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In vitro chemoresistance and chemosensitivity assays have been developed to provide information about the characteristics of an individual patient’s malignancy to predict potential responsiveness of their cancer to specific drugs. These assays are sometimes used by oncologists to select treatment regimens for an individual patient. Several assays have been developed that differ with respect to processing of biologic samples and detection methods. However, all involve similar principles and share protocol components including: (1) isolation of cells and establishment in an in vitro medium (sometimes in soft agar); (2) incubation of the cells with various drugs; (3) assessment of cell survival; and (4) interpretation of the result.
A variety of chemosensitivity and chemoresistance assays have been clinically evaluated in human trials. All assays use characteristics of cell physiology to distinguish between viable and nonviable cells to quantify cell kill following exposure to a drug of interest. With few exceptions, drug doses used in the assays are highly variable depending on tumor type and drug class, but all assays require drug exposures ranging from several-fold below physiologic relevance to several-fold above physiologic relevance. Although a variety of assays exist to examine chemosensitivity or chemoresistance, only a few are commercially available. Available assays are outlined as follows.
Methods Using Differential Staining/Dye Exclusion
Differential Staining Cytotoxicity Assay
The Differential Staining Cytotoxicity assay relies on dye exclusion of live cells after mechanical disaggregation of cells from surgical or biopsy specimens by centrifugation. Cells are then established in culture and treated with the drugs of interest at three dose levels: the middle (relevant) dose, which could be achieved in therapy; a 10-fold lower than the physiologically relevant dose; and a 10-fold higher dose. Exposure time ranges from 4-6 days; then, cells are restained with fast green dye and counterstained with hematoxylin and eosin (H&E). The fast green dye is taken up by dead cells, and hematoxylin and eosin differentiates tumor cells from normal cells. The intact cell membrane of a live cell precludes staining with the green dye. Drug sensitivity is measured by the ratio of the number of live cells in the treated samples to the number of live cells in the untreated controls.
The EVA/PCD assay (Rational Therapeutics, Long Beach, CA) relies on ex vivo analysis of programmed cell death, as measured by differential staining of cells after apoptotic and nonapoptotic cell death markers in tumor samples exposed to chemotherapeutic agents. Tumor specimens obtained through biopsy or surgical resection are disaggregated using DNase and collagenase IV to yield tumor clusters of the desired size (50-100 cell spheroids). Because these cells are not proliferated, these microaggregates are believed to more closely approximate the human tumor microenvironment. These cellular aggregates are treated with the dilutions of the chemotherapeutic drugs of interest and incubated for 3 days. After drug exposure is completed, a mixture of nigrosin B & fast green dye with glutaraldehyde-fixed avian erythrocytes is added to the cellular suspensions. The samples are then agitated and cytospin-centrifuged and, after air drying, are counterstained with H&E. The end point of interest for this assay is cell death, as assessed by observing the number of cells differentially stained due to changes in cellular membrane integrity.
Fluorometric Microculture Cytotoxicity Assay
The fluorometric microculture cytotoxicity assay is another cell viability assay that relies on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate to fluorescein in viable cells. Cells from tumor specimens are incubated with cytotoxic drugs; drug resistance is associated with higher levels of fluorescence.
Methods Using Incorporation of Radioactive Precursors by Macromolecues in Viable Cells
Tritiated thymine incorporation measures uptake of tritiated thymidine by DNA of viable cells. Using proteases and DNase to disaggregate the tissue, samples are seeded into single-cell suspension cultures on soft agar. They are then treated with the drug(s) of interest for four days. After three days, tritiated thymidine is added. After 24 hours of additional incubation, cells are lysed, and radioactivity is quantified and compared with a blank control consisting of cells that were treated with sodium azide. Only cells that are viable and proliferating will take up the radioactive thymidine. Therefore, there is an inverse relationship between update of radioactivity and sensitivity of the cells to the agent(s) of interest.
Extreme Drug Resistance Assay
The Extreme Drug Resistance assay (EDR; Exiqon Diagnostics, Tustin, CA; no longer commercially available) is methodologically similar to the thymidine incorporation assay, using metabolic incorporation of tritiated thymidine to measure cell viability; however, single cell suspensions are not required, so the assay is simpler to perform. Tritiated thymidine is added to the cultures of tumor cells, and uptake is quantified after various incubation times. Only live (resistant) cells will incorporate the compound. Therefore, the level of tritiated thymidine incorporation is directly related to chemoresistance. The interpretation of the results is unique in that resistance to the drugs is evaluated, as opposed to evaluation of responsiveness. Tumors are considered to be highly resistant when thymidine incorporation is at least one standard deviation above reference samples.
Methods That Quantify Cell Viability Using Colorimetric Assay
Histoculture Drug Resistance Assay
The Histoculture Drug Resistance Assay (HDRA; AntiCancer Inc., San Diego, CA) evaluates cell growth after chemotherapy treatment based on a colorimetric assay that relies on mitochondrial dehydrogenases in living cells. Drug sensitivity is evaluated by quantification of cell growth in the 3-dimensional collagen matrix. There is an inverse relationship between the drug sensitivity of the tumor and cell growth. Concentrations of drug and incubation times are not standardized and vary depending on drug combination and tumor type.
Methods Using Incorporation of Chemoluminescent Precursors by Macromolecules in Viable Cells
Adenosine Triphosphate Bioluminescence Assay
The adenosine triphosphate (ATP) bioluminescence assay relies on measurement of ATP to quantify the number of viable cells in a culture. Single cells or small aggregates are cultured, then exposed to drugs. Following incubation with drug, the cells are lysed and the cytoplasmic components are solubilized under conditions that will not allow enzymatic metabolism of ATP. Luciferin and firefly luciferase are added to the cell lysis product. This catalyzes the conversion of ATP to adenosine di- and monophospate, and light is emitted proportionally to metabolic activity. This is quantified with a luminometer. From the measurement of light, the number of cells can be calculated. A decrease in ATP indicates drug sensitivity, whereas no loss of ATP suggests that the tumor is resistant to the agent of interest.
The ChemoFX (Helomics Corp., previously called Precision Therapeutics, Pittsburgh, PA) assay also relies on quantifying ATP based on chemoluminescence. Cells must be grown in a monolayer rather than in a three-dimensional matrix.
Methods That Use Differential Optical Density
Similar to the EVA/PCD assay, the CorrectChemo (previously called the Microculture Kinetic [MiCK]) assay; DiaTech Oncology, Franklin, TN) assay relies on measures of programmed cell death. In the assay, tumor cells are exposed to multiple concentrations of drugs and cultured. The optical density of the cells is measured over time, to create a density-by-time curve. A sudden increase in optical density is associated with cell apoptosis; the extent of drug-induced apoptosis is a measure of the cell’s sensitivity to that agent. As of March 2016, DiaTech no longer offers the CorrectChemo assay commercially.
The rationale for chemosensitivity assays is strongest when there are a variety of therapeutic options and there are no clear selection criteria for any particular regimen in an individual patient.
Clinical laboratories may develop and validate tests in-house and market them as a laboratory service; laboratory-developed tests (LDTs) must meet the general regulatory standards of the Clinical Laboratory Improvement Amendments (CLIA). Chemoresistance and chemosensitivity assays discussed in this policy are available under the auspices of CLIA. Laboratories that offer LDTs must be licensed by CLIA for high-complexity testing. To date, the U.S. Food and Drug Administration has chosen not to require any regulatory review of this test.
POLICYIn vitro chemosensitivity assays, including, but not limited to, the Histoculture Drug Response Assay, a fluorescent cytoprint assay, the ChemoFx assay, or the CorrectChemo assay, are considered investigational.
In vitro chemoresistance assays, including, but not limited to, Extreme Drug Resistance Assays, are considered investigational.
The coverage guidelines outlined in the Medical Policy Manual should not be used in lieu of the Member's specific benefit plan language.
Investigative is defined as the use of any treatment procedure, facility, equipment, drug, device, or supply not yet recognized as a generally accepted standard of good medical practice for the treatment of the condition being treated and; therefore, is not considered medically necessary. For the definition of Investigative, “generally accepted standards of medical practice” means standards that are based on credible scientific evidence published in peer-reviewed medical literature generally recognized by the relevant medical community, and physician specialty society recommendations, and the views of medical practitioners practicing in relevant clinical areas and any other relevant factors. In order for equipment, devices, drugs or supplies [i.e, technologies], to be considered not investigative, the technology must have final approval from the appropriate governmental bodies, and scientific evidence must permit conclusions concerning the effect of the technology on health outcomes, and the technology must improve the net health outcome, and the technology must be as beneficial as any established alternative and the improvement must be attainable outside the testing/investigational setting.
POLICY HISTORY8/1998: Approved by Medical Policy Advisory Committee (MPAC)
12/18/2000: "and extreme drug resistance (EDR)" added to third paragraph under "description" section.
2/8/2002: Investigational definition added
5/1/2002: Type of Service and Place of Service deleted
9/2/2004: Code Reference section completed, non-covered table added
8/5/2005: Code Reference section reviewed, no changes
10/17/2006: Policy reviewed, no changes
1/4/2007: Policy reviewed, policy statement rewritten for clarity
2/19/2008: Policy reviewed, no changes
4/24/2009: Policy reviewed, no changes
04/27/2010: Policy title changed from “In Vitro Chemotherapeutic Drug Assays” to “In Vitro Chemoresistance and Chemosensitivity Assays” to reflect the scope of the policy. Policy description extensively re-written to add information on available tests and how they are performed. Policy statement unchanged. Deleted outdated references from the Sources section.
06/21/2011: Policy reviewed; no changes.
04/26/2012: Policy reviewed; no changes.
08/07/2013: Policy reviewed; no changes.
06/09/2014: Policy description updated regarding the following: methods using differential staining/dye exclusion; methods using incorporation of radioactive precursors by macromolecules in viable cells; methods to quantify cell viability by colorimetric assay; methods using incorporation of chemoluminescent precursors by macromolecules in viable cells; and methods using differential optical density. Policy statement unchanged.
07/30/2015: Code Reference section updated for ICD-10.
09/11/2015: Policy description updated regarding commercially available assays. Policy statement for in vitro chemosensitivity assays updated to add the ChemoFx assay and the CorrectChemo assay as investigational. Investigative definition updated in the Policy Guidelines section.
06/06/2016: Policy number added.
08/19/2016: Policy description updated to add section headings and information regarding laboratory-developed tests. Policy statements unchanged.
SOURCE(S)Blue Cross Blue Shield Association policy #2.03.01
CODE REFERENCEThis may not be a comprehensive list of procedure codes applicable to this policy.