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  #1  
Old 07-27-2012, 02:51 AM
gdpawel gdpawel is offline
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Default Laboratory Oncology

Larry Weisenthal, M.D., PhD.

The tumor holds the key to a patient's clinical outcome and survival. Each specimen must be individualized. Performing cell function analysis deserves the same degree of professional time and attention as major extirpative or debulking surgery or radiotherapy.

All sorts of specimens, from nice, sterile, viable sugar-cubed size pieces of tumor tissue from a sterile site to mucinous, contaminated low viability specimens from inside the colon lumen to several liters of bloody fluid to fried liver (from electrocautery biopsies of liver tumors) to small needle biopsies to bone marrow and blood specimens.

For solid tumors, testing is done with three-dimensional (3D) clusters (microclusters). It takes a lot of work to glean viable tumor cells and get a quantitative yield and separate tumor cells from normal and dead cells and get rid of mucin, and then to isolate the viable cell clusters from the discohesive, single cells and so on. Two specimens are seldom alike.

Not infrequently though, patients have a fairly major, invasive surgery primarily to get tumor for testing, so failure (an inevaluable assay) is not an option. Going after a surgical/biopsy specimen has a role in eliminating ineffective agents and avoid unnecessary toxicity and in directing "correct" therapy.

There would be a huge advantage to the patient to receive a "positive/sensitive" drug, compared to a "negative/resistant" drug. The time and energy required to conduct an excisional biopsy pales in comparison to the time, energy and lost opportunities associated with months of ineffective, toxic therapy.

Reliable, sensitive and specific cell-death endpoints are needed in a functional cytometric profiling assay. At least three different cell-death endpoints are used for every specimen (of the five that are immediately at disposal). You've got to make sure that the signal that is being measured is really from tumor and not normal cells and different endpoints have different advantages and disadvantages, depending on the type of specimen.

In certain instances, one cell-death endpoint is biologically more valid than another. When you get the same result with multiple endpoints, there is confidence in the results. When there is disagreement, and there is no readily understandable reason for the disagreement, much more caution is done in using this information for treatment recommendations.

Functional cytometric profiling is not a simple, turn-key solution. It is more a professional service, more than a simple laboratory test. It is sometimes thought of as a practicing the specialty of "Laboratory Oncology." I am an early member of an emerging new medical specialty, which I refer to as Laboratory Oncology. The function of the laboratory oncologist is to utilize available forms of laboratory testing of tumor biopsies to best individualize (personalize) cancer treatment with drugs, radiation, and/or surgery.

These forms of laboratory testing are based on multiple approaches, including traditional anatomic pathology, molecular genetics, and cell biology (typically through the application of cell culture methodologies). The importance of Laboratory Oncology is that there is an exploding growth in the number of anti-cancer drugs, which tend to be of only partial and unpredictable efficacy, which are often toxic, and which are extremely expensive. There is a huge need for existing and improved methodologies to best match treatment to the patient.

Dr. Weisenthal is Medical and Laboratory Director, Weisenthal Cancer Group, Huntington Beach, California

[url]http://www.medpedia.com/users/110

Dr. Weisenthal thinks that one day there will be residencies for subspecialty training in Laboratory Oncology. Doing it properly requires either an M.D./PhD oncologist with at least a street education in pathology or else a collaboration between an oncologist, pathologist and PhD cell biologist.
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Last edited by gdpawel : 01-27-2013 at 09:06 PM. Reason: correct url address
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  #2  
Old 09-29-2012, 07:01 PM
gdpawel gdpawel is offline
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Default Flow Cytometry

Flow Cytometry is a modern method of studying cells individually in population to determine their multiple physical and biological properties. Cell suspensions are required for flow cytometic analysis. These are prepared from the blood and other body fluids, or in solid tissue, can be minced and passed through a graded series of nylon meshes to prepare a cell syspension, which can be subsequently analyzed by the flow cytometry.

The flow cytometer is a versatile tool with enormous potential for the study of cells and particles. Because of its unique analytic capabilities, the flow cytometer has become an integral part of the medical research laboratory. No other laboratory instrument provides multiparametric analysis at the single cell level, and the flow cytometer will become more valuable as medical diagnosis and therapy changes.

Flow cytometry is a technique of quantitative single cell analysis. It was developed in the 1970's and became an essential instrument for the biologic sciences, spurred by the HIV pandemic and a plethora of discoveries in hematology. The major clinical application of flow cytometry are diagnosis of hematologic malignancy, reticulocyte enumeration and cell function analysis.

Present "state-of-the-art" flow cytometers are capable of analysing up to 13 parameters (forward scatter, side scater, 11 colors of immunofluorescence) per cell at rates up to 100,000 cells per second. Automation and robotics is increasingly being applied to flow cytometry to reduce analytic cost and improve efficiency.

Prepared single cell or particle suspensions are necessary for flow cytometric analysis. Various immunoflurescent dyes or antibodies can be attached to the antigen or protein of interest. The suspension of cells or particles is aspirated into a flow cell where, surrounded by a narrow fluid stream, they pass one at a time through a focused laser beam. The light is either scattered or absorbed when it strikes a cell.

Absorbed light of the appropriate wavelength may be re-emitted as fluorescence if the cell contains a naturally fluorescent substance or one or more fluorochrome-labeled antibodies are attached to surface or internal cell structures. Light scatter is dependent on the internal structure of the cell and its size and shape.

Fluorescent substances absorb light of an appropriate wavelength and reemit light of a different wavelength. Fluorescein isothiocyanate (FITC), Texas red, and phycoerythrin (PE) are the most common fluorescent dyes used in the biomedical sciences. Light and/or fluorescence scatter signals are detected by a series of photodiodes and amplified. Optical filters are essential to block unwanted light and permit light of the desired wave-length to reach the photodetector. The resulting electrical pulses are digitized, and the data is stored, analyzed, and displayed through a computer system.

The end result is quantitative information about every cell analyzed. Since large numbers of cells are analyzed in a short period of time (>1,000/sec), statistically valid information about cell populations is quickly obtained.

The significant medical discovery of understanding the human cell cycle in combination with flow cytometric technology allowed for the development of DNA analysis of neoplasia by flow cytometry.
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Old 09-29-2012, 07:02 PM
gdpawel gdpawel is offline
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Default Tissue culture: unlocking the mysteries of viruses and cancer

Tissue culture methods have played a major part in the work of more than a third of the winners of the Nobel prize for medicine since 1953 and have made gene therapy and stem cell research possible.

Tissue culture entered mainstream medicine in 1949 when the US scientists John Enders, Thomas Weller, and Frederick Robbins reported that they had grown polio virus in cultured human embryonic skin and muscle cells. This achievement soon led to methods for measuring immunity to polio and to the award of the Nobel prize for medicine to the three scientists in 1953.

Fifty years on we are on the brink of eradicating polio by using vaccines derived from cell cultures, and cells are grown on an industrial scale to yield vaccines, antibodies, and other biological products such as recombinant factor VIII for hemophilia.

In his Nobel lecture Enders described the technical difficulties encountered before the second world war in efforts to grow viruses in culture and how, after the war, antibiotics were used to keep bacterial contamination at bay. The new accessibility of tissue culture methods ushered in the golden era of virus discovery.

It also revived many previously unattainable ambitions in medical science, having a crucial role in no fewer than 18 of the 52 subsequent Nobel prize winning discoveries, including RNA interference (the 2006 winner), the nature of oncogenes (1989), growth factors (1986), monoclonal antibodies (1984), tumor viruses (1975), and virus genetics (1965).

An Old Dream is Realized

Although short term survival outside the body of the beating heart and twitching muscle was known to the ancients, serious attempts to achieve lengthy tissue survival in vitro were possible only in the second half of the 19th century.

Among the pioneers were embryologists, who studied the early development of amphibian and avian eggs and began to experiment on "organizers," soluble messengers that directed organ development. With the advent of cell culture the nature of these growth factors could be elucidated. Modern stem cell research is the most exciting and controversial descendent of this work.

Surgeons had dreamt of organ transplantation since the Middle Ages. In the 1920s and 1930s Alexis Carrel, a French surgeon working at the Rockefeller Institute in New York, collaborated with the aviator Charles Lindberg to overcome the technical challenges of organ perfusion. Their tissue survival studies attracted enormous public interest, fuelled by newspaper reports such as "birthday" notices for one culture of chick embryo cardiac muscle cells.

Carrel's earlier Nobel prize for work on surgical anastomoses and his philosophical writings added to the mystique of cell culture, which was reinforced by the extreme precautions needed against contamination.

Only the most determined groups succeeded. Before the second world war Thomas Strangeways and Honor Fell in Cambridge used cell culture as part of their multidisciplinary approach to bone and joint disease, paving the way for the recognition of tissue specific markers, which are now so widely used in diagnostic pathology.

After the second world war the serial subculture of cells was achieved through the use of trypsin (a serine protease found in the digestive system where it breaks down proteins) to produce single cell suspensions, antibiotics to control contamination, and better growth media, such as the famous "199" with its 64 ingredients.

The finite number of divisions achievable in the culture of normal cells contrasted with the "immortality" of cancer cell lines. HeLa cell, the most famous of these, was derived from the cervical cancer that killed Henrietta Lacks in 1951. Continuous lines were used to develop convenient in vitro methods for testing the efficacy of potential anticancer drugs and the carcinogenic effects of drugs and chemicals.

The demonstration of integrated viral genes in many tumours and of similar homologues in normal cells revolutionized concepts of growth regulation. The discovery of mutations in these homologues (the cellular "oncogenes") in cancer tissues and in the normal cells of family members with an inherited risk of cancer had applications in cancer diagnosis and screening.

By the 1960s, cell culture technology was well established in virology and cancer research. The time was right for the interaction between cell biology and genetics that gave birth to molecular biology.

Study of the chromosomes of dividing cultured cells spawned the new discipline of cytogenetics, while work on gene expression began to explain the mechanisms involved in differentiation, which could now be observed in vitro. This ultimately produced skin cultures that could be used for grafting, but its more profound consequences resulted from elucidating the functions of T cells as they proliferated in vitro after stimulation with antigens.

Exquisitely Specific Antibodies

Fusing cultured benign and malignant cells provided important insight into the control of cell division, and the technique was spectacularly exploited to generate "hybridomas" (fused cells) between myeloma cells and B cells to produce monoclonal antibodies. Immortalized cell lines now provided a potentially unlimited source of antibodies of exquisite specificity for enzyme linked immunosorbent assay and radioimmunoassay, and monoclonal antibodies are now being used in treatment.

The ability to transfect (introducing foreign DNA into a cell) cultured cells with DNA gene sequences has allowed us to assign functions to different genes and understand the mechanisms that activate or redress their function. Gene therapy has yet to fulfil its promise, but it may ultimately overtake the many other medical applications of cell culture.

Without cell culture we would lack vaccines against measles, mumps, and rubella and would still be dependent on much more expensive and reactogenic vaccines for polio, rabies, and yellow fever.

We would be unable to karyotype (standardized arrangement of all the chromosomes of a cell) patients with suspected genetic disorders or to perform in vitro fertilization.

Antibodies for diagnostic or therapeutic use would be derived from immunization of whole animals, with much greater variation in titre (the unit in which the analytical detection of many substances is expressed) and specificity (a measure of a test's effectiveness) than products derived from cells.

Our concepts of growth, differentiation, biological ageing, and malignant transformation would be simplistic; and gene therapy and the use of stem cells to repopulate damaged organs or clone individuals would be beyond imagination.

Source: Yvonne Cossart, Bosch professor of infectious diseases, Department of Infectious Diseases and Immunology, University of Sydney, Australia BMJ 2007; 334 : s18 doi: 10.1136/bmj.39034.719942.94
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Old 10-10-2012, 12:53 AM
gdpawel gdpawel is offline
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Default The microenvironment contributes critically to drug response

What about the fibroblast matrix, the lymphatic vessels, the infiltrating monocytes, the T-cells, the B-cells and neutrophils: the vast complexities of the human tumor microenvironment? Real-life cancers grow as a complex organism that includes both malignant and non-malignant components.

It may include fibrous tissue, mesothelial cells, fibroblasts, endothelial cells, etc. In order to exhibit its most characteristic behavior patterns, a cancer cell needs to be surrounded by a colony of other cells, both normal and malignant.

By examining drug-induced cell death events in native-state 3D (three dimensional) microclusters, the functional profiling platform has the unique capacity to capture stromal, cytokines (chemokines), macrophages, lymphocytes, vascular and inflammatory cell interactions with tumor cells, known to be crucial for clinical response prediction.

The microclusters recapitulate the human tumor environment, while the "3D" advancement recreates the extracellular matrix (metalloproteinases). The platform studies cancer response to drugs within this microenvironment, enabling it to provide clinically relevant predictions to cancer patients. It is this capacity to study human tumor microenvironments that distinguishes it from other platforms in the field.

Tumors are very complex organisms. Ignoring this complexity, most studies of human cancer in culture have focused upon individual tumor cells that have been removed from their complex microenvironment.

Some previous methods of assays limited their analysis only to isolated tumor cells and failed to incorporate the crucial contribution of non-tumorous elements to the cancer phenomenon. Each of these microspheres contains all the complex elements of tumor biosystems that are found in the human body and which can impact clinical response.
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