussion on the differential roles of macroautophagy/autophagy in disease. This one-day symposium was held at the BC Cancer Research Centre in Vancouver, BC, bringing together experts in cell biology, protein biochemistry and medicinal chemistry across several different disease models and model organisms. The Vancouver Autophagy Symposium featured 2 keynote speakers that are well known for their seminal contributions to autophagy research, Dr. David Rubinsztein (Cambridge Institute for Medical Research) and Dr. Kay F. Macleod (University of Chicago). Key discussions included the context-dependent roles and mechanisms of dysregulation of autophagy in diseases and the corresponding need to consider context-dependent autophagy modulation strategies. Additional highlights included the differential roles of bulk autophagy versus selective autophagy, novel autophagy regulators, and emerging chemical tools to study autophagy inhibition. Interdisciplinary discussions focused on addressing questions such as which stage of disease to target, which type of autophagy to target and which component to target for autophagy modulation. : AD: Alzheimer disease; AMFR/Gp78: autocrine motility factor receptor; CCCP: carbonyl cyanide -chlorophenylhydrazone; CML: chronic myeloid leukemia; CVB3: coxsackievirus B3; DRPLA: dentatorubral-pallidoluysian atrophy; ER: endoplasmic reticulum; ERAD: ER-associated degradation; FA: focal adhesion; HCQ: hydroxychloroquine; HD: Huntingtin disease; HIF1A/Hif1α: hypoxia inducible factor 1 subunit alpha; HTT: huntingtin; IM: imatinib mesylate; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; NBR1: neighbour of BRCA1; OGA: O-GlcNAcase; PDAC: pancreatic ductal adenocarcinoma; PLEKHM1: pleckstrin homology and RUN domain containing M1; polyQ: poly-glutamine; ROS: reactive oxygen species; RP: retinitis pigmentosa; SNAP29: synaptosome associated protein 29; SPCA3: spinocerebellar ataxia type 3; TNBC: triple-negative breast cancer.