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Single-Cell Sequencing | August 20-21, 2014

CSHL, UCLA & Einstein to Lead Roundtable Discussions on Single-Cell Sequencing

Interactive discussions on three of the key questions researchers are facing when considering single-cell analysis will be held on the second day of the Single-Cell Sequencing Conference at Next Generation Dx Summit, taking place August 20-21, 2014 in Washington, DC. For full program details and to register, please visit NextGenerationDx.com/Single-Cell-Sequencing.

Making Single-Cell Analysis Cost Effective for Clinical Use
Moderator: James Hicks, Ph.D., Research Professor, Cancer Genomics, Cold Spring Harbor Laboratory

  • Methods for capture: What are the tradeoffs?
  • Combining RNA, DNA and protein analysis
  • What genomic assays are most informative?
  • Can assays be certifiable?

Finding a Needle in a Haystack: Towards Diagnosing Rare Soft Tissue Cancer Stem Cells (CSCs)
Moderator: Michael Masterman-Smith, Ph.D., Entrepreneurial Scientist, UCLA California NanoSystems Institute

  • Rethinking companion diagnostics for cancer to incorporate analysis of CSCs
  • Current direct methodologies of CSC detection/isolation
  • Current proxy methodologies of CSC detection/isolation
  • The hope and promise of single-cell assay tools and technologies

Why Single-Cell Sequencing?
Moderator: Jan Vijg, Ph.D., Professor and Chairman, Genetics, Albert Einstein College of Medicine
Sample limitations, e.g., prenatal diagnostics and CTCs

  • Sample limitations, e.g., prenatal diagnostics and CTCs
  • To study cell-to-cell variation, e.g., in tumors as well as normal tissues
  • To overcome technological constraints, e.g., detecting somatic mutations
  • Cell-to-cell fluctuations in gene expression can easily impair function, yet can be undetectable by measuring averages
  • How many different cell types are there?
View Brochure    |   Register (Advance Registration Ends July 18)  |   NextGenerationDx.com/Single-Cell-Sequencing


About the Conference

Sequencing data from bulk DNA or RNA from multiple cells provide global information on average states of cell populations. But with whole-genome amplification and NGS, researchers can detect variation in individual cancer cells and dissect tumor evolution. Such cancer genome sequencing will improve oncology by detecting rare tumor cells early, measuring intra-/intertumor heterogeneity, guiding chemotherapy and controlling drug resistance. The Single-Cell Sequencing conference explores the latest strategies, data analyses and clinical considerations that influence and aid cancer diagnosis, prognosis and prediction and will lead to individualized cancer therapy.

Sessions include presentations spanning the opportunities of clinical single-cell analysis from:

  • Sunney Xie, Ph.D., Mallinckrodt Professor. Chemistry and Chemical Biology, Harvard University
  • Maximilian Diehn, M.D., Ph.D., Assistant Professor, Radiation Oncology, Stanford Cancer Institute, Institute for Stem Cell Biology & Regenerative Medicine, Stanford University
  • Denis Smirnov, Associate Scientific Director, US Biomarker Oncology, Janssen R&D US
  • James Hicks, Ph.D., Research Professor, Cancer Genomics, Cold Spring Harbor Laboratory
  • Jan Vijg, Ph.D., Professor and Chairman, Genetics, Albert Einstein College of Medicine
  • John F. Zhong, Ph.D., Associate Professor, Pathology, University of Southern California School of Medicine
  • Mark Hills, Ph.D., Research Scientist, Peter M. Lansdorp Laboratory, BC Cancer Research Centre
  • Michael Masterman-Smith, Ph.D., Entrepreneurial Scientist, UCLA California NanoSystems Institute
  • Parveen Kumar, Research Scientist, Thierry Voet Laboratory, Human Genetics, University of Leuven
  • Peter Nemes, Ph.D., Assistant Professor, Chemistry, George Washington University
  • Theresa Zhang, Ph.D., Vice President, Research Services, Personal Genome Diagnostics
  • Yong Wang, Ph.D., Senior Postdoctoral Fellow, Nicholas E. Navin Laboratory, Genetics, Bioinformatics, MD Anderson Cancer Center
  • Zivana Tezak, Ph.D., Associate Director, Science and Technology, Personalized Medicine, Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD), Center for Devices and Radiological Health (CDRH), FDA

 


Recommended Pre-Conference Courses

NGS Data Analysis – Determining Clinical Utility of Genome Variants
Monday, August 18 | 9:00am – 12:00pm
This course will explore the strategies of genomic data analysis and interpretation, an emergent discipline that seeks to deliver better answers from NGS data so that patients and their physicians can determine informed healthcare decisions. View Details

NGS as a Diagnostics Platform
Monday, August 18 | 2:00pm – 5:00pm
The focus of this short course will be on understanding the use of NGS in clinical diagnosis, practical implementation of NGS in clinical laboratories and analysis of large data sets by using bioinformatics tools to parse and interpret data in relation to the clinical phenotype. The concluding presentation will be dedicated to quality and standardization of NGS assays. View Details

Register   |   View Agenda   | NextGenerationDx.com

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Next Generation Dx Summit 2014
Cambridge Healthtech Institute, 250 First Avenue, Suite 300, Needham, MA 02494
www.healthtech.com

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Cancer Metastasis

Author: Tilda Barliya PhD

Metastasis, a complex process that involves the spread of tumor cells, accounts for more than 90%of cancer-related mortality (1,2). A metastatic tumor cell has a treacherous journey to go through:

  • local invasion and intravasation
  • survival in the circulation
  • homing and extravasation into the parenchyma of distant organs
  • adaptation to the new environment
  • outgrowth of secondary lesions

Although tumor cells that are shed from the primary tumor disseminate throughout the body, they tend to colonize select organs, with characteristically different periods of latency and efficiency depending on tumor type or subtype (2).

Steven Paget’s century-old ‘seed and soil’ hypothesis (2, 9) likened tumor cells to ‘seeds’ that are systemically distributed, but that only inhabit particular environments, or ‘soils’, which are supportive to their sustained growth. Understanding the molecular complexity of this process is difficult and we’ll try to unravel some of the pathogenesis and cellular basis that support the metastatic process.

Progression models:

There are two major tumor progression models (2) :

  • Linear –  primary tumour cells undergo successive rounds of mutation and selection35, giving rise to a biologically heterogeneous cellular population in which a subset of malignant clones have accumulated genetic alterations, necessary for metastasis.
  • Parallel –  tumor cells may disseminate very early in malignant progression, colonize multiple secondary sites at different times and ultimately accumulate genetic changes independently from those incurred by the primary tumor.

While both theories are possible, the linear model is validated by both clinical evidence and animal models, the parallel model is mainly based on animal models and still under investigation for clinical clues.

Meera Saxena. Molecular Oncology
Volume 7, Issue 2 , Pages 283-296, April 2013

Drivers of metastasis

During the past few years several methods and studies have been used to find and correlate between a specific gene and it’s homing target.

These genes, which were found using next-generation sequencing and their equivalents, were also validated their actual functional consequences.

Figure 1 (Meera Saxena et al) represent some of the genes that were associated with organ-specific translocation (additional genes were recently identified and included in table 1 – Sethi N et all). Herein, we generally show the gene to organ-specific homing, yet we will not discuss each and every one of them.  An example of specific gene to organ will be further discussed in detail in follow up article.

Signalling pathways in cancer metastasis have been extensively studied at the level of individual proteins or as a linear cascade of proteins but they have been less frequently evaluated through a network approach (2). Understanding the different variables in the gene-metastasis network may be crucial for drug development.

For example;  the drug–gene–phenotype Connectivity Map approach was successfully used to identify the mTOR inhibitor rapamycin as an effective agent for overcoming dexamethasone resistance in acute lymphoblastic leukaemia (2, 4).

Microenvironment

“Non-neoplastic stromal cells have a function in the development of tumor metastasis. Stromal cells as important regulators of metastasis through their ability to influence cancer cell functions such as chemotaxis and invasion, as well as microenvironment properties. It should not come as a surprise that tumor angiogenesis was among the initial findings that supported a role for stromal cells in cancer metastasis; the poor vascular integrity of newly synthesized blood vessels within the tumour allows for the escape of malignant cells with the potential of distant spread” (2). Such cells include:

  • Tumour-associated macrophages,
  • Leukocytes and other immune cells,
  • Mesenchymal cells that reside in breast tissue
  • Mesenchymal cells and neuroendocrine cells

Although some of the molecular pathway was discovered, the molecular components that facilitate communication between tumour cells and individual stromal cells of the primary tumor have yet to be fully understood.

Circulating Tumor cells (CTC)

“Essential to cancer metastasis is the ability of primary tumor cells to enter the vasculature and to use these fluid ‘highways’ as a means to reach distant organs”. Tight vascular wall barriers, unfavorable conditions for survival in distant organs, and a rate-limiting acquisition of organ colonization functions are just some of the impediments to the formation of distant metastasis (2,5,6 ).

Despite their clear prognostic importance, the diagnostic value of CTCs is largely unknown and fairly unexplored. Research challenges both in detection and interpretation render their ability to  be clinically accepted. Additional research is needed to fully explore CTCs’ potential in to predict clinical response to therapy would also help to guide disease management.

Colonization

The colonization and outgrowth of tumor cells in a secondary organ is often considered the rate-limiting, as well as the most poorly delineated, step in the metastatic cascade. Understanding the functional involvement of the tumor stromal cells of the secondary site may be crucial to understanding their ability to colonize.

The pre-metastatic niche model shows that, preceding the arrival of  disseminated tumour cells (DTCs), bone marrow-derived haematopoietic stem cells are mobilized by tumour-derived factors and are recruited to the secondary site where they negotiate a more hospitable microenvironment to foster the survival and expansion of metastatic lesions. Inflammatory cytokines have emerged as crucial mediators of the pre-metastatic niche and self-seeding and include IL-6, SRC and NF-kB.

After surviving the adjustment to the secondary site, tumor cells must sustain their growth to develop overt metastases. Developmental pathways have emerged as important players in tumor progression and metastasis. These include: transforming growth factor-β (TGFβ), bone morphogenetic protein (BMP), WNT and Hedgehog.  These genes will trigger additional genes that will affect downstream steps of the colonization process.

Clinical Aspect

“As most metastatic cancers are inoperable, systemic treatments using chemotherapeutic or targeted therapy is often the only option to slow tumor growth or to relieve metastasis-associated morbidity”.  Genes and pathways that have crucial roles in primary tumour growth and metastasis are ideal targets for therapeutic inventions. One example is the oncogenic BRAF:  potent inhibitors of mutant BRAF, had initial clinical results which suggest dramatic efficacy in the treatment of metastatic malignant melanoma.  It is important to keep in mind that many cancers develop resistance to BRAF inhibitor and require used of next-generation drugs. More so,  the mechanism of resistance will be discussed elsewhere.

A sound framework of normal homeostatic mechanisms can improve our ability to understand and target tumor–stromal interactions in metastasis.

Summary:

“Despite recognizing the devastating consequences of metastasis, we are not yet able to effectively treat cancer that has spread to vital organs” .  Despite our increasing knowledge about metastatic colonization, we still hold little understanding of how metastatic tumour cells behave as solitary disseminated entities. Understanding the genomics of metastatic cancer cells and the complexity of the metastasis process will enable us to develop a better target-therapeutic drugs.

 

References:

1. Naure Review: Cancer: focus on metastasis. http://www.nature.com/nrc/focus/metastasis/index.html

2. Nilay Sethi and Yibin Kang. Unravelling the complexity of metastasis — molecular understanding and targeted therapies. Nature Reviews Cancer 2011; 11:732- 748. http://www.nature.com/nrc/journal/v11/n10/abs/nrc3125.html

3. Meera Saxena and Gerhard Christophor. Rebuilding cancer metastasis in the mouse. Molecular Oncology 2013, 7(2):283-296. http://www.moloncol.org/article/S1574-7891(13)00033-1/abstract

4. Lamb, J. et al. The Connectivity Map: using geneexpression signatures to connect small molecules, genes, and disease. Science 2006 313, 1929–1935. http://www.sciencemag.org/content/313/5795/1929.short

5. Chiang AC and Massagué J. Molecular basis of metastasis. N Engl J Med. 2008 Dec 25;359(26):2814 23 ;http://www.ncbi.nlm.nih.gov/pubmed/19109576

6. By: Ritu Saxena PhD. In focus: Circulating Tumor Cells. https://pharmaceuticalintelligence.com/2013/06/24/in-focus-circulating-tumor-cells/

7.   Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 2002, 417, 949–954. http://www.nature.com/nature/journal/v417/n6892/full/nature00766.html

8. Arozarena, I. et al. Oncogenic BRAF induces melanoma cell invasion by downregulating the cGMP specific phosphodiesterase PDE5A. Cancer Cell 19, 45–57 (2011). http://www.ncbi.nlm.nih.gov/pubmed/21215707

9. Isaiah J. Fidler. The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited. Nature Review Cancer. 2003 June. 3(6):453-8. http://www.ncbi.nlm.nih.gov/pubmed/12778135

10. Christoph A. Klein. Parallel progression of primary tumours and metastases.   Nat Rev Cancer. 2009 Apr;9(4):302-12  http://www.ncbi.nlm.nih.gov/pubmed/19308069 http://prometheus.fmrp.usp.br/biocelmolcancer/Klein.pdf

 

Other related articles published on this Open Access Scientific Journal, include the following:

I. By: Ritu Saxena PhD. In focus: Circulating Tumor Cells. https://pharmaceuticalintelligence.com/2013/06/24/in-focus-circulating-tumor-cells/

II. By: Ritu Saxena PhD. Scientists use natural agents for prostate cancer bone metastasis treatment. https://pharmaceuticalintelligence.com/2012/09/17/natural-agents-for-prostate-cancer-bone-metastasis-treatment/

III. By: Prabodh Kandala, PhD. All Cancer Cells Are Not Created Equal: Some Cell Types Control Continued Tumor Growth, Others Prepare the Way for Metastasis. https://pharmaceuticalintelligence.com/2012/05/17/all-cancer-cells-are-not-created-equal-some-cell-types-control-continued-tumor-growth-others-prepare-the-way-for-metastasis/

IV. By: Aviva Lev-Ari PhD RN. MIT Scientists Identified Gene that Controls Aggressiveness in Breast Cancer Cells. https://pharmaceuticalintelligence.com/2013/07/03/mit-scientists-identified-gene-that-controls-aggressiveness-in-breast-cancer-cells/

V. By: Demet Sag PhD CRA, GCP.  The Magic of the Pandora’s Box : Epigenetics and Stemness with Long non-coding RNAs (lincRNA). https://pharmaceuticalintelligence.com/2013/06/30/the-magic-of-the-pandoras-box-epigenetics-and-stemmness-with-long-non-coding-rnas-lincrna/

 

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