Leukaemias and other cancers possess a rare population of cells capable of the limitless self-renewal necessary for cancer initiation and maintenance. Eradication of these cancer stem cells is probably a critical part of any successful anti-cancer therapy, and may explain why conventional cancer therapies are often effective in reducing tumour burden, but are only rarely curative. Given that both normal and cancer stem cells are capable of self-renewal, the extent to which cancer stem cells resemble normal tissue stem cells is a critical issue if targeted therapies are to be developed. However, it remains unclear whether cancer stem cells must be phenotypically similar to normal tissue stem cells or whether they can retain the identity of committed progenitors. Here we show that leukaemia stem cells (LSC) can maintain the global identity of the progenitor from which they arose while activating a limited stem-cell- or self-renewal-associated programme. We isolated LSC from leukaemias initiated in committed granulocyte macrophage progenitors through introduction of the MLL-AF9 fusion protein encoded by the t(9;11)(p22;q23). The LSC were capable of transferring leukaemia to secondary recipient mice when only four cells were transferred, and possessed an immunophenotype and global gene expression profile very similar to that of normal granulocyte macrophage progenitors. However, a subset of genes highly expressed in normal haematopoietic stem cells was re-activated in LSC. LSC can thus be generated from committed progenitors without widespread reprogramming of gene expression, and a leukaemia self-renewal-associated signature is activated in the process. Our findings define progression from normal progenitor to cancer stem cell, and suggest that targeting a self-renewal programme expressed in an abnormal context may be possible.
Drug resistance remains a major obstacle to successful cancer treatment. A database of drug-associated gene expression profiles was screened for molecules whose profile overlapped with a gene expression signature of glucocorticoid (GC) sensitivity/resistance in acute lymphoblastic leukemia (ALL) cells. The screen indicated that the mTOR inhibitor rapamycin profile matched the signature of GC sensitivity. We tested the hypothesis that rapamycin would induce GC sensitivity in lymphoid malignancy cells and found that it sensitized to GC-induced apoptosis via modulation of antiapoptotic MCL1. These data indicate that MCL1 is an important regulator of GC-induced apoptosis and that the combination of rapamycin and glucocorticoids has potential utility in lymphoid malignancies. Furthermore, this approach represents a strategy for identification of promising combination therapies for cancer.
The lack of established standards to describe and annotate biological assays and screening outcomes in the domain of drug and chemical probe discovery is a severe limitation to utilize public and proprietary drug screening data to their maximum potential. We have created the BioAssay Ontology (BAO) project (http://bioassayontology.org) to develop common reference metadata terms and definitions required for describing relevant information of low-and high-throughput drug and probe screening assays and results. The main objectives of BAO are to enable effective integration, aggregation, retrieval, and analyses of drug screening data. Since we first released BAO on the BioPortal in 2010 we have considerably expanded and enhanced BAO and we have applied the ontology in several internal and external collaborative projects, for example the BioAssay Research Database (BARD). We describe the evolution of BAO with a design that enables modeling complex assays including profile and panel assays such as those in the Library of Integrated Network-based Cellular Signatures (LINCS). One of the critical questions in evolving BAO is the following: how can we provide a way to efficiently reuse and share among various research projects specific parts of our ontologies without violating the integrity of the ontology and without creating redundancies. This paper provides a comprehensive answer to this question with a description of a methodology for ontology modularization using a layered architecture. Our modularization approach defines several distinct BAO components and separates internal from external modules and domain-level from structural components. This approach facilitates the generation/extraction of derived ontologies (or perspectives) that can suit particular use cases or software applications. We describe the evolution of BAO related to its formal structures, engineering approaches, and content to enable modeling of complex assays and integration with other ontologies and datasets.
Gene expression profiling has identified several potentially useful gene signatures for predicting outcome or for selecting targeted therapy. However, these signatures have been developed in fresh or frozen tissue, and there is a need to apply them to routinely processed samples. Here, we demonstrate the feasibility of a potentially high-throughput methodology combining automated in situ hybridization with quantum dot-labeled oligonucleotide probes followed by spectral imaging for the detection and subsequent deconvolution of multiple signals. This method is semiautomated and quantitative and can be applied to formalin-fixed, paraffin-embedded tissues. We have combined dual in situ hybridization with immunohistochemistry, enabling simultaneous measurement of gene expression and cell lineage determination. The technique achieves levels of sensitivity and specificity sufficient for the potential application of known expression signatures to biopsy specimens in a semiquantitative way, and the semiautomated nature of the method enables application to highthroughput studies.
In this paper we present some lessons learned from building viz s la, the keyword search and topic classification system used on the largest Hungarian portal, [ origo .hu]. Based on a simple statistical language model, and the large-scale supporting evidence from vizsla, we argue that in topic classification only positive evidence matters.
Quantum dots (QDs) are fluorescent semiconductor nanocrystals (2–10-nm core diameter) possessing the unique properties of extremely high fluorescence efficiency, lack of photobleaching and long fluorescence lifetime, making them an ideal tool for bioimaging. We have developed a novel technique for in situ hybridisation (ISH) using biotinylated oligonucleotides conjugated to streptavidin coated QD, and used them in this study to label bone marrow trephine samples. 50-mer long oligonucleotide probes were conjugated to QDs prior to ISH and conjugation efficiency was demonstrated by gel electrophopresis. ISH conditions and molar ratio of QDs to probe were optimised using a polyT probe. Images were captured using a CRI Nuance spectral imaging system and signal intensity was semi-quantitated using IPLab software. Specific oligonucleotide hybridisation was demonstrated using a probe for myeloperoxidase (MPO) in 40 bone marrow sections infiltrated by acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML) as well as reactive bone marrow. In each case hybridisation signal was consistent with the distribution of MPO by standard immunohistochemistry - MPO was strongly expressed by myeloid blasts and absent in lymphoid blasts; in CML, most, but not all, cells were positive for MPO, in comparison to many fewer positive cells in reactive marrow. Duplex ISH was demonstrated using a probe for bcl-2 together with MPO in 5 bone marrow sections infiltrated by follicular lymphoma (FL). Strong hybridisation signal for bcl-2 was detected in all cells of the paratrabecular aggregates of FL but showed only scattered positivity in the remainder of the bone marrow. Conversely, MPO was absent in the paratrabecular aggregates and present in the myeloid cells in the remainder of the marrow. This pattern was present in both single and duplex ISH for MPO and bcl-2 in the marrow infiltrated by FL. Duplex ISH was performed both by sequential hybridisation with bcl-2 followed by MPO, and simultaneously with a mix of bcl-2 and MPO probes. As negative controls, scrambled oligonucleotide probes for the corresponding genes were used in each case and did not show hybridisation. In summary, we have developed a generic method for QD labelling and semi-quantitative detection of oligonucleotide ISH in routinely processed clinical tissue samples. Although, in this study we primarily used bone marrow trephine samples, this technique can be applied to any tissue. It has the potential to facilitate transfer of microarray-identified gene signatures to clinical research and diagnostics, whilst the ability of spectral imaging to resolve multiple signals offers the possibility of multiplexed probe detection.
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