Affiliation : Professor of Oncology and Director at Department of Flow & Image Cytometry, Roswell Park Comprehensive Cancer Center, USA
Title of the Talk/Lab :Proliferation by Many Other Names: Monitoring Cell Cycle Progression and Cell Division by Flow Cytometry AND Flow Cytometric Detection, Diagnosis, and Monitoring of Multiple Myeloma
Paul Wallace, Professor Oncology and Director Department of Flow & Image Cytometry at Roswell Park Comprehensive Cancer Center, is recognized for his expertise in clinical flow cytometry with a strong background in immunology and current research interests in myeloid cell biology and measuring minimal residual disease by high dimensional flow cytometry. He is a former President of the International Society for the Advancement of Cytometry (ISAC), a past Councilor of the International Clinical Cytometry Society (ICCS) and recipient of their Wallace H. Coulter award for lifetime achievement in clinical cytometry. Flow and Image Cytometry at Roswell Park offers a strong combination of clinical and research missions and under Dr. Wallace’s direction actively works to build translational synergies between them. The clinical laboratory is focused on the diagnosis and monitoring of patients with leukemia and lymphoma with a recent emphasis on minimal residual disease. Before joining Roswell Park, Dr. Wallace was an Assistant Professor of Immunology at Dartmouth Medical School, Lebanon, NH (1993-2003); a cofounder of Zynaxis Cell Science, Inc., Malvern PA (1988-1991) the company that developed the PKH tracking dyes, and supervisor of Microbiology, Immunology, Serology, and Flow Cytometry at SmithKline Clinical Laboratories. He obtained his PhD from the Medical College of Pennsylvania in 1993 and his Masters from Idaho State University in 1979.
Lecture and Lab: Proliferation by Many Other Names: Monitoring Cell Cycle Progression and Cell Division by Flow Cytometry: Cytometric definitions of “proliferation” vary, but the study of normal growth processes and how they go awry in tumors has been of interest to cytometrists since the inception of the field. Measurements of cell proliferation by flow cytometry can be broadly divided into two different categories – methods that measure aspects of DNA content or cell cycle and methods that track cell division by dye-dilution. Cell cycle analysis is a very common flow cytometric application, but its accurate measurement can test the limits of both technique and the flow cytometer. By using a DNA-specific stain, one can determine a DNA profile and thus determine the cell’s ploidy and percentage of cells in G0/G1, S, and G2/M. This information can be used to, for example, to monitor the aggressiveness of a tumor or the effect of an anticancer treatment. Measurement of the S-phase fraction based on DNA content tells us what proportion of cells in a sample are preparing for cell division at a given point in time. It does not, however, allow us to say how many divisions a given cell may have undergone in response to a stimulus, how much a particular cell subset may have expanded during that response or what fraction of a starting cell population went on to divide during the response. Flow cytometry can, however, be used to monitor the extent of cell division by: 1) staining cells with bright, stable, non-toxic fluorescent dyes that label bulk cell proteins or membranes; and 2) following the decrease in intensity (dye dilution) as the dyes are partitioned between daughter cells at each successive mitosis. The dye-dilution method has been used to monitor proliferation in vitro, to enumerate antigen specific cells, and to identify quiescent stem cells to name just a few applications. The principles of cell staining, analysis strategies, practical problems and specific applications for each of the two major cytometric approaches will be covered.
Lecture: Flow Cytometric Detection, Diagnosis, and Monitoring of Multiple Myeloma: Plasma cells (PC) are terminally differentiated and non-dividing immune cells arising from B cells whose primary function is to secrete antibodies to fight infection. PCs like all other leukocytes are susceptible to transformation. Most plasma cell dyscrasias (PCDs) develop after affinity maturation has occurred in the germinal center, exhibit phenotypic features similar to those of long-lived PCs, and are usually distributed in multiple compartments of the bone marrow. While flow cytometric immunophenotyping is considered mandatory for the diagnostic characterization of the vast number of hematological malignancies, there had been a reluctance to utilize this technology for PCD disorders. This was mainly attributed to the lack of PC‐specific markers and the lower PC frequencies usually detected in bone marrow samples by flow cytometry versus morphological approaches. Despite this, immunophenotyping has been clearly shown to provide accurate assessment of the expression of multiple markers and to easily discriminate between aberrant and both normal and reactive PC in >95% of cases. Recently, intense efforts to understand the biology and the clinical aspects of PCD have led to the development of novel regimens, drugs, and therapeutic approaches which have significantly increased complete response (CR) rates for MM. With better CR rates, strategies to better detect and define low levels of residual disease were needed. This led to more sensitive molecular and flow cytometric methods to measure minimal residual disease which can detect now one malignant cell in 100,000 to 1,000,000 leukocytes. These in turn were found to closely correlate with progression free and overall survival resulting in assays that can reliably predict long term outcomes in as few as 100 days post treatment. This lecture will focus on how immunophenotyping can be helpful in the diagnosis, prognosis and disease monitoring of PCDs. Specifically, the methodological considerations involved during panel design, including the selection and validation of assays, and data analysis and interpretation will be elaborated.
Cell Cycle and Cell Division