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Experimental Oncology

Linda Pilarski

Professor Emerita
Experimental Oncology
Department of Oncology
University of Alberta
Cross Cancer Institute
11560 University Avenue
Edmonton, Alberta T6G 1Z2
Tel: 780.432.8925
lpilarsk@ualberta.ca

Profile

Canada Research Chair in Biomedical Nanotechnology and
Professor, University of Alberta and Cross Cancer Institute, Winner of the 2009 ASTech Award for Outstanding Leadership in Alberta Technology
 
Linda Pilarski is Professor in the Department of Oncology and Tier I Canada Research Chair in Biomedical Nanotechnology She is a Senior Scientist of the Alberta Cancer Board.  She is the Scientific Director of the newly established AHFMR Interdisciplinary Team "Improving Health Care Access and Sustainability With Microfluidic Platforms for Short AHFMR Team Microfluidics".
 
Research in the Pilarski lab focuses on understanding cancer as it occurs in cancer patients themselves, and on translating her work to the clinic, for the benefit of patients. She works closely with Dr Andrew Belch, her long time research partner. Together they comprise a very effective basic/clinical team for translational research. They have been pioneers in developing approaches to analyze the biological processes underlying human cancer using fresh malignant cells from patients. Her lab is located at the Cross Cancer Institute on the University of Alberta campus. She specializes in cancer of the human immune system and novel technology focused on better diagnosis and monitoring tools for the clinic. Her research is funded by CIHR, AHFMR and the Alberta Cancer Research Institute (ACRI). She was an Eleanor Roosevelt International Cancer Research Fellow in 1987/88, an Associate Editor of the Journal of Immunology from 1995-2000, and was Vice-President and then President of the Canadian Society of Immunology from 1993-1997. She was selected as YWCA Woman of Distinction for Science and Technology in 1998, and has been the recipient of McCalla and Killam Professorships at the University of Alberta.
 
In 2002, she received the Cinader Award for research excellence and contributions to immunology, awarded by the Canadian Society for Immunology. She sits on the Scientific Advisory Board of the International Myeloma Foundation (Los Angeles, CA). She has published over 200 papers. She holds 6 issued and 8 pending patents. One patent family has been licensed to Orhrist Bio (Calgary) for comercialization. She is a member of the U Alberta Nano Council Executive and sits on the advisory board of the Microsystems Technology Research Institute (MSTRI) a funding body dedicated to closing the gap between research and commercialization.
 
 
Dr. Pilarski is the Editor of Daily/Haiga, an edited online journal featuring contemporary and traditional haiga (http://www.dailyhaiga.org/).
 
Research Interests 
 
Research in the Pilarski lab focuses on B lineage malignancies and on adapting clinically important cancer and biomedical testing to nanotechnology platforms for use in the clinic.
 
Biology and biomarkers for B lineage malignancies
 
Multiple myeloma (MM) is an incurable cancer that accounts for 1% of all cancers, 10% of all hematological malignancies and 19% of deaths from hematological malignancies. It kills over 15,000 North Americans each year, and the median survival is 3-4 years. MM is characterized by the presence of monoclonal immunoglobulin (Ig) in the blood, lytic bone lesions, and monoclonal plasma cells in the bone marrow. MM may arise through progressive acquisition of diverse genetic abnormalities, and is characterized by complex chromosomal abnormalities. The MM clone is definitively identified by expression of a unique IgH VDJ gene rearrangement, termed clonotypic. A variety of work leads to the idea that the generative compartment in MM includes B lineage cells in the bone marrow (BM), the blood, or both, at a stage of differentiation preceding that of plasma cells. Our work shows that MM is a hierarchy of B lineage cells that includes circulating drug-resistant malignant B cells with stem cell-like properties. The B cell compartments of the MM clone are clinically relevant: a) Xenografted MM B cells give rise to lytic bone disease, clonotypic progeny and are self-renewing in immunodeficient mice; b) Clonotypic MM cells with the phenotype of hematopoietic progenitors xenograft human MM to immunodeficient mice and c) aberrant HAS1 splicing within the circulating B cell compartment correlates with reduced survival.
 
Monoclonal gammopathy of undetermined significance (MGUS): Clinically, MGUS is a frequent, presumptively non-malignant condition, found in 3% of the total population over age 70. About 25% of MGUS eventually progress to overt MM. It is currently very difficult to clinically distinguish between early MM and advanced stage MGUS. MGUS patients have <10g/L of mIg and <10% plasma cells in their BM. MGUS patients have no clinical evidence of malignant disease. The consistent incidence of disease transformation from MGUS to MM dictates a pre-screening approach, and this in turn dictates the need for a cost-effective testing platform. MM is a disease continuum, with MGUS, followed by smoldering or indolent MM as the least, and plasma cell leukemia as the most aggressive. Approaches for improved stratification of MM prognostic groups and earlier detection of patients at risk for transformation to MM or for relapse are urgently needed and are a research priority for the Pilarski lab. . Identification of early changes would enable early monitoring in MGUS, potentially aimed at preventing transformation from MGUS to MM. The development of clinically feasible routine screening tests, e.g. as proposed here to develop tests for inherited and acquired HAS1 mutations, is essential for this approach. The implementation of rapid and inexpensive genetic testing on point of care “lab on a chip” devices that employ microfluidics, to detect important biomarkers that could be used for allow screening of large numbers of patients at multiple time points during their disease to identify predisposing and disease-promoting mutations. A similar strategy for risk assessment can be developed around indolent/responsive MM and aggressive/resistant MM.
 
Hyaluronan Synthase 1 (HAS1) and RHAMM in MM and WM Hyaluronan (HA) molecules are synthesized by HASs, integral transmembrane proteins with multiple enzymatic activities and a probable pore-like structure. We have recently identified a family of aberrant splice variants of HAS1 expressed in MM and WM, but absent from healthy cells. Two of these are intronic splice variants, a splicing event that characterized some cancers but is not seen in healthy tissues. Bioinformatic and functional analysis suggests that the aberrant HAS1 variants retain the ability to synthesize HA. Aberrantly spliced HAS1 variants appear to be essential for synthesis of HA by malignant B cells. More than a dozen human cancers are associated with abnormalities in alternative splicing, particularly when intronic sequences are abnormally retained in the transcript. One cause of aberrant splicing is genetic variation (mutation and/or SNPs) in or near splice donor an/or acceptor sites and cis-splicing elements, the consequences of which are exon skipping and/or intron retention in the transcript. Our working hypothesis is that aberrant HAS1 splice variants lead to mitotic abnormalities and genetic instability. A highly significant correlation was seen between poor survival and expression of the intronic splice variant HAS1Vb (p=0.002), with a strong trend towards clinical correlations with poor outcome for HAS1Va (p=0.05). The strong association between HAS1Vb and survival, taken together with the rare detection of HAS1Vb in the bone marrow, suggests that HAS1Vb may be preferentially upregulated in the circulating compartment of malignant cells.
 
HAS1 and Genetic Instability: HAS1 gene expression may promote genetic instability. Our work provides evidence for genetic instability in the malignant MM B cells that overexpress HAS1 and its variants. Regardless of mechanism, the significant correlation between poor survival and the expression of HAS1 and its splice variants by circulating B cells suggests a key role for expression of HASs by “stem cell” compartments of the MM clone that circulate in the blood and mediate malignant spread to distant bone marrow sites. A variety of evidence leads to the speculation that aberrant HAS1 indirectly regulates mitotic events, thus contributing to the generation of increasingly aggressive clones in MM and WM.
For many cancers, inherited polymorphisms appear to be predisposing biomarkers that predict risk of developing cancer. As detailed below, inherited and somatic genetic variations (GVs) have been reported to influence gene splicing and oncogenesis, and aberrant hypermutation characterizes B cell cancers. Broadly speaking, there are two approaches to identify genetic determinants of cancer. The most common is to perform e.g. genome wide screening (GWS), to identify polymorphisms that may predict risk, but no functional or mechanistic analysis is known for the risk predictors identified. Further confirmation of GWS risk predictors will require painstaking analysis of identified genes to confirm their importance and screening of very large cohorts to ensure that the identified genes are clinically relevant. An alternate approach is to determine the genetic basis for abnormal gene products having a clinically significant influence on outcome in a given cancer(s), as done here. We have shown that the hyaluronan synthase 1 (HAS1) gene is aberrantly spliced in multiple myeloma (MM), that HAS1 aberrant splicing predicts significantly reduced survival in MM, and that aberrant HAS1 splice variants are transforming. To determine the genetic basic for aberrant HAS1 splicing, we sequenced the HAS1 gene in multiple cell types from multiple patients and controls. We have identified a large series of genetic changes in MM and WM patients, including inherited single nucleotide polymorphisms (SNPs) in HAS1, insertions/deletions and substitutions (collectively termed GVs). We detected acquired GVs that originated in hematopoietic progenitor cells and were passed to their T, B and plasma cell (PC) progeny, and numerous tumor specific HAS1 GVs in malignant B and PC. These were absent from B-CLL or healthy donors. Some novel GVs were independently acquired in multiple unrelated patients, termed “recurrent”. We have shown that HAS1 GVs direct the aberrant splicing of HAS1 that correlates with poor outcome, confirming the clinical relevance of aberrant HAS1 splicing. Our work shows that the impact of inherited and acquired GVs on aberrant splicing is manifested in the context of accompanying tumor-specific HAS1 GVs. In combination, inherited and acquired HAS1 GVs give rise to clinically significant aberrant splicing of HAS1. Altogether, HAS1 hypermutation profiling may provide a powerful risk stratification strategy for MM.
 
Research Priorities for the Translational Cancer Biology Projects in the Pilarski Lab:
  1. Analysis of chromosomal abnormalities in the heterogeneous compartments that comprise the myeloma clone.
  2. Analysis and characterization of  compartments of the MM clone that escape therapy and mediate relapse.
  3. Characterization of the MM progenitor compartments.
  4. Evaluate inherited polymorphisms (SNPs) that may  predispose to  MM and WM.
  5. Evaluate the significance of acquired somatic mutations in HAS1 that accumulate in splicing sites of tumor cells in MM and WM.
  6. Evaluate the value of HAS1 mutation patterns for risk assessment in MGUS, MM and WM.
Microfluidic Devices for Cancer, Infectious Disease and Pharmacogenetics
 
Microfluidic devices hold promise for widespread and cost-effective delivery of otherwise difficult and expensive genetic testing for diseases such as cancer, for infectious agents and for detection of genetic polymorphisms that impact on disease susceptibility, drug metabolism and response to therapy. PCR on a microfluidic chip effectively amplifies gene products known to be important in disease, and PCR products are readily detectable when analyzed using capillary electrophoresis (CE) on chip. Many cancers are characterized by unique molecular signatures; both genomic DNA and mRNA transcripts encoding such signatures can be amplified on chip from groups of cells or from individual cells and detected on chip with a sensitivity equal to that of conventional technology. In kidney transplantation, viral nephropathy can cause graft dysfunction and failure. Amplification of viral DNA from raw urine by on-chip PCR and CE, provides efficient detection of as few as 2-4 viral templates. This confirms the utility of microfluidic devices for detecting and containing spread of infectious agents such as e.g. influenza. Pharmacogenetics for avoiding adverse drug events is currently too expensive for routine use. Single nucleotide polymorphisms can be detected by on chip PCR, restriction digestion and analysis of restriction. These devices enable identification of high risk genetic profiles, of emerging infections threats as soon as they arise, and by identification of key genetic polymorphisms in at risk populations, could facilitate avoidance of adverse drug events that are a significant cost to health care systems. When fully automated, these applications will be feasible in any health care centre, in urban or rural locations, without a need for highly skilled operators. The development of cost-effective miniaturized diagnostic/monitoring tools is likely to have broad application for maintaining public health and safety as well as for more effective treatment of disease. These applications offer strong commercialization possibilities. With the availability of a commercialized product, microfluidic based testing is likely to have a profound impact on the way health care is delivered by enabling more informed medical decision making for personalized therapies.

 

Publications

Adamia S, Maxwell CA, Pilarski LM, Hyaluronan and Hyaluronan Synthases: Potential Therapeutic Targets in Cancer. Current Drug Targets: Cardiovascular and Hematological Disorders 5:3-14, 2005.
 
Maxwell CA, Keats JJ, Belch AR, Pilarski LM, Reiman T. Receptor for hyaluronan mediated motility (RHAMM) correlates with centrosome abnormalities in multiple myeloma and maintains mitotic integrity. Cancer Research. 65:850-860, 2005.
 
Keats JJ, Maxwell CA, Taylor BJ, Hendzel MJ, Chesi M, Bergsagel PL, Larratt L, Mant MJ, Reiman T, Belch AR, Pilarski LM. Overexpression of transcripts originating from the MMSET Locus characterizes all t(4;14)(p16;q32) positive multiple myeloma patients.,
 
Adamia S, Reiman T, Crainie, M, Mant MJ, Belch AR, Pilarski LM. Intronic splicing of hyaluronan synthase 1 (HAS1): a biologically relevant indicator of poor outcome in multiple myeloma. BLOOD 105:4836-4844
 
Pilarski LM, Lauzon J, Strachan E, Adamia S, Atrazhev A, Belch AR, Backhouse CJ. Sensitive Detection Using Microfluidics Technology of Single Cell PCR Products from High and Low Abundance IgH VDJ Templates in Multiple Myeloma. J. Immunological Methods. 305:94-105, 2005.
 
Adamia S, Steven P. Treon, Tony Reiman, Olivier Tournilhac, Carrie McQuarrie, Michael J. Mant, Andrew R. Belch and Linda M. Pilarski. Single nucleotide polymorphism of hyaluronan synthase 1 gene and aberrant splicing in Waldenstroms macroglobulinemia. Clinical Lymphoma. 5:253-256, 2005. 
 
Taylor BJ, Kriangkum J, Strachan E, Wizniak J, Pilarski LM. Identification of clonotypic IgH VDJ sequences in multiple myeloma, R Brown and PJ Ho, Editors. Humana Press. Totowa, NJ. Methods Mol Med. 2005;113:121-44.
 
Kriangkum, J, Taylor B.J, Strachan, E., Mant, M.J. Reiman, T. Belch A.R., Pilarski L.M. Impaired class switch recombination (CSR) in Waldenstrom macroglobulinemia (WM) despite apparently normal CSR machinery. BLOOD, Blood 2006 107: 2920-2927 
 
Kaigala GV, Huskins RJ, Preiksaitis J, Pang XL, Pilarski LM, Backhouse CJ. Automated screening using microfluidic chip-based PCR and product detection to assess risk of BK virus-associated nephropathy in renal transplant recipients. Electrophoresis, 27:3753-63.  2006. (Online publication on September 6,2006).
 
Keats JJ, T Reiman T, Belch AR, Pilarski LM. Ten Years and Counting: So what do we know about t(4;14)(p16q;32) Multiple Myeloma? Leukemia and Lymphoma. 47:2289-2300. 2006
 
Kriangkum J, Taylor BJ, Treon SP, Mant MJ, Reiman T, Belch AR, Pilarski LM. Molecular characterization of Waldenstrom’s macroglobulinemia reveals frequent occurrence of two B cell clones having distinct IgH VDJ sequences. Clinical Cancer Research. 13:2006-2013, 2007.
 
VanDijken J, Kaigala G, Lauzon J, Astrazhev A, Adamia S, Taylor BJ, Reiman T, Belch AR, Backhouse CJ, Pilarski LM. Microfluidic Chips for Detecting the t(4;14) Translocation and Monitoring Disease During Treatment Using RT-PCR Analysis of IgH-MMSET Hybrid Transcripts. Journal Molecular Diagnostics. 9:358-367. 2007 
 
Chowdhury,J., Kaigala,G.V., Pushpakom,S., Lauzon, J. , Makin,A., Atrazhev, A., Stickel,A., Newman, WG, Backhouse,C.J. and Pilarski,L.M. Microfluidic platforms for point of care SNP genotyping of the thiopurine s-methlytransferase gene to evaluate risk of adverse drug reactions,  J Mol Diagn.2007; 9: 521-529.
 
Sieben V, Debes-Marun C, Pilarski PM, Kaigala GV, Pilarski LM, Backhouse CJ. FISH and chips: chromosomal analysis on microfluidic platforms. IET Nanobiotechnology, 1:27-35, 2007. 
 
Kaigala GV, Hoang VG, Lauzon J, Pilarski LM, Backhouse CJ. Fully portable and inexpensive microchip-based genetic analysis platform for integrated RT-PCR and capillary elecrophoresis. The Analyst. 133:331-338, 2008.
 
Multiple myeloma includes CD20+ B and plasma cells that persist in patients treated with rituximab. LM Pilarski, E Baigorri, MJ Mant, PM Pilarski, P Adamson, H Zola, AR Belch. Clinical Medicine: Oncology, 2275-287. 2008. 
 
Taylor BJ, Kriangkum J, Pittman JA, Mant MJ, Reiman T, Belch AR, Pilarski, LM. Analysis Of Clonotypic Switch Junctions Reveals Multiple Myeloma Originates From A Single Class Switch Event With Ongoing Mutation In The Isotype Switched Progeny. Blood 112:1894-1903, 2008.
 
Kirshner J, Thulien KJ, Martin LD, Debes Marun C, Reiman T, Belch AR, Pilarski LM. A Unique 3-D Model for Evaluating the Impact of Therapy on Multiple Myeloma. Blood. In press, 2008. Blood First Edition- June 5th online. Blood 2008 112: p. 2935-2945. 
 
Adamia S, Reichert AA, Kuppusamy H, Kriangkum J, Ghosh A, Hodges JJ, Pilarski PM, Treon SP, Mant MJ, Reiman T, Belch AR, Pilarski LM. Inherited and acquired variations in hyaluronan synthase 1 gene may contribute to disease progression in multiple myeloma and Waldenstrom’s macroglobulinemia. Blood 112: 511-5121, 2008.
 
Ghosh A, Kuppusamy H, Pilarski LM. Aberrant splice variants of hyaluronan synthase 1 (HAS1) multimerize with and modulate normally spliced HAS1 protein: A potential mechanism promoting human cancer. J Biol Chem. 284: 18840-18850, 2009.