WO2015171848A2 - Synergism and antagonism between multiple anti-cancer agents determined by mick assay - Google Patents
Synergism and antagonism between multiple anti-cancer agents determined by mick assay Download PDFInfo
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- WO2015171848A2 WO2015171848A2 PCT/US2015/029589 US2015029589W WO2015171848A2 WO 2015171848 A2 WO2015171848 A2 WO 2015171848A2 US 2015029589 W US2015029589 W US 2015029589W WO 2015171848 A2 WO2015171848 A2 WO 2015171848A2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7023—(Hyper)proliferation
- G01N2800/7028—Cancer
Definitions
- the present disclosure relates to use of a spectrophotometric apoptosis (MiCK) assay to determine synergism and/or antagonism between multiple anti-cancer agents.
- MiCK spectrophotometric apoptosis
- Apoptosis is a mechanism by which a cell disassembles and packages itself for orderly disposal by the body. Apoptosis is commonly used by the body to discard cells when they are no longer needed, are too old, or have become damaged or diseased. In fact, some cells with dangerous mutations that might lead to cancer, and even some early-stage cancerous cells, may undergo apoptosis as a result of natural processes.
- Apoptosis generally occurs after one of several triggers sends a signal to the cell that it should undergo apoptosis. In many cancer cells, this message system does not work correctly because the cell cannot detect the trigger, fails to send a signal properly after the trigger is received, or fails to act on the signal, or the cell may even have combinations of these problems. The overall effect is a resistance to undergoing apoptosis in some cancer cells.
- Cancer includes all cancers or malignancies, both hematologic and non- hematologic, as well as myelodysplastic syndromes (MDS). This contemplates the four major categories for all blood/ marrow cancers, solid tumors, and effusions: leukemia, lymphomas, epithelial malignancies, and mesenchymal malignancies.
- chemotherapeutic drugs with different mechanisms of action.
- the Microculture Kinetic Assay (MiCK assay), described in U.S. Patent 6,077,684 and U.S. Patent 6,258,553, is currently used to detect whether cancer cells from a patient undergo apoptosis in response to a particular drug known to be effective against specific cancer types.
- MiCK assay cancer cells from a patient are placed in a suspension of a given
- OD versus time data may also be used to calculate kinetic units, the units which can be used to measure apoptosis, which similarly correlate with the suitability of a therapy for the patient.
- the present application relates to methods of using the MiCK assay to determine the most effective drug candidate or combination of drug candidates for an individual patient.
- the method may include placing a single-cell suspension of viable cancer cells obtained from a tumor site in an individual patient in at least one well of a plate suitable to be read by a spectrophotometer, wherein the cancer cells are in a concentration sufficient to form a monolayer of cells on the bottom of the well, adding at least one drug candidate to the well in an amount sufficient to achieve a target drug candidate concentration, measuring the optical density of the well at a wavelength of approximately 600 nm using a spectrophotometer at selected time intervals for a selected duration of time, determining a kinetic units (KU) value from the optical density and time measurements, and correlating the KU value with an ability of the anti-cancer drug candidate to induce apoptosis in the cancer cells if the KU value is positive, or an inability of the anti-cancer drug candidate to induce apoptosis in the cancer cells if the KU value
- a method for determining the most effective drug candidate or combination of drug candidates for an individual patient, wherein a sample from a primary tumor site or a metastasis of the primary tumor site is tested using the MiCK assay.
- a method of identifying a drug candidate that decreases the effectiveness of another drug candidate in an individual patient may include placing a single-cell suspension of viable cancer cells obtained from a
- a drug candidate to one well in an amount sufficient to achieve a target drug candidate concentration
- adding a drug combination to a different well in an amount sufficient to achieve a target drug candidate concentration measuring the optical density of the wells at a wavelength of approximately 600 nm using a spectrophotometer at selected time intervals for a selected duration of time, determining a kinetic units (KU) value for each well from the optical density and time measurements, and correlating the KU value with an ability of the anti-cancer drug candidate or anti-cancer drug combination to induce apoptosis in the cancer cells if the KU value is positive, or an inability of the anti-cancer drug candidate or anti-cancer drug combination to induce apoptosis in the cancer cells if the KU value is not positive.
- a method of identifying a drug candidate that increases the effectiveness of another drug candidate in an individual patient may include placing a single-cell suspension of viable cancer cells obtained from a tumor site in an individual patient in at least three wells of a plate suitable to be read by a spectrophotometer, , adding a drug candidate to one well in an amount sufficient to achieve a target drug candidate concentration, adding a different drug candidate to a different well in an amount sufficient to achieve a target drug candidate concentration, adding a drug combination to a different well in an amount sufficient to achieve a target drug candidate concentration, measuring the optical density of the wells at a wavelength of approximately 600 nm using a spectrophotometer at selected time intervals for a selected duration of time, determining a kinetic units (KU) value for each well from the optical density and time measurements, and correlating the KU value with an ability of the anti-cancer drug candidate or anti-cancer drug combination to induce apoptosis in the cancer cells if the KU value is
- the drug candidate is an individual anti-cancer drug.
- the drug candidate is a combination of anti-cancer drugs.
- the cancer cells are from a blood cancer.
- the blood cancer is selected from the group consisting of acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and non-Hodgkin's lymphoma (NHL).
- the cancer cells are from a solid tumor.
- the solid tumor is selected from the group consisting of breast cancer, lung cancer, ovarian cancer, gastric cancer, and uterine cancer.
- MiCK microculture kinetic
- FIG. 1 shows a time sequences photomicrograph of a cancer cell moving through the stages of apoptosis.
- the first panel of the left (1) shows the cell prior to apoptosis.
- the middle panel (2) shows the cell during apoptosis and blebbing is apparent.
- the last panel on the right (3) shows the cell after apoptosis is complete or nearly complete.
- FIG. 2 is a graph showing representative curves for induction of apoptosis, drug resistance, and control cells without drug in a MiCK assay.
- the curve labeled “B12” shows data representative of cells in which the drug induces apoptosis.
- the curve labeled “F3” shows data representative of cells that are resistant to the drug.
- the curve labeled “G5" shows data representative of control cells that did not receive any drug.
- FIG. 3 is a graph showing representative data for induction of apoptosis or necrosis in a MiCK assay.
- the curve labeled "D2" shows data representative of cells in which the drug induces
- the curve labeled "D7" shows data representative of cells in which the drug induces necrosis or which otherwise undergoes necrosis during the course of the assay.
- FIG. 4 is a graph showing representative data for general non-drug-induced cell death in a MiCK assay.
- the curve labeled "C4" shows data representative of spontaneous cell death during the course of the assay.
- the data provided herein demonstrate substantial variability between individual patients regarding the drug candidate(s) that induce the highest degree of apoptosis in the individual patients' cancer cells. Additionally, it is commonly thought that combining drug candidates creates at least an additive effect on apoptosis.
- certain anti-cancer drugs can produce synergistic effects in combination with other anti-cancer drugs in individual patients.
- the same anti-cancer drug can produce an antagonistic effect in combination with a different anti-cancer drug in the same individual patient.
- the subject disclosure features, in one aspect, methods of using an assay similar to the microculture kinetic (MiCK) assay, as disclosed in U.S. Patent 6,077,684 and U.S. Patent 6,258,553, both incorporated by reference herein, to determine the most effective
- chemotherapeutic drug or combination of chemotherapeutic drugs for an individual patient.
- the assay may proceed by selecting an anti-cancer drug candidate and selecting at least one tumor on which to test the drug.
- Purified cancer cells are obtained from the tumor and the cancer cells may be suspended as a single-cell suspension in culture medium, such as RPMI.
- a "single cell suspension” is a suspension of one or more cells in a liquid in which the cells are separated as individual cells or in clumps of usually 10 cells or fewer.
- the culture medium may contain other components, such as fetal- bovine serum or components specifically required by the cancer cells. These components may be limited to those necessary to sustain the cells for the duration of the assay, typically at least 24 hours and not longer than 120 hours.
- Suspended cells may be tested by placing samples in wells of a spectrophotometric plate.
- the cells may be suspended at any concentration such that during the spectrophotometric measurements of OD, the beam of the plate reader normally passes through only one cell layer at a time. For most cells, a concentration of between 2 X 10 5 and 1 X 10 6 cells/mL may be used. Concentration may be increased for small cells and decreased for large cells.
- the volume of cell suspension to be used in drug candidate test samples may be added to at least one concentration test well of the plate. If the well will be prefilled with additional medium during testing of the drug candidates, then the concentration test well may similarly be prefilled with additional medium.
- the plate may be centrifuged (e.g. for 30 sec to 2 min at 500 RPM) to settle the cells on the bottom of the well. If the cell concentration is appropriate for the assay, the cells should form a monolayer without overlapping. Cell concentration may be adjusted as appropriate until this result is achieved. Multiple concentrations of cells may be tested at one time using different concentration test wells.
- the cell concentration may be adjusted to initially achieve less than a monolayer to allow for growth such that sufficient cells for a monolayer will be present when the drug candidate assay commences.
- the cancer cells may be in an exponential or a non-exponential growth phase. In a specific embodiment, particularly when the cancer cells are from a cancer cell line, they may be in an exponential growth phase.
- the drug-candidate assay may proceed by filling test and control wells in the plate with an appropriate volume of medium and
- the well may be partially pre-filled with medium alone.
- the cells may be allowed to adjust to the plate conditions for a set period of time, such as at least 12 hours, at least 16 hours, at least 24 hours, or 12-16 hours, 12-24 hours, or 16- 24 hours.
- the adjustment period is typically short enough such that the cells do not experience significant growth during the time.
- the adjustment period may vary depending on the type of cancer cells used in the drug candidate assay. Adjustment may take place under conditions suitable to keep the cells alive and healthy.
- the plate may be placed in a humidified incubator at 37°C under 5% C0 2 atmosphere.
- the plate may be centrifuged (e.g. for 30 sec to 2 minutes at 500 RPM) to settle the cells on the bottom of the wells.
- the drug candidate and any control drugs or other control samples may be added to the wells after the adjustment period.
- the drug candidate will be added in a small volume of medium or other liquid as compared to the total volume of liquid in the well.
- the volume of drug added may be less than 10% of the total volume of liquid in the well.
- Drug candidates may be added in multiple dilutions to allow determination of any concentration effects. Although many drug candidates may be water-soluble, drug candidates that are not readily soluble in water may also be tested. Such candidates may be mixed with any appropriate carrier. Such candidates may preferably be mixed with carriers anticipated for actual clinical use. Viscous drug candidates may require substantial dilution in order to be tested. Drug candidates with a strong color may benefit from monitoring of OD in test wells containing only the drug candidate and subtraction of this OD from measurements for the test sample wells.
- the cells may be allowed another short period of adjustment, for example of 15 minutes or 30 minutes.
- the cells may be placed under conditions suitable to keep the cells alive and healthy.
- the plate may be placed in a humidified incubator at 37°C under 5% C0 2 atmosphere. After this short adjustment period, a layer of mineral oil may be placed on top of each well to maintain C0 2 in the medium and prevent evaporation.
- the plate may then be placed in a spectrophotometer configured to measure the OD at a wavelength of 600 nm for each well at a given time interval for a given total period of time.
- OD for each well may be measured periodically over a time frame of seconds, minutes, or hours for a period of between 24 and 120 hours. For certain cells, measurements for a period of as little as 12 hours may be sufficient. In specific embodiments, measurements
- the spectrophotometer may have an incubated chamber to avoid spontaneous death of the cells.
- Spectrophotometric data may be converted to kinetic units.
- Kinetic units are determined by the slope of the curve created when the change in the OD at 600 nm caused by cell blebbing is plotted as a function of time. Specific information regarding the calculation of kinetic units is provided in Kratsov, Vladimir D. et al., Use of the Microculture Kinetic Assay of Apoptosis to Determine Chemosensitivities of Leukemias, Blood 92:968-980 (1998), incorporated by reference herein.
- Optical density for a given drug candidate at a given concentration may be plotted against time. This plot gives a distinctive increasing curve if the cells are undergoing apoptosis.
- FIGs 2 and 3 An example of the curve obtained when cells undergo apoptosis is shown in FIGs 2 and 3.
- the curve is similar to that obtained for a control sample with no drug or drug candidate (FIG. 2).
- Cell death due to reasons other than apoptosis can also be determined by the current assay and is useful in eliminating false positives from drug candidate screening. For example, cell necrosis produces a distinctive downward sloping curve easily distinguishable from the apoptosis-related curve as seen in FIG. 3. Further, general cell death also causes a downward curve as seen in FIG. 4.
- the effectiveness of a drug candidate may be determined by the value of the kinetic units it produces in a modified MiCK assay using a known cell line.
- Kinetic units may be determined as follows:
- Apoptosis (KU) (Vmax Drug Candidate Treated - Vmax Control ) x 60 x X/(OD control - OD blank )
- the KU is a calculated value for quantifying apoptosis.
- the optical densities (OD) from each well are plotted against time.
- the maximum slope of the apoptotic curve (V max) is calculated for each plot of drug-treated microculture. It is then compared to the Vmax of a control well without drug (calculated at the same time as the Vmax of the drug exposed cells). For convenience, the Vmax is multiplied by 60 to convert the units from mOD/minute to mOD/hour.
- the coefficient is a calculated value for normalizing the amount of cells per well when measuring apoptosis and quantifying said apoptosis in Kinetic Units.
- X optimal optical density value for the cell type tested (determined empirically).
- OD control average optical density of all the control wells.
- OD blank average optical density of all the blank wells.
- a coefficient of 1.000 means that the cell concentration is optimal.
- a coefficient value below 1.000 means that the cell concentration is higher than the optimal concentration. If the coefficient value is above 1.000, it means that the cell concentration in the well is suboptimal.
- the acceptable coefficient values for an optimal MiCK assay are between 0.8 and 1.5. If the value is under 0.8, the coefficient will erroneously reduce the value of the calculated KU. If the value is above 1.5, there will not be enough cells per well to detect the signal of apoptosis.
- the "X" in the formula will vary depending on the cell type. For solid tumor specimens, this value is 0.09. For most of the leukemias, the value is 0.15. For CLL (chronic lymphocytic leukemias) and the lymphomas, the value is 0.21.
- This "X" value is adapted to the tumor type and determined empirically.
- the coefficient is developed by trial and error, using different concentrations of cells and by checking them under a microscope while looking for complete proper coverage in the well. The proper well is read by a reader and the OD becomes the new X value. Further information regarding this equation may be found in Kravtsov et al. (Blood, 92:968-980), which was previously incorporated herein by reference.
- kinetic unit values generated using the current assay may be compared to determine if a particular drug candidate may also be performed and may give general indications of appropriate dosage. Occasionally some drugs may perform less well at higher concentrations than lower concentrations in some cancers. Comparison of kinetic unit values for different concentrations of drug candidates may identify drug candidates with a similar profile.
- evaluation of an anti-cancer drug candidate may include any determination of the effects of that drug candidate on apoptosis of a cancer cell. Effects may include, but are not limited to induction of apoptosis, degree of induction of apoptosis as compared to known cancer drugs, degree of induction of apoptosis at different drug candidate concentrations, and failure to induce apoptosis.
- the anti-cancer drug evaluation assay may also be able to detect non-drug-related or non-apoptotic events in the cancer cells, such as cancer cell growth during the assay or cell necrosis.
- threshold kinetic unit values may be set to distinguish drug candidates able to induce clinically relevant levels of apoptosis in cancer cells.
- the threshold amount may be 1.5, 2 or 3 kinetic units.
- the actual threshold selected for a particular drug candidate or concentration of drug candidate may depend on a number of factors.
- a lower threshold such as 1.5 or 2 may be acceptable for a drug candidate able to induce apoptosis in cancer types that do not respond to other drugs or respond only to drugs with significant negative side effects.
- a lower threshold may also be acceptable for drug candidates that exhibit decreased efficacy at higher concentrations or which themselves are likely to have significant negative side effects.
- a higher threshold such as 3, may be acceptable for drug candidates able to induce apoptosis in cancer types for which there are already suitable treatments.
- the KU value is ⁇ 7, more preferably the KU value is ⁇ 8, even more preferably the KU value is ⁇ 9, and most preferably the KU value is ⁇ 10.
- These ranges were established based on a statistical analysis of cancer cells. The ranges establish a baseline for relative comparison of chemotherapeutic drugs being tested on a specific cell type. Test outcomes may be affected by extenuating factors such as: time elapsed from obtaining sample to testing; quantity of viable cells available to test; microbial contamination of specimen; quality or viability of cells being tested; cell type; and recent treatment such as chemotherapy or radiation therapy.
- Clinical sensitivity to chemotherapy drugs is not completely limited to outcomes as forecast in the above ranges.
- the KU measurement of drug-induced apoptosis in the assay may be used by physicians to develop an individual patient treatment regimen along with other important factors such as patient history, prior treatment results, overall patient health, patient
- the particular ranges of KU value utilized will be dependent upon context. That is, depending upon the particular type of tumor cell being tested, the particular drug being utilized, and the particular patient or patient population under analysis.
- the KU value therefore represents a dependable and flexible analytical variable that can be tailored by the practitioner of the disclosed methods to create a suitable metric by which to evaluate a given drug's effect.
- the anti-cancer drug candidates may be any chemical, chemicals, compound, compounds, composition, or compositions to be evaluated for the ability to induce apoptosis in cancer cells.
- These candidates may include various chemical or biological entities such as chemotherapeutics, other small molecules, protein or peptide-based drug candidates, including antibodies or antibody fragments linked to a chemotherapeutic molecule, nucleic acid-based therapies, other biologies, nanoparticle-based candidates, and the like.
- Drug candidates may be in the same chemical families as existing drugs, or they may be new chemical or biological entities.
- Drug candidates are not confined to single chemical, biological or other entities. They may include combinations of different chemical or biological entities, for example, proposed combination therapies. Further, although many examples herein relate to an assay in which a single drug candidate is applied, assays may also be conducted for multiple drug candidates in combination. It is also important to understand that embodiments of the invention may utilize the metabolites of the various drug candidates in a method as described. Embodiments of the invention are able to test all manner of anti-cancer drug candidates.
- anti-cancer drug candidates can be tested by the disclosed methods: Abraxane, Afatinib, Alimta, Amsacrine, Asparaginase, BCNU, Bendamustine, Bleomycin, Bosutinib, Caelyx (Doxil), Carboplatin, Carmustine, CCNU, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Cytarabine, Cytoxan (4HC), dacarbazine, Dactinomycin, Dasatinib, Daunorubicin, Decitabine, Dexamethasone, Doxorubicin, Epirubicin, Estramustine, Etoposide, Fludarabine, 5- Fluorouracil, Gemcitabine, Gleevec (imatinib), Hexamethylmelamine, Hydroxyurea, Idarubicin, Ifosfamide (4HI), Interferon-2a, Irinotecan, Ixabepil
- Methotrexate Mitomycin, Mitoxantrone, Nilotinib, Nitrogen Mustard, Oxaliplatin, Pentostatin, Sorafenib, Streptozocin, Sunitinib, Tarceva, Taxol, Taxotere, Temozolomide, Temsirolimus, Teniposide, Thalidomide, Thioguanine, Topotecan, Tretinoin, Velcade, Vidaza, Vinblastine, Vincristine, Vinorelbine, Vorinostat, Xeloda (5DFUR), Everolimus, Lapatinib, Lanalidomide, Rapamycin, and Votrient (Paxopanib).
- anti-cancer drug candidates including but not limited to other nonchemotherapy drugs and/ or chemicals which can produce apoptosis or which are examined for their ability to produce apoptosis, are also able to be tested by the disclosed methods.
- the methods of the present invention are not strictly applicable to anti-cancer drug candidates, but rather embodiments of the disclosed methods can be utilized to test any number of potential drug candidates for a whole host of diseases.
- More than one drug candidate, concentration of drug candidate, or combination of drugs or drug candidates may be evaluated in a single assay using a single plate. Different test samples may be placed in different wells.
- the concentration of the drug candidate tested may be, in particular embodiments, any concentration in the range from 0.1 to ⁇ , ⁇ , or any concentration in the range from 0.01 to 10,000 ⁇ , or any concentration in the range from 0.001 to 100,000 ⁇ , for example.
- the concentration tested may vary by drug type, and the aforementioned example concentrations are not to be considered as limiting, for the skilled artisan will understand how to construct the appropriate concentration for utilization with the taught methods and assays, depending upon the particular anti-cancer drug tested.
- the plate and spectrophotometer may be selected such that the spectrophotometer may read the plate.
- the diameter of the bottom of each well is no smaller than the diameter of the light beam of the spectrophotometer.
- the diameter of the bottom of each well is no more than twice the diameter of the light beam of the spectrophotometer. This helps ensure that the OD at the measured wavelength, 600 nm for example, of a representative portion of the cells in each well is accurately read.
- the spectrophotometer may make measurements at wavelengths of than 600 nm.
- the wavelength may be +/- 5 or +/- 10.
- other wavelengths may be selected so as to be able to distinguish blebbing.
- Spectrophotometers may include one or more computers or programs to operate the equipment or to record the results.
- the spectrophotometer may be functionally connected to one or more computers able to control the measurement process, record its results, and display or transmit graphs plotting the optical densities as a function of time for each well.
- Plates designed for tissue culture may be used, or other plates may be sterilized and treated to make them compatible with tissue culture. Plates that allow cells to congregate in areas not accessible to the spectrophotometer, such as in corners, may work less well than plates that avoid such congregation. Alternatively, more cancer cells may be added to these plates to ensure the presence of a monolayer accessible to the spectrophotometer during the assay. Plates with narrow bottoms, such as the Corning Costar ® half area 96 -well plate, may also assist in encouraging formation of a monolayer at the bottom of the well without requiring inconveniendy low sample volumes. Other plates, such as other 96-well plates of smaller well plates, such as 384-well plates, may also be used.
- a modified MiCK assay protocol has recendy been developed as described in International Patent Application Publication WO 2013/ 172955, incorporated by reference herein.
- This modified assay protocol is particularly suitable for the study of solid tumors. Specifically, adherence of cancer cells to the well bottom is required for testing cancers and sarcomas that are not of blood or bone marrow origin because these cells require a permanent close contact with each other due to the nature of solid tumors. Accordingly, in a preferred embodiment, a method of determining the most effective drug candidate or combination of drug candidates for an individual patient, wherein a sample from a primary or metastatic site is tested using a MiCK assay that has been modified as described in this section.
- the MiCK assay may be modified, for example, by: a. overnight incubation for solid tumor sample specimens;
- cell isolation and plating can be modified as follows:
- a cell count from a pure cell suspension is used to adjust the cell concentration to 1 x 10 6 cells/mL.
- a test well is plated to observe the cell distribution. If the cells are not in good shape, more cells are added to each well. If the test well seems adequate (monolayer of uniformly distributed cells that covers the bottom of the well), one proceeds to the next step (plating). If the test well is not adequate, adjustment of the cell concentratio(en.g. ., diluting the cells or concentrating the cells) and retesting a new well is repeated until the cell distribution in the well is satisfactory.
- the stock solution is ready to be plated into additional wells in the plate until the cells are depleted.
- the cell suspension is distributed in the plate into as many wells as possible, retaining enough cells to do at least 1 cytospin and immunocytochemistry (ICC) if possible.
- An automated pipettor is used to distribute the cells while avoiding the edge wells of the plates; the edge wells are filled with media.
- Optimal liquid dispensing parameters were developed to prevent air bubble formation while the drugs are added to the wells. This feature is important as it eliminates the formation of bubbles in the media during the assay which artificially elevate the slope values which leads to markedly elevated KU values.
- the plate Once the plate has undergone the aforementioned steps, it is ready for overnight incubation (approximately 15 hour); allowing time for the cells to adhere to the bottom of the wells as well as to stabilize metabolically. After the incubation plate is removed from the incubator, the cell distribution and viability are evaluated from an observation of the plate with an inverted microscope. A photomicrograph of a representative well is taken and the plate is then ready for addition of the drugs ., po(es.gs.ible anti-cancer agents) by the automated pipettor. Drugs are selected by the treating oncologist (for example), and NCCN panels, then off panel drugs (off label).
- each well of the plate comprises a different anti-cancer drug candidate.
- the method also contemplates embodiments in which a different concentration of the anti-cancer drug candidate is contained in each well. Therefore, the present disclosure may relate to high-throughput assays by which multiple potential drug candidates at multiple potential concentration strengths may be simultaneously tested.
- the potential anti-cancer drug candidate concentration which may be loaded into each well of the assay will vary depending upon the manufacturer's recommended dosage and the corresponding dilutions required to achieve the concentration in the well that would correspond to this dosage.
- the target drug concentration in each well is determined by molarity and can range from 0.01 to 10,000 ⁇ , or 0.001 to 100,000 ⁇ , or 0.1 to 10,000 ⁇ for example, but could also deviate from these disclosed example ranges or comprise any integer contained within these ranges.
- One skilled in the art will understand how to achieve a target drug concentration by utilizing the manufacturer's recommended blood level concentrations, which may vary plus or minus one serial dilution if enough specimen cells are present.
- a trained observer may assess cytologic characteristics of cells at all stages of purification.
- a trained observer may also analyze ranking of drugs; analyze best drugs or combinations; and analyze most active drug candidates (may also include analyzing drug metabolites) and other developed drugs or agents.
- the cancer is a sarcoma, lymphoma, carcinoma, germ cell tumor and/or blastoma.
- the cancer is a lung cancer, a breast cancer, a liver cancer, a colon cancer, a pancreatic cancer, a colorectal cancer, an ovarian cancer, a uterine cancer, a testicular cancer, a prostate cancer, a central nervous system cancer, a cancer of the head and neck, an
- the cancer may be a solid tumor such as, but not limited to, follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to, colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer.
- follicular lymphomas such as, but not limited to, follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to, colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma
- the cancer may be a solid tumor including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma,
- chondrosarcoma osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
- adenocarcinomas cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
- hemangioblastoma hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
- Embodiments of the present invention may be utilized to test a wide variety of malignancies.
- the present disclosure may be used to test the following carcinomas:
- Ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometroid carcinoma), Ovarian granulosa cell tumor, Fallopian tube adenocarcinoma, Peritoneal carcinoma, Uterine (endometrial) adenocarcinoma, sarcomatoid carcinoma, Cervical squamous cell carcinoma, Endocervical adenocarcinoma, Vulvar carcinoma, Breast carcinoma, primary and metastatic (ductal carcinoma, mucinous carcinoma, lobular carcinoma, malignant phyllodes tumor), Head and neck carcinoma, Oral cavity carcinoma including tongue, primary and metastatic, Esophageal carcinoma, squamous cell carcinoma and adenocarcinoma, Gastric adenocarcinoma, malignant lymphoma, GIST, Primary small bowel carcinoma, Colonic adenocarcinoma, primary and metastatic (adenocarcinoma, mucinous carcinoma, large cell
- hepatocellular carcinoma, cholangiocarcinoma Metastatic carcinoma to the liver, Lung cancer, primary and metastatic (squamous cell, adenocarcinoma, adenosquamous carcinoma, giant cell carcinoma, non-small cell carcinoma, NSCLC, small cell carcinoma, neuroendocrine carcinoma, large cell carcinoma, bronchoalveolar carcinoma), Renal cell (kidney) carcinoma, primary and metastatic, Urinary bladder carcinoma, primary and metastatic, Prostatic adenocarcinoma, primary and metastatic, Brain tumors, primary and metastatic (glioblastoma, multiforme, cerebral neuroectodermal malignant tumor, neuroectodermal tumor, oligodendroglioma, malignant astrocytoma), Skin tumors (malignant melanoma, sebaceous cell carcinoma), Thyroid carcinoma (papillary and follicular), Thymic carcinoma, Shenoidal carcinoma, Carcinoma of unknown origin, primary and metastatic, Neuroendocrine carcinoma,
- the present disclosure may be used to test the following malignant lymphomas, for example: Large cell malignant lymphoma, Small cell lymphoma, Mixed large and small cell lymphoma, Malt lymphoma, Non Hodgkins malignant lymphoma, T cell malignant lymphoma, chronic myelogenous (or myeloid) leukemia (CML), myeloma, other leukemias, mesothelioma, mantle cell lymphomas, marginal cell lymphomas, lymphomas not otherwise specified as to type, and others.
- malignant lymphomas for example: Large cell malignant lymphoma, Small cell lymphoma, Mixed large and small cell lymphoma, Malt lymphoma, Non Hodgkins malignant lymphoma, T cell malignant lymphoma, chronic myelogenous (or myeloid) leukemia (CML), myeloma, other leukemias, mesothelioma, mantle cell lymphomas,
- the present disclosure may be used to test the following sarcomas, for example:
- Leimyosarcoma (uterine sarcoma), GIST-gastrointestinal stromal tumor, primary and metastatic (stomach, small bowel, colon), Liposarcoma, Myxoid sarcoma, Chondrosarcoma,
- Osteosarcoma Ewings sarcoma/PNET, Neuroblastoma, Malignant peripheral nerve sheath tumor, Spindle cell carcinoma, Embryonal rhabdomyosarcoma, Mesothelioma, and others. Cancer cell preparation
- cancer cells from solid tumor sites may be prepared by a method comprising: a. obtaining a tumor specimen;
- Patient A was a 70 year old male diagnosed with CLL (plasmatic differentiation) with no previous therapy.
- the sample collected was from whole blood.
- Patient B was a 66 year old male diagnosed with AML, in relapse at the time the sample was collected.
- the sample collected was from blood.
- Patient C was a 57 year old male diagnosed with stage IV NHL-low grade/ CLL, in relapse at the time the sample was collected.
- Patient C had previously been treated with Leukeran, MINE-Rituxan, Fludarabine-Cytoxan-Rituxan, Campath, Bendamustine, and arsenic.
- the sample collected was from peripheral blood.
- chemotherapy drugs were added to the wells of the 96 -well plate in 5 ⁇ L aliquots or to the wells of a 384-well plate in 2.5 ⁇ L aliquots using an automated pipettor.
- the number of drugs or drug combinations and the number of concentrations tested depended on the number of viable malignant cells that were isolated from the tumor specimen.
- the drug concentrations, determined by molarity, were those indicated by the manufacturer as the desired blood level concentration plus or minus one serial dilution if enough cells were available.
- Patient D was a 64 year old female diagnosed with metastatic breast cancer, in relapse at the time the sample was collected. The sample collected was obtained from liver and/or lung metastatic sites.
- Patient E was a 54 year old female diagnosed with invasive ductal carcinoma of the right breast, in subde progression at the time the sample was collected. The sample collected was obtained from primary uterine CA/pleural effusion. Patient E had previously been treated with Carboplatin, Taxol, cytarabine, Cisplatin, Navelbine, Abraxane and Avastin.
- Patient F was a 60 year old male diagnosed with lung adenocarcinoma with no previous chemotherapy. The sample collected was obtained from the lung.
- Patient G was a 79 year old female diagnosed with small cell lung carcinoma with no previous chemotherapy. The sample collected was obtained from the lung.
- Cisplatin alone produced a KU value of 4.3.
- a KU value of 5.6 was detected.
- Cisplatin was combined with Alimta a KU value of 2.9 was detected.
- Alimta is combined with Paclitaxel, a KU value of 4.7 is detected, which is greater than the predicted additive effect of Alimta (0.7 KU) and Paclitaxel (3.3 KU).
- Table 8 The results for all chemotherapeutics tested in this patient are included in Table 8.
- Cisplatin alone produced a KU value of 6.4.
- a KU value of 8.3 was detected.
- Pemetrexed alone produced a 1.4 KU value, indicating that the addition of Pemetrexed to Cisplatin produces more than just an additive effect.
- Adding Paclitaxel to Pemetrexed completely eliminated the effectiveness of Pemetrexed, as indicated by the 0.0 KU value detected for this combination despite Paclitaxel resulting in a KU value of 1.7 as a single agent.
- Patient H was a 55 year old female diagnosed with ovarian cancer. The sample collected was obtained from the uterus+ovaries.
- Patient I was a 66 year old female diagnosed with ovarian/ tubal cancer with no previous chemotherapy.
- Cisplatin alone produced a KU value of 9.5.
- a KU value of > 10.0 was detected.
- Gemcitabine alone is inactive (0.5 KU).
- gemcitabine is combined with Carboplatin
- Cisplatin alone produced a KU value of 5.4. However, when Cisplatin was combined with Gemcitabine, a KU value of 7.9 was detected. Gemcitabine alone produced a
- a patient with gastric cancer had a tumor sent independently for drug-induced apoptosis analysis as described (Salom et a/., ] Trans Med 2012; 10:162).
- Purified tumor cells were cultured for 48 hours with individual drugs, and drug-induced apoptosis was measured using the MiCK assay as described in Example 1. Data were obtained optically using Mie light-scattering.
- Patient J was a 64 year old female diagnosed with linitis plastic gastric adenocarcinoma, in relapse at the time the sample was collected. The sample collected was obtained from primary gastric CA/ ascitic fluid. Patient J had previously been treated with 5-Fu and XRT.
- chemotherapeutic combinations in an individual patient could be detected using the MiCK® assay in additional solid tumors.
- a patient with uterine cancer had a tumor sent independendy for drug-induced apoptosis analysis as described (Salom et a/., ] Trans Med 2012; 10:162).
- Purified tumor cells were cultured for 48 hours with individual drugs, and drug-induced apoptosis was measured using the MiCK assay as described in Example 1. Data were obtained optically using Mie light-scattering.
- Patient K was a female diagnosed with stage III clear cell uterine cancer, in relapse at the time the sample was collected.
- the sample collected was obtained from metastatic abdominal wall tissue.
- Patient K had previously been treated with carboplatin and Taxol, which resulted in complete response.
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Abstract
Provided are methods of using the MiCK assay to determine the most effective drug candidate(s) for an individual patient. Additionally, methods of identifying a drug candidate that increases or decreases the effectiveness of another drug candidate in an individual patient is provided, wherein an increase or a decrease in apoptosis produced by the drug combination compared to the apoptosis produced by the drug candidate alone is indicative of an increase or decrease in effectiveness, respectively.
Description
SYNERGISM AND ANTAGONISM BETWEEN MULTIPLE ANTI-CANCER AGENTS DETERMINED BY MiCK ASSAY
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of U.S. Provisional Application No. 61 /990,268, filed May 8, 2014, which is hereby incorporated by reference herein in its entirety.
BACKGROUND
1. Field
The present disclosure relates to use of a spectrophotometric apoptosis (MiCK) assay to determine synergism and/or antagonism between multiple anti-cancer agents.
2. Description of Related Art
Cell death may occur in a variety of ways, but most successful anti-cancer drugs tend to cause death of cancer cells by the very specific process of apoptosis. Apoptosis is a mechanism by which a cell disassembles and packages itself for orderly disposal by the body. Apoptosis is commonly used by the body to discard cells when they are no longer needed, are too old, or have become damaged or diseased. In fact, some cells with dangerous mutations that might lead to cancer, and even some early-stage cancerous cells, may undergo apoptosis as a result of natural processes.
During apoptosis, the cell cuts and stores DNA, condenses the nucleus, discards excess water, and undergoes various changes to the cell membrane, such as blebbing, the formation of irregular bulges in the cell membrane. {See FIG. 1) Apoptosis generally occurs after one of several triggers sends a signal to the cell that it should undergo apoptosis. In many cancer cells, this message system does not work correctly because the cell cannot detect the trigger, fails to send a signal properly after the trigger is received, or fails to act on the signal, or the cell may even have combinations of these problems. The overall effect is a resistance to undergoing apoptosis in some cancer cells.
Cancer, as used herein, includes all cancers or malignancies, both hematologic and non- hematologic, as well as myelodysplastic syndromes (MDS). This contemplates the four major
categories for all blood/ marrow cancers, solid tumors, and effusions: leukemia, lymphomas, epithelial malignancies, and mesenchymal malignancies.
Although many effective cancer drugs can induce cancerous cells to undergo apoptosis despite their resistance to the apoptotic process, no drug works against all types of cancer cells and no test predicts the relative efficacy of these drugs based on kinetic unit measurements of apoptosis. Accordingly, there is a need to detect whether a particular drug candidate can cause apoptosis in various types of cancer cells and also to determine the drug candidate's effectiveness as compared to other drugs or drug candidates, especially with regard to individual patients.
Traditionally, decisions about cancer treatment have been based on defining the anti-cancer drug that is most effective against most individuals with a certain type of cancer, resulting in everyone with a particular cancer receiving the same treatment. However, this ignores that many individuals do not respond to standard chemotherapy. Given the consequences of treating an individual with chemotherapeutics that ultimately will not benefit that individual, there is a need for methods to determine the most effective chemotherapeutic drug for an individual prior to initiating a treatment protocol.
Additionally, some patients benefit from combinations of chemotherapeutic drugs with different mechanisms of action. However, there is no reliable method available to identify, for an individual tumor, which combinations of chemotherapeutic drugs will be effective. Since chemotherapeutics are known to cause significant side effects, it is essential to only administer chemotherapeutic combinations that will ultimately benefit the patient treated.
The Microculture Kinetic Assay (MiCK assay), described in U.S. Patent 6,077,684 and U.S. Patent 6,258,553, is currently used to detect whether cancer cells from a patient undergo apoptosis in response to a particular drug known to be effective against specific cancer types. In the MiCK assay, cancer cells from a patient are placed in a suspension of a given
concentration of single cells or small cell clusters and allowed to adjust to conditions in multiple wells of a microtiter plate. Control solutions or solutions with various concentrations of known anti-cancer drugs, typically those drugs recommended for the patient's cancer type, are introduced into the wells with one test sample per well. The optical density of each well is then measured periodically, typically every few minutes, for a period of hours to days. As a cell undergoes apoptosis-related blebbing, its optical density increases in a detectable and specific fashion. If the cell does not undergo apoptosis or dies from other causes, its optical density does not change in this manner. Thus, if a plot of optical density (OD) versus time for a well
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yields a straight line curve having a positive slope over the time, followed by a plateau and/ or a negative slope, then the anti-cancer drug in that well induces apoptosis of the patient's cancer cells and might be a suitable therapy for that patient. OD versus time data may also be used to calculate kinetic units, the units which can be used to measure apoptosis, which similarly correlate with the suitability of a therapy for the patient. One of ordinary skill in the art will be familiar with the aforementioned general description of the MiCK assay. Further, the contents of U.S. Patent 6,077,684 and U.S. Patent 6,258,553, are herein incorporated by reference in their entirety for all purposes, and provide a more detailed description of the MiCK assay.
The solution to this technical problem is provided by the embodiments characterized in the claims.
BRIEF SUMMARY
The present application relates to methods of using the MiCK assay to determine the most effective drug candidate or combination of drug candidates for an individual patient. The method may include placing a single-cell suspension of viable cancer cells obtained from a tumor site in an individual patient in at least one well of a plate suitable to be read by a spectrophotometer, wherein the cancer cells are in a concentration sufficient to form a monolayer of cells on the bottom of the well, adding at least one drug candidate to the well in an amount sufficient to achieve a target drug candidate concentration, measuring the optical density of the well at a wavelength of approximately 600 nm using a spectrophotometer at selected time intervals for a selected duration of time, determining a kinetic units (KU) value from the optical density and time measurements, and correlating the KU value with an ability of the anti-cancer drug candidate to induce apoptosis in the cancer cells if the KU value is positive, or an inability of the anti-cancer drug candidate to induce apoptosis in the cancer cells if the KU value is not positive.
According to additional embodiments, a method is provided for determining the most effective drug candidate or combination of drug candidates for an individual patient, wherein a sample from a primary tumor site or a metastasis of the primary tumor site is tested using the MiCK assay.
According to a more specific embodiment, a method of identifying a drug candidate that decreases the effectiveness of another drug candidate in an individual patient is provided. The method may include placing a single-cell suspension of viable cancer cells obtained from a
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tumor site in an individual patient in at least three wells of a plate suitable to be read by a spectrophotometer, , adding a drug candidate to one well in an amount sufficient to achieve a target drug candidate concentration, adding a different drug candidate to a different well in an amount sufficient to achieve a target drug candidate concentration, adding a drug combination to a different well in an amount sufficient to achieve a target drug candidate concentration, measuring the optical density of the wells at a wavelength of approximately 600 nm using a spectrophotometer at selected time intervals for a selected duration of time, determining a kinetic units (KU) value for each well from the optical density and time measurements, and correlating the KU value with an ability of the anti-cancer drug candidate or anti-cancer drug combination to induce apoptosis in the cancer cells if the KU value is positive, or an inability of the anti-cancer drug candidate or anti-cancer drug combination to induce apoptosis in the cancer cells if the KU value is not positive. A decrease in effectiveness of the drug candidate is indicated by a decrease in the KU value of the drug combination compared to the KU value of the drug candidate alone.
According to an additional specific embodiment, a method of identifying a drug candidate that increases the effectiveness of another drug candidate in an individual patient is provided. The method may include placing a single-cell suspension of viable cancer cells obtained from a tumor site in an individual patient in at least three wells of a plate suitable to be read by a spectrophotometer, , adding a drug candidate to one well in an amount sufficient to achieve a target drug candidate concentration, adding a different drug candidate to a different well in an amount sufficient to achieve a target drug candidate concentration, adding a drug combination to a different well in an amount sufficient to achieve a target drug candidate concentration, measuring the optical density of the wells at a wavelength of approximately 600 nm using a spectrophotometer at selected time intervals for a selected duration of time, determining a kinetic units (KU) value for each well from the optical density and time measurements, and correlating the KU value with an ability of the anti-cancer drug candidate or anti-cancer drug combination to induce apoptosis in the cancer cells if the KU value is positive, or an inability of the anti-cancer drug candidate or anti-cancer drug combination to induce apoptosis in the cancer cells if the KU value is not positive. An increase in effectiveness of the drug candidate is indicated by an increase in the KU value of the drug combination compared to the KU value of the drug candidate alone.
According to a further specific embodiment, the drug candidate is an individual anti-cancer drug.
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According to a further specific embodiment, the drug candidate is a combination of anti-cancer drugs.
According to a further specific embodiment, the cancer cells are from a blood cancer. In a preferred embodiment, the blood cancer is selected from the group consisting of acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and non-Hodgkin's lymphoma (NHL).
According to a further specific embodiment, the cancer cells are from a solid tumor. In a preferred embodiment, the solid tumor is selected from the group consisting of breast cancer, lung cancer, ovarian cancer, gastric cancer, and uterine cancer.
The following abbreviations and terms are used commonly throughout this Specification:
OD— optical density.
MiCK— microculture kinetic.
KU— kinetic unit.
BRIEF DESCRIPTION OF THE DRAWINGS
For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements.
FIG. 1 shows a time sequences photomicrograph of a cancer cell moving through the stages of apoptosis. The first panel of the left (1) shows the cell prior to apoptosis. The middle panel (2) shows the cell during apoptosis and blebbing is apparent. The last panel on the right (3) shows the cell after apoptosis is complete or nearly complete.
FIG. 2 is a graph showing representative curves for induction of apoptosis, drug resistance, and control cells without drug in a MiCK assay. The curve labeled "B12" shows data representative of cells in which the drug induces apoptosis. The curve labeled "F3" shows data representative of cells that are resistant to the drug. The curve labeled "G5" shows data representative of control cells that did not receive any drug.
FIG. 3 is a graph showing representative data for induction of apoptosis or necrosis in a MiCK assay. The curve labeled "D2" shows data representative of cells in which the drug induces
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apoptosis. The curve labeled "D7" shows data representative of cells in which the drug induces necrosis or which otherwise undergoes necrosis during the course of the assay.
FIG. 4 is a graph showing representative data for general non-drug-induced cell death in a MiCK assay. The curve labeled "C4" shows data representative of spontaneous cell death during the course of the assay.
DETAILED DESCRIPTION
Before the subject disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments of the disclosure described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present disclosure will be established by the appended claims.
In this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.
Surprisingly, the data provided herein demonstrate substantial variability between individual patients regarding the drug candidate(s) that induce the highest degree of apoptosis in the individual patients' cancer cells. Additionally, it is commonly thought that combining drug candidates creates at least an additive effect on apoptosis. However, it is shown herein that certain anti-cancer drugs can produce synergistic effects in combination with other anti-cancer drugs in individual patients. Furthermore, the same anti-cancer drug can produce an antagonistic effect in combination with a different anti-cancer drug in the same individual patient. These effects are different in different individuals, even in individuals with related cancers. Therefore, there is a substantial need for methods to identify the most effective drug candidate or drug combination in an individual patient prior to initiation of treatment.
The subject disclosure features, in one aspect, methods of using an assay similar to the microculture kinetic (MiCK) assay, as disclosed in U.S. Patent 6,077,684 and U.S. Patent 6,258,553, both incorporated by reference herein, to determine the most effective
chemotherapeutic drug or combination of chemotherapeutic drugs for an individual patient.
General MiCK Assay Protocol
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According to one specific embodiment, the assay may proceed by selecting an anti-cancer drug candidate and selecting at least one tumor on which to test the drug. Purified cancer cells are obtained from the tumor and the cancer cells may be suspended as a single-cell suspension in culture medium, such as RPMI. As used herein, a "single cell suspension" is a suspension of one or more cells in a liquid in which the cells are separated as individual cells or in clumps of usually 10 cells or fewer. The culture medium may contain other components, such as fetal- bovine serum or components specifically required by the cancer cells. These components may be limited to those necessary to sustain the cells for the duration of the assay, typically at least 24 hours and not longer than 120 hours.
Suspended cells may be tested by placing samples in wells of a spectrophotometric plate. The cells may be suspended at any concentration such that during the spectrophotometric measurements of OD, the beam of the plate reader normally passes through only one cell layer at a time. For most cells, a concentration of between 2 X 105 and 1 X 106 cells/mL may be used. Concentration may be increased for small cells and decreased for large cells. To more precisely determine the appropriate cell concentration, the volume of cell suspension to be used in drug candidate test samples may be added to at least one concentration test well of the plate. If the well will be prefilled with additional medium during testing of the drug candidates, then the concentration test well may similarly be prefilled with additional medium. After the concentration test well is filled, the plate may be centrifuged (e.g. for 30 sec to 2 min at 500 RPM) to settle the cells on the bottom of the well. If the cell concentration is appropriate for the assay, the cells should form a monolayer without overlapping. Cell concentration may be adjusted as appropriate until this result is achieved. Multiple concentrations of cells may be tested at one time using different concentration test wells.
According to embodiments where the cells may grow significantly overnight or during another period of time between placement of the cells in the plate and commencement of the drug candidate assay, the cell concentration may be adjusted to initially achieve less than a monolayer to allow for growth such that sufficient cells for a monolayer will be present when the drug candidate assay commences.
The cancer cells may be in an exponential or a non-exponential growth phase. In a specific embodiment, particularly when the cancer cells are from a cancer cell line, they may be in an exponential growth phase.
After the appropriate cell concentration has been determined, the drug-candidate assay may proceed by filling test and control wells in the plate with an appropriate volume of medium and
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an appropriate number of cells. In other embodiments, the well may be partially pre-filled with medium alone.
After filling, the cells may be allowed to adjust to the plate conditions for a set period of time, such as at least 12 hours, at least 16 hours, at least 24 hours, or 12-16 hours, 12-24 hours, or 16- 24 hours. The adjustment period is typically short enough such that the cells do not experience significant growth during the time. The adjustment period may vary depending on the type of cancer cells used in the drug candidate assay. Adjustment may take place under conditions suitable to keep the cells alive and healthy. For example, the plate may be placed in a humidified incubator at 37°C under 5% C02 atmosphere. For some cell types, particularly cell types that do not undergo an adjustment period, the plate may be centrifuged (e.g. for 30 sec to 2 minutes at 500 RPM) to settle the cells on the bottom of the wells.
The drug candidate and any control drugs or other control samples may be added to the wells after the adjustment period. Typically the drug candidate will be added in a small volume of medium or other liquid as compared to the total volume of liquid in the well. For example, the volume of drug added may be less than 10% of the total volume of liquid in the well. Drug candidates may be added in multiple dilutions to allow determination of any concentration effects. Although many drug candidates may be water-soluble, drug candidates that are not readily soluble in water may also be tested. Such candidates may be mixed with any appropriate carrier. Such candidates may preferably be mixed with carriers anticipated for actual clinical use. Viscous drug candidates may require substantial dilution in order to be tested. Drug candidates with a strong color may benefit from monitoring of OD in test wells containing only the drug candidate and subtraction of this OD from measurements for the test sample wells.
After addition of the drug candidate, the cells may be allowed another short period of adjustment, for example of 15 minutes or 30 minutes. The cells may be placed under conditions suitable to keep the cells alive and healthy. For example, the plate may be placed in a humidified incubator at 37°C under 5% C02 atmosphere. After this short adjustment period, a layer of mineral oil may be placed on top of each well to maintain C02 in the medium and prevent evaporation.
The plate may then be placed in a spectrophotometer configured to measure the OD at a wavelength of 600 nm for each well at a given time interval for a given total period of time. For example, OD for each well may be measured periodically over a time frame of seconds, minutes, or hours for a period of between 24 and 120 hours. For certain cells, measurements for a period of as little as 12 hours may be sufficient. In specific embodiments, measurements
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may be taken every 5 to 10 minutes. The spectrophotometer may have an incubated chamber to avoid spontaneous death of the cells.
Spectrophotometric data may be converted to kinetic units. Kinetic units are determined by the slope of the curve created when the change in the OD at 600 nm caused by cell blebbing is plotted as a function of time. Specific information regarding the calculation of kinetic units is provided in Kratsov, Vladimir D. et al., Use of the Microculture Kinetic Assay of Apoptosis to Determine Chemosensitivities of Leukemias, Blood 92:968-980 (1998), incorporated by reference herein. Optical density for a given drug candidate at a given concentration may be plotted against time. This plot gives a distinctive increasing curve if the cells are undergoing apoptosis. An example of the curve obtained when cells undergo apoptosis is shown in FIGs 2 and 3. In comparison, if the drug candidate has no effect on the cells(e.g. . they are resistant), then the curve is similar to that obtained for a control sample with no drug or drug candidate (FIG. 2). Cell death due to reasons other than apoptosis can also be determined by the current assay and is useful in eliminating false positives from drug candidate screening. For example, cell necrosis produces a distinctive downward sloping curve easily distinguishable from the apoptosis-related curve as seen in FIG. 3. Further, general cell death also causes a downward curve as seen in FIG. 4.
Kinetic Units of Apoptosis (KU)
The effectiveness of a drug candidate may be determined by the value of the kinetic units it produces in a modified MiCK assay using a known cell line. Kinetic units may be determined as follows:
Apoptosis (KU) = (VmaxDrug Candidate Treated - VmaxControl) x 60 x X/(ODcontrol - ODblank)
The KU is a calculated value for quantifying apoptosis. The optical densities (OD) from each well are plotted against time. The maximum slope of the apoptotic curve (V max) is calculated for each plot of drug-treated microculture. It is then compared to the Vmax of a control well without drug (calculated at the same time as the Vmax of the drug exposed cells). For convenience, the Vmax is multiplied by 60 to convert the units from mOD/minute to mOD/hour. The data are normalized with a coefficient (coefficient = X / (ODcontrol— ODblank), which is discussed below.
Coefficient
As stated above, the coefficient is a calculated value for normalizing the amount of cells per well when measuring apoptosis and quantifying said apoptosis in Kinetic Units.
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The coefficient is calculated as follows:
Coefficient = X / (ODcontrol - ODblank)
X = optimal optical density value for the cell type tested (determined empirically).
ODcontrol = average optical density of all the control wells.
ODblank = average optical density of all the blank wells.
A coefficient of 1.000 means that the cell concentration is optimal. A coefficient value below 1.000 means that the cell concentration is higher than the optimal concentration. If the coefficient value is above 1.000, it means that the cell concentration in the well is suboptimal. The acceptable coefficient values for an optimal MiCK assay are between 0.8 and 1.5. If the value is under 0.8, the coefficient will erroneously reduce the value of the calculated KU. If the value is above 1.5, there will not be enough cells per well to detect the signal of apoptosis. The "X" in the formula will vary depending on the cell type. For solid tumor specimens, this value is 0.09. For most of the leukemias, the value is 0.15. For CLL (chronic lymphocytic leukemias) and the lymphomas, the value is 0.21.
This "X" value is adapted to the tumor type and determined empirically. Thus, the coefficient is developed by trial and error, using different concentrations of cells and by checking them under a microscope while looking for complete proper coverage in the well. The proper well is read by a reader and the OD becomes the new X value. Further information regarding this equation may be found in Kravtsov et al. (Blood, 92:968-980), which was previously incorporated herein by reference.
In addition to allowing determinations of whether or not a drug candidate causes apoptosis, kinetic unit values generated using the current assay may be compared to determine if a particular drug candidate may also be performed and may give general indications of appropriate dosage. Occasionally some drugs may perform less well at higher concentrations than lower concentrations in some cancers. Comparison of kinetic unit values for different concentrations of drug candidates may identify drug candidates with a similar profile.
Overall, evaluation of an anti-cancer drug candidate may include any determination of the effects of that drug candidate on apoptosis of a cancer cell. Effects may include, but are not limited to induction of apoptosis, degree of induction of apoptosis as compared to known cancer drugs, degree of induction of apoptosis at different drug candidate concentrations, and failure to induce apoptosis. The anti-cancer drug evaluation assay may also be able to detect
non-drug-related or non-apoptotic events in the cancer cells, such as cancer cell growth during the assay or cell necrosis.
Any statistically significant positive kinetic unit value above 1.0 KU may indicate some tendency of a drug candidate to induce apoptosis of a cancer cell. For many clinical purposes, however, drug candidates or concentrations of drugs only able to induce very low levels of apoptosis are not of interest. Accordingly, in certain embodiments of the disclosure, threshold kinetic unit values may be set to distinguish drug candidates able to induce clinically relevant levels of apoptosis in cancer cells. For example, the threshold amount may be 1.5, 2 or 3 kinetic units. The actual threshold selected for a particular drug candidate or concentration of drug candidate may depend on a number of factors. For example, a lower threshold, such as 1.5 or 2, may be acceptable for a drug candidate able to induce apoptosis in cancer types that do not respond to other drugs or respond only to drugs with significant negative side effects. A lower threshold may also be acceptable for drug candidates that exhibit decreased efficacy at higher concentrations or which themselves are likely to have significant negative side effects. A higher threshold, such as 3, may be acceptable for drug candidates able to induce apoptosis in cancer types for which there are already suitable treatments.
In another embodiment, the following threshold ranges can be utilized:
0- 1 KU = non-sensitive;
1- 2 KU = low sensitivity;
2-3 KU = low/ moderate sensitivity;
3-5 KU = moderate sensitivity; and
>5 KU = sensitive.
Preferably, the following threshold ranges can be utilized:
0-1 KU = non- sensitive;
1-2.6 KU = low sensitivity;
2.6-4.2 KU = low/moderate sensitivity;
4.2-5.8 KU = moderate sensitivity; and
>5.8 KU = very sensitive.
Preferably, the KU value is≥ 7, more preferably the KU value is≥ 8, even more preferably the KU value is≥ 9, and most preferably the KU value is≥ 10.
These ranges were established based on a statistical analysis of cancer cells. The ranges establish a baseline for relative comparison of chemotherapeutic drugs being tested on a specific cell type. Test outcomes may be affected by extenuating factors such as: time elapsed from obtaining sample to testing; quantity of viable cells available to test; microbial contamination of specimen; quality or viability of cells being tested; cell type; and recent treatment such as chemotherapy or radiation therapy.
These factors suggest some elasticity in the predictive values of the kinetic response reported. Clinical sensitivity to chemotherapy drugs is not completely limited to outcomes as forecast in the above ranges. The KU measurement of drug-induced apoptosis in the assay may be used by physicians to develop an individual patient treatment regimen along with other important factors such as patient history, prior treatment results, overall patient health, patient
comorbidities, patient preferences, as well as other clinical factors.
Therefore, the particular ranges of KU value utilized will be dependent upon context. That is, depending upon the particular type of tumor cell being tested, the particular drug being utilized, and the particular patient or patient population under analysis. The KU value therefore represents a dependable and flexible analytical variable that can be tailored by the practitioner of the disclosed methods to create a suitable metric by which to evaluate a given drug's effect.
Drug Candidates
According to a specific embodiment, the anti-cancer drug candidates may be any chemical, chemicals, compound, compounds, composition, or compositions to be evaluated for the ability to induce apoptosis in cancer cells. These candidates may include various chemical or biological entities such as chemotherapeutics, other small molecules, protein or peptide-based drug candidates, including antibodies or antibody fragments linked to a chemotherapeutic molecule, nucleic acid-based therapies, other biologies, nanoparticle-based candidates, and the like. Drug candidates may be in the same chemical families as existing drugs, or they may be new chemical or biological entities.
Drug candidates are not confined to single chemical, biological or other entities. They may include combinations of different chemical or biological entities, for example, proposed combination therapies. Further, although many examples herein relate to an assay in which a single drug candidate is applied, assays may also be conducted for multiple drug candidates in combination. It is also important to understand that embodiments of the invention may utilize the metabolites of the various drug candidates in a method as described.
Embodiments of the invention are able to test all manner of anti-cancer drug candidates. For example, the following anti-cancer drug candidates can be tested by the disclosed methods: Abraxane, Afatinib, Alimta, Amsacrine, Asparaginase, BCNU, Bendamustine, Bleomycin, Bosutinib, Caelyx (Doxil), Carboplatin, Carmustine, CCNU, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Cytarabine, Cytoxan (4HC), Dacarbazine, Dactinomycin, Dasatinib, Daunorubicin, Decitabine, Dexamethasone, Doxorubicin, Epirubicin, Estramustine, Etoposide, Fludarabine, 5- Fluorouracil, Gemcitabine, Gleevec (imatinib), Hexamethylmelamine, Hydroxyurea, Idarubicin, Ifosfamide (4HI), Interferon-2a, Irinotecan, Ixabepilone, Melphalan, Mercaptopurine,
Methotrexate, Mitomycin, Mitoxantrone, Nilotinib, Nitrogen Mustard, Oxaliplatin, Pentostatin, Sorafenib, Streptozocin, Sunitinib, Tarceva, Taxol, Taxotere, Temozolomide, Temsirolimus, Teniposide, Thalidomide, Thioguanine, Topotecan, Tretinoin, Velcade, Vidaza, Vinblastine, Vincristine, Vinorelbine, Vorinostat, Xeloda (5DFUR), Everolimus, Lapatinib, Lanalidomide, Rapamycin, and Votrient (Paxopanib).
However, many other anti-cancer drug candidates, including but not limited to other nonchemotherapy drugs and/ or chemicals which can produce apoptosis or which are examined for their ability to produce apoptosis, are also able to be tested by the disclosed methods.
Further still, the methods of the present invention are not strictly applicable to anti-cancer drug candidates, but rather embodiments of the disclosed methods can be utilized to test any number of potential drug candidates for a whole host of diseases.
More than one drug candidate, concentration of drug candidate, or combination of drugs or drug candidates may be evaluated in a single assay using a single plate. Different test samples may be placed in different wells. The concentration of the drug candidate tested may be, in particular embodiments, any concentration in the range from 0.1 to ΙΟ,ΟΟΟμΜ, or any concentration in the range from 0.01 to 10,000μΜ, or any concentration in the range from 0.001 to 100,000μΜ, for example. The concentration tested may vary by drug type, and the aforementioned example concentrations are not to be considered as limiting, for the skilled artisan will understand how to construct the appropriate concentration for utilization with the taught methods and assays, depending upon the particular anti-cancer drug tested.
Plate and Spectrophotometer Systems
In specific embodiments, the plate and spectrophotometer may be selected such that the spectrophotometer may read the plate. For example, when using older spectrophotometers, one may use plates with larger wells because the equipment is unable to read smaller-well plates. Newer spectrophotometers may be able to read a plate with smaller wells. In one embodiment,
the diameter of the bottom of each well is no smaller than the diameter of the light beam of the spectrophotometer. In a more specific embodiment, the diameter of the bottom of each well is no more than twice the diameter of the light beam of the spectrophotometer. This helps ensure that the OD at the measured wavelength, 600 nm for example, of a representative portion of the cells in each well is accurately read. The spectrophotometer may make measurements at wavelengths of than 600 nm. For example, the wavelength may be +/- 5 or +/- 10. However, other wavelengths may be selected so as to be able to distinguish blebbing.
Spectrophotometers may include one or more computers or programs to operate the equipment or to record the results. In one embodiment, the spectrophotometer may be functionally connected to one or more computers able to control the measurement process, record its results, and display or transmit graphs plotting the optical densities as a function of time for each well.
Plates designed for tissue culture may be used, or other plates may be sterilized and treated to make them compatible with tissue culture. Plates that allow cells to congregate in areas not accessible to the spectrophotometer, such as in corners, may work less well than plates that avoid such congregation. Alternatively, more cancer cells may be added to these plates to ensure the presence of a monolayer accessible to the spectrophotometer during the assay. Plates with narrow bottoms, such as the Corning Costar® half area 96 -well plate, may also assist in encouraging formation of a monolayer at the bottom of the well without requiring inconveniendy low sample volumes. Other plates, such as other 96-well plates of smaller well plates, such as 384-well plates, may also be used.
ModiGed MiCK Assay Protocol
A modified MiCK assay protocol has recendy been developed as described in International Patent Application Publication WO 2013/ 172955, incorporated by reference herein. This modified assay protocol is particularly suitable for the study of solid tumors. Specifically, adherence of cancer cells to the well bottom is required for testing cancers and sarcomas that are not of blood or bone marrow origin because these cells require a permanent close contact with each other due to the nature of solid tumors. Accordingly, in a preferred embodiment, a method of determining the most effective drug candidate or combination of drug candidates for an individual patient, wherein a sample from a primary or metastatic site is tested using a MiCK assay that has been modified as described in this section.
In particular, the MiCK assay may be modified, for example, by:
a. overnight incubation for solid tumor sample specimens;
b. use of low volume wells since solid tumors usually give fewer cells than blood samples; c. adjusting cell concentration via visual interpretation;
d. allowing cells to adhere to the bottom of the wells and spread/ stretch overnight;
e. utilization of a special incubation chamber to diffuse heat evenly;
f. avoiding the edges of the plates when one loads the cells into the wells;
g. utilization of an automated pipettor to plate the cells, media (e.g.., RPMI + 10% Fetal Bovine Serum + Penstrep) and drugs; and
h. utilization of proprietary code created to translate template in a format that a robot can understand.
In addition, cell isolation and plating can be modified as follows:
A cell count from a pure cell suspension is used to adjust the cell concentration to 1 x 106 cells/mL. A test well is plated to observe the cell distribution. If the cells are not in good shape, more cells are added to each well. If the test well seems adequate (monolayer of uniformly distributed cells that covers the bottom of the well), one proceeds to the next step (plating). If the test well is not adequate, adjustment of the cell concentratio(en.g. ., diluting the cells or concentrating the cells) and retesting a new well is repeated until the cell distribution in the well is satisfactory.
After the aforementioned steps, the stock solution is ready to be plated into additional wells in the plate until the cells are depleted. Using the selected cell concentration, the cell suspension is distributed in the plate into as many wells as possible, retaining enough cells to do at least 1 cytospin and immunocytochemistry (ICC) if possible.
An automated pipettor is used to distribute the cells while avoiding the edge wells of the plates; the edge wells are filled with media. Optimal liquid dispensing parameters were developed to prevent air bubble formation while the drugs are added to the wells. This feature is important as it eliminates the formation of bubbles in the media during the assay which artificially elevate the slope values which leads to markedly elevated KU values.
Once the plate has undergone the aforementioned steps, it is ready for overnight incubation (approximately 15 hour); allowing time for the cells to adhere to the bottom of the wells as well as to stabilize metabolically.
After the incubation plate is removed from the incubator, the cell distribution and viability are evaluated from an observation of the plate with an inverted microscope. A photomicrograph of a representative well is taken and the plate is then ready for addition of the drugs ., po(es.gs.ible anti-cancer agents) by the automated pipettor. Drugs are selected by the treating oncologist (for example), and NCCN panels, then off panel drugs (off label).
In some embodiments, each well of the plate comprises a different anti-cancer drug candidate. Further, the method also contemplates embodiments in which a different concentration of the anti-cancer drug candidate is contained in each well. Therefore, the present disclosure may relate to high-throughput assays by which multiple potential drug candidates at multiple potential concentration strengths may be simultaneously tested.
The potential anti-cancer drug candidate concentration which may be loaded into each well of the assay will vary depending upon the manufacturer's recommended dosage and the corresponding dilutions required to achieve the concentration in the well that would correspond to this dosage. For example, the target drug concentration in each well is determined by molarity and can range from 0.01 to 10,000 μΜ, or 0.001 to 100,000 μΜ, or 0.1 to 10,000 μΜ for example, but could also deviate from these disclosed example ranges or comprise any integer contained within these ranges. One skilled in the art will understand how to achieve a target drug concentration by utilizing the manufacturer's recommended blood level concentrations, which may vary plus or minus one serial dilution if enough specimen cells are present.
Once the drug candidates are added to the wells, an incubation of 30 minutes at 37°C and 5% C02 is done to allow for pH equilibration; oil is added to every well to prevent air exchange and evaporation; the plate is placed in a reader and the assay is started; the assay automatically terminates after 576 reads (48 hours, 5 min intervals). These settings can be adjusted as needed and the assay can be manually terminated if all the reactions are deemed to have been completed prior to the 48 hours.
A trained observer may assess cytologic characteristics of cells at all stages of purification. A trained observer may also analyze ranking of drugs; analyze best drugs or combinations; and analyze most active drug candidates (may also include analyzing drug metabolites) and other developed drugs or agents.
Cancer cells
The methods described herein may be used to determine the most effective drug candidate for the treatment of an individual cancer patient. In a preferred embodiment, the cancer is a
sarcoma, lymphoma, carcinoma, germ cell tumor and/or blastoma. In a further preferred embodiment, the cancer is a lung cancer, a breast cancer, a liver cancer, a colon cancer, a pancreatic cancer, a colorectal cancer, an ovarian cancer, a uterine cancer, a testicular cancer, a prostate cancer, a central nervous system cancer, a cancer of the head and neck, an
endothelioma, an osteoblastoma, an osteoclastoma, Ewing's sarcoma and/or Kaposi's sarcoma. In a specific embodiment, the cancer may be a solid tumor such as, but not limited to, follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to, colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer.
In a further preferred embodiment , the cancer may be a solid tumor including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Embodiments of the present invention may be utilized to test a wide variety of malignancies. For example, the present disclosure may be used to test the following carcinomas:
Ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometroid carcinoma), Ovarian granulosa cell tumor, Fallopian tube adenocarcinoma, Peritoneal carcinoma, Uterine (endometrial) adenocarcinoma, sarcomatoid carcinoma, Cervical squamous cell carcinoma, Endocervical adenocarcinoma, Vulvar carcinoma, Breast carcinoma, primary and metastatic (ductal carcinoma, mucinous carcinoma, lobular carcinoma, malignant phyllodes
tumor), Head and neck carcinoma, Oral cavity carcinoma including tongue, primary and metastatic, Esophageal carcinoma, squamous cell carcinoma and adenocarcinoma, Gastric adenocarcinoma, malignant lymphoma, GIST, Primary small bowel carcinoma, Colonic adenocarcinoma, primary and metastatic (adenocarcinoma, mucinous carcinoma, large cell neuroendocrine carcinoma, colloid carcinoma), Appendiceal adenocarcinoma, Colorectal carcinoma, Rectal carcinoma, Anal carcinoma (squamous, basaloid), Carcinoid tumors, primary and metastatic (appendix, small bowel, colon), Pancreatic carcinoma, Liver carcinoma
(hepatocellular carcinoma, cholangiocarcinoma), Metastatic carcinoma to the liver, Lung cancer, primary and metastatic (squamous cell, adenocarcinoma, adenosquamous carcinoma, giant cell carcinoma, non-small cell carcinoma, NSCLC, small cell carcinoma, neuroendocrine carcinoma, large cell carcinoma, bronchoalveolar carcinoma), Renal cell (kidney) carcinoma, primary and metastatic, Urinary bladder carcinoma, primary and metastatic, Prostatic adenocarcinoma, primary and metastatic, Brain tumors, primary and metastatic (glioblastoma, multiforme, cerebral neuroectodermal malignant tumor, neuroectodermal tumor, oligodendroglioma, malignant astrocytoma), Skin tumors (malignant melanoma, sebaceous cell carcinoma), Thyroid carcinoma (papillary and follicular), Thymic carcinoma, Shenoidal carcinoma, Carcinoma of unknown origin, primary and metastatic, Neuroendocrine carcinoma, Testicular malignancies (seminoma, embryonal carcinoma, malignant mixed tumors), and others.
The present disclosure may be used to test the following malignant lymphomas, for example: Large cell malignant lymphoma, Small cell lymphoma, Mixed large and small cell lymphoma, Malt lymphoma, Non Hodgkins malignant lymphoma, T cell malignant lymphoma, chronic myelogenous (or myeloid) leukemia (CML), myeloma, other leukemias, mesothelioma, mantle cell lymphomas, marginal cell lymphomas, lymphomas not otherwise specified as to type, and others.
Further still, the present disclosure may be used to test the following sarcomas, for example:
Leimyosarcoma (uterine sarcoma), GIST-gastrointestinal stromal tumor, primary and metastatic (stomach, small bowel, colon), Liposarcoma, Myxoid sarcoma, Chondrosarcoma,
Osteosarcoma, Ewings sarcoma/PNET, Neuroblastoma, Malignant peripheral nerve sheath tumor, Spindle cell carcinoma, Embryonal rhabdomyosarcoma, Mesothelioma, and others. Cancer cell preparation
In a further embodiment, cancer cells from solid tumor sites may be prepared by a method comprising:
a. obtaining a tumor specimen;
b. mincing, digesting, and filtering the specimen;
c. optionally removing non-viable cells by density gradient centrifugation;
d. incubating the cell suspension to remove macrophages by adherence;
e. performing positive, negative, and/ or depletion isolation to isolate the cells of interest; f. removing any remaining macrophages, if necessary, using CD14 antibody conjugated magnetic beads; and
g. plating and testing the final cell suspension as described herein.
EXAMPLES
The following Examples describe exemplary embodiments of the invention. These Examples should not be interpreted to encompass the entire breadth of the invention.
EXAMPLE 1 - Detection of Synergistic and Antagonistic Chemotherapeutic
Combinations in Blood Cancers.
Background:
Blinded clinical trials have demonstrated higher response rates and longer survival in groups of patients with acute myelocytic leukemia and epithelial ovarian cancer who have been treated with drugs that show high apoptosis as determined by the MiCK assay (Bosserman et al., Cancer Res 2012; 72:3901-3905). Thus, the MiCK assay provides great potential to guide treatment decisions in individual patients. This study aimed to detect the most effective combinations of chemotherapeutic drugs in an individual patient.
Methods:
Summary: Patients with different blood cancers had blood samples sent independently for drug-induced apoptosis analysis as described (Salom et a/., J Trans Med 2012; 10:162). Purified tumor cells were cultured for 48 hours with individual drugs, and drug-induced apoptosis was measured using the MiCK assay as described herein. Data were obtained optically using Mie light- scattering. Results from paired tumor sites in individual patients were compared and analyzed statistically. Active apoptosis (sensitivity) was defined as >= 1.0 kinetic units (KU),
and no apoptosis (resistance) was < 1.0 KU, and 2 standard deviations (SD) in the assay were 1.14 KU.
Patients: Three patients with different blood cancers (AML, CLL, and NHL) were included in this study. Patient A was a 70 year old male diagnosed with CLL (plasmatic differentiation) with no previous therapy. The sample collected was from whole blood. Patient B was a 66 year old male diagnosed with AML, in relapse at the time the sample was collected. The sample collected was from blood. Patient C was a 57 year old male diagnosed with stage IV NHL-low grade/ CLL, in relapse at the time the sample was collected. Patient C had previously been treated with Leukeran, MINE-Rituxan, Fludarabine-Cytoxan-Rituxan, Campath, Bendamustine, and arsenic. The sample collected was from peripheral blood.
MiCK Assay protocol:
The MiCK assay procedure was adapted from the method described in U.S. Patent No.
6,077,684 and U.S. Patent No. 6,258,553, both patents incorporated herein by reference in their entirety. Also, the MiCK assays described in: Kravtsov V. et al. "Use of the Microculture Kinetic Assay of apoptosis to determine chemosensitivities of leukemias." Blood 1998;
92:9680980, is incorporated herein by reference in its entirety for all purposes.
After overnight incubation, chemotherapy drugs were added to the wells of the 96 -well plate in 5 μL aliquots or to the wells of a 384-well plate in 2.5 μL aliquots using an automated pipettor. The number of drugs or drug combinations and the number of concentrations tested depended on the number of viable malignant cells that were isolated from the tumor specimen. The drug concentrations, determined by molarity, were those indicated by the manufacturer as the desired blood level concentration plus or minus one serial dilution if enough cells were available.
Following drug addition, the plate was incubated for 30 min at 37°C in a 5% carbon dioxide humidified atmosphere incubator. Each well was then overlayed with sterile mineral oil, and the plate was placed into the incubator chamber of a microplate spectrophotometric reader. The optical density at 600 nanometers was read and recorded every 5 minutes over a period of 48 hours. Optical density increases, which correlates with apoptosis, were converted to kinetic units (KU) of apoptosis by a proprietary software ProApo with a formula described in the Kravtsov reference incorporated by reference (i.e. Kravtsov V. et al. "Use of the Microculture Kinetic Assay of apoptosis to determine chemosensitivities of leukemias." Blood 1998; 92:968- 980). Active apoptosis was indicated as > 1.0 KU. A drug producing≤ 1 KU was described as inactive, or that the tumor was resistant to that drug based on previous laboratory correlations
of KU with other markers of drug-induced cytotoxicity (growth in culture, thymidine uptake). Two standard deviations (SD) in the assay were 1.14 KU.
Results:
In Patient A, Doxorubicin alone produced a KU value of 3.5, indicating that it would be moderately effective for this patient. However, when Doxorubicin was combined with 4HC+Etoposide, a KU value of 7.1 was detected. Surprisingly, the combination of
4HC+Etoposide without Doxorubicin resulted in a 0.0 KU value. Therefore in this patient, the combination of a moderately effective chemotherapeutic and an ineffective chemotherapeutic resulted in a very effective treatment option for this individual patient. Furthermore, when Doxorubicin was combined with 4HC alone, a KU value of 0.7 was detected, indicating the addition of 4HC nearly obliterated the effect of Doxorubicin to produce apoptosis in cancer cells from this individual. The results for all chemotherapeutics tested in this patient are included in Table 3.
In Patient B, Daunorubicin alone produced a KU value of 5.1. However, when Daunorubicin was combined with 4HC, a KU value of 9.8 was detected. Adding Cytarabine to Daunorubicin, however, decreased the effectiveness of Daunorubicin, as indicated by the 2.4 KU value detected for this combination. The results for all chemotherapeutics tested in this patient are included in Table 4.
In Patient C, 4HC alone produced a KU value of 3.2. However, when Vincristine was combined with 4HC, a KU value of 6.6 was detected. Surprisingly, Vincristine alone produced a value of 2.2. Therefore, the effect of combining Vincristine with 4HC was more than the expected additive effect. However, when Fludarabine was combined with 4HC, a KU value of 2.1 was detected. Thus, adding a chemotherapeutic with little to no effect on apoptosis by itself (Fludarabine alone = 0.4 KU) to a moderately effective chemotherapeutic in this patient had an antagonistic effect. Furthermore, in Patient C, Carboplatin alone produced a KU value of 0.6; Etoposide alone produced a KU value of 0.2; and 4HI (Ifosfamide) alone produced a KU value of 3.3. However, the combination of Carboplatin+Etoposide+4HI produced a KU value of 6.6, which is much greater than would be expected based on the individual values. The results for all chemotherapeutics tested in this patient are included in Table 5.
EXAMPLE 2 - Detection of Synergistic and Antagonistic Chemotherapeutic
Combinations in Breast Cancer.
Background:
Given the results of drug-induced apoptosis as determined by the MiCK assay in leukemia described in Example 1, we sought to determine whether the synergism and antagonism between different chemotherapeutic combinations in an individual patient could be detected using the MiCK® assay in solid tumors.
Methods:
Patients with breast cancer had tumors sent independendy for drug-induced apoptosis analysis as described (Salom et a/., J Trans Med 2012; 10:162). Purified tumor cells were cultured for 48 hours with individual drugs, and drug-induced apoptosis was measured using the MiCK assay as described in Example 1. Data were obtained optically using Mie light- scattering.
Patients: Two patients with breast cancer were included in this study. Patient D was a 64 year old female diagnosed with metastatic breast cancer, in relapse at the time the sample was collected. The sample collected was obtained from liver and/or lung metastatic sites. Patient E was a 54 year old female diagnosed with invasive ductal carcinoma of the right breast, in subde progression at the time the sample was collected. The sample collected was obtained from primary uterine CA/pleural effusion. Patient E had previously been treated with Carboplatin, Taxol, cytarabine, Cisplatin, Navelbine, Abraxane and Avastin.
Results:
In Patient D, 4HC alone produced a KU value of 5.7. However, when 4HC was combined with 5-Fu+Leuco+Epirubicin, a KU value of 8.3 was detected. Surprisingly, the combination of 5- Fu+Leuco without 4HC or Epirubicin resulted in a 1.2 KU value. Epirubicin, as a single agent produced a KU value of 2.4. Furthermore, when 4HC was combined with Paclitaxel, a KU value of 1.0 was detected, despite Paclitaxel resulting in a KU value of 2.2 as a single agent. Thus, the combination of 4HC and Paclitaxel resulted in less apoptosis in this individual's cancer cells than either agent alone. The results for all chemotherapeutics tested in this patient are included in Table 6.
In Patient E, 4HC alone produced a KU value of 2.2. However, when 4HC was combined with Doxorubicin+Docetaxel, a KU value of 7.9 was detected. The combination of
Doxorubicin+Docetaxel without 4HC produced a 4.7, indicating that the addition of 4HC to Doxorubicin+Docetaxel produces more than just an additive effect. Adding Methotrexate+5- Fu to 4HC, however, decreased the effectiveness of 4HC, as indicated by the 1.2 KU value detected for this combination. The results for all chemotherapeutics tested in this patient are included in Table 7.
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EXAMPLE 3 - Detection of Synergistic and Antagonistic Chemotherapeutic
Combinations in Lung Cancer.
Background:
Given the results of drug-induced apoptosis as determined by the MiCK assay in leukemia and breast cancer described in Examples 1 and 2, we sought to determine whether the synergism and antagonism between different chemotherapeutic combinations in an individual patient could be detected using the MiCK® assay in additional solid tumors.
Methods:
Patients with lung cancer had tumors sent independently for drug-induced apoptosis analysis as described (Salom et al., J Trans Med 2012; 10:162). Purified tumor cells were cultured for 48 hours with individual drugs, and drug-induced apoptosis was measured using the MiCK assay as described in Example 1. Data were obtained optically using Mie light- scattering.
Patients: Two patients with lung cancer were included in this study. Patient F was a 60 year old male diagnosed with lung adenocarcinoma with no previous chemotherapy. The sample collected was obtained from the lung. Patient G was a 79 year old female diagnosed with small cell lung carcinoma with no previous chemotherapy. The sample collected was obtained from the lung.
Results:
In Patient F, Cisplatin alone produced a KU value of 4.3. However, when Cisplatin was combined with Docetaxel, a KU value of 5.6 was detected. Furthermore, when Cisplatin was combined with Alimta, a KU value of 2.9 was detected. Interestingly, when Alimta is combined with Paclitaxel, a KU value of 4.7 is detected, which is greater than the predicted additive effect of Alimta (0.7 KU) and Paclitaxel (3.3 KU). The results for all chemotherapeutics tested in this patient are included in Table 8.
In Patient G, Cisplatin alone produced a KU value of 6.4. However, when Cisplatin was combined with Pemetrexed, a KU value of 8.3 was detected. Pemetrexed alone produced a 1.4 KU value, indicating that the addition of Pemetrexed to Cisplatin produces more than just an additive effect. Adding Paclitaxel to Pemetrexed, however, completely eliminated the effectiveness of Pemetrexed, as indicated by the 0.0 KU value detected for this combination despite Paclitaxel resulting in a KU value of 1.7 as a single agent. Thus, the combination of
Pemetrexed and Paclitaxel resulted in less apoptosis in this individual's cancer cells than either agent alone. The results for all chemotherapeutics tested in this patient are included in Table 9.
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EXAMPLE 4 - Detection of Synergistic and Antagonistic Chemotherapeutic
Combinations in Ovarian Cancer.
Background:
Given the results of drug-induced apoptosis as determined by the MiCK assay in leukemia, breast cancer, and lung cancer described in Examples 1-3, we sought to determine whether the synergism and antagonism between different chemotherapeutic combinations in an individual patient could be detected using the MiCK® assay in additional solid tumors.
Methods:
Patients with ovarian cancer had tumors sent independently for drug-induced apoptosis analysis as described (Salom et a/., J Trans Med 2012; 10:162). Purified tumor cells were cultured for 48 hours with individual drugs, and drug-induced apoptosis was measured using the MiCK assay as described in Example 1. Data were obtained optically using Mie light- scattering.
Patients: Two patients with ovarian cancer were included in this study. Patient H was a 55 year old female diagnosed with ovarian cancer. The sample collected was obtained from the uterus+ovaries. Patient I was a 66 year old female diagnosed with ovarian/ tubal cancer with no previous chemotherapy.
Results:
In Patient H, Cisplatin alone produced a KU value of 9.5. However, when Cisplatin was combined with Gemcitabine, a KU value of > 10.0 was detected. Surprisingly, Gemcitabine alone is inactive (0.5 KU). Interestingly, when Gemcitabine is combined with Carboplatin
(which has the same mechanism of action as Cisplatin), a KU value of 3.5 is detected, indicating that Gemcitabine has a negative effect on Carboplatin in cancer cells from this individual, despite Gemcitabine increasing the effectiveness of Cisplatin in the same individual. The same dramatic parallel as seen with Cisplatin and Gemcitabine can be observed with Doxorubicin and 4HC (Cytoxan). Doxorubicin as a single agent has a KU value of >10.0. 4HC (Cytoxan) as a single agent shows low to neutral activity with a KU value of 1.1. However, when 4HC (Cytoxan) is used in combination with Doxorubicin, the antagonistic effect on Doxorubicin is substantial (1.8 KU). The results for all chemotherapeutics tested in this patient are included in Table 10.
In Patient I, Cisplatin alone produced a KU value of 5.4. However, when Cisplatin was combined with Gemcitabine, a KU value of 7.9 was detected. Gemcitabine alone produced a
0.7 KU value, indicating that the addition of Gemcitabine to Cisplatin produces more than an
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additive effect. Adding Docetaxel to Cisplatin produced a KU value of 3.7. Additionally, in Patient I, 4HC alone produced a value of 9.3 KU. The addition of Doxorubicin to 4HC resulted in a KU value of 7.2. Therefore, adding a chemotherapeutic with a minor effect on apoptosis by itself (Doxorubicin alone = 1.4 KU) to a highly effective chemotherapeutic in this patient had an antagonistic effect. The results for all chemotherapeutics tested in this patient are included in Table 11.
EXAMPLE 5 - Detection of Synergistic and Antagonistic Chemotherapeutic
Combinations in Gastric Cancer.
Background:
Given the results of drug-induced apoptosis as determined by the MiCK assay in leukemia, breast cancer, lung cancer, and ovarian cancer described in Examples 1-4, we sought to determine whether the synergism and antagonism between different chemotherapeutic combinations in an individual patient could be detected using the MiCK® assay in additional solid tumors.
Methods:
A patient with gastric cancer had a tumor sent independently for drug-induced apoptosis analysis as described (Salom et a/., ] Trans Med 2012; 10:162). Purified tumor cells were cultured for 48 hours with individual drugs, and drug-induced apoptosis was measured using the MiCK assay as described in Example 1. Data were obtained optically using Mie light-scattering.
Patients: One patient with gastric cancer was included in this study. Patient J was a 64 year old female diagnosed with linitis plastic gastric adenocarcinoma, in relapse at the time the sample was collected. The sample collected was obtained from primary gastric CA/ ascitic fluid. Patient J had previously been treated with 5-Fu and XRT.
Results:
In Patient J, 5-Fu alone produced a KU value of 0.0. However, when 5-Fu is combined with Paclitaxel, a KU value of 2.3 was detected. Interestingly, Paclitaxel alone produced a KU value of 5.2, indicating that 5-Fu has a negative effect on Paclitaxel in cancer cells from this individual. A similar effect is seen when 5-Fu is combined with Irinotecan and Oxaliplatin. However, when 5-Fu is combined with Cisplatin, a KU value of 4.7 is detected, indicating that 5-Fu increased the effectiveness of Cisplatin (3.7 KU as single agent). Additionally, when 5-Fu is combined with both Paclitaxel and Cisplatin, a KU value of 6.5 is detected. The results for all chemotherapeutics tested in this patient are included in Table 12.
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EXAMPLE 6 - Detection of Synergistic and Antagonistic Chemotherapeutic
Combinations in Uterine Cancer.
Background:
Given the results of drug-induced apoptosis as determined by the MiCK assay in leukemia, breast cancer, lung cancer, ovarian cancer, and gastric cancer described in Examples 1-5, we sought to determine whether the synergism and antagonism between different
chemotherapeutic combinations in an individual patient could be detected using the MiCK® assay in additional solid tumors.
Methods:
A patient with uterine cancer had a tumor sent independendy for drug-induced apoptosis analysis as described (Salom et a/., ] Trans Med 2012; 10:162). Purified tumor cells were cultured for 48 hours with individual drugs, and drug-induced apoptosis was measured using the MiCK assay as described in Example 1. Data were obtained optically using Mie light-scattering.
Patients: One patient with uterine cancer was included in this study. Patient K was a female diagnosed with stage III clear cell uterine cancer, in relapse at the time the sample was collected. The sample collected was obtained from metastatic abdominal wall tissue. Patient K had previously been treated with carboplatin and Taxol, which resulted in complete response.
Results:
In Patient K, the combination that previously resulted in complete response (carboplatin and Taxol) produced a KU value of 4.2. However, Cisplatin (alone or in combination with
Paclitaxel, Docetaxel, Doxorubicin, or Doxorubicin+Paclitaxel) all produced KU values >10.0, indicating Cisplatin would be beneficial for this patient. The results for the combination of Carboplatin+Paclitaxel are also interesting because the KU value produced is less than that obtained with Paclitaxel alone (5.1 KU). Additionally, when 4HI is combined with Paclitaxel, the resulting KU value is approximately 50% lower than the KU value produced by 4HI alone (5.2 KU vs. > 10.0 KU, respectively. The results for all chemotherapeutics tested in this patient are included in Table 13.
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TABLE 1. Enzyme Utilization Dependent Upon Tumor Type of Specimen
TABLE 2. Final Cell Suspension Plating Protocol
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TABLE 3. Results from Patient A, diagnosed with CLL
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TABLE 4. Results from Patient B, diagnosed with AML
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TABLE 5. Results from Patient C, diagnosed with stage IV NHL-low grade/ CLL.
TABLE 6. Results from Patient D, diagnosed with metastatic breast cancer.
TABLE 7. Results from Patient E, diagnosed with invasive ductal carcinoma of the right breast.
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TABLE 8. Results from Patient F, diagnosed with lung adenocarcinoma.
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TABLE 9. Results from Patient G, diagnosed with small cell lung carcinoma.
-34-
TABLE 10. Results from Patient H, diagnosed with ovarian cancer.
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TABLE 11. Results from Patient I, diagnosed with ovarian /tubal cancer.
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TABLE 12. Results from Patient J, diagnosed with linitis plastic gastric adenocarcinoma.
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TABLE 13. Results from Patient K, diagnosed with stage III clear cell uterine cancer.
All references cited in this specification are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entided to antedate such reference by virtue of prior invention.
It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The
- 38 -
foregoing embodiments are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.
- 39 -
Claims
1. A method of identifying a drug candidate that reduces the effectiveness of another drug candidate in an individual patient comprising:
a. plating a single-cell suspension of viable cancer cells obtained from a tumor site in said subject in at least three wells of a plate suitable to be read by a spectrophotometer,
b. adding a drug candidate to one well in an amount sufficient to achieve a target drug candidate concentration,
c. adding a different drug candidate to a different well in an amount sufficient to achieve a target drug candidate concentration,
d. adding the drug candidate of step (b) and the drug candidate of step (c) to form a drug combination to a different well in an amount sufficient to achieve a target drug candidate concentration,
e. measuring, at selected time intervals for a selected duration of time, the optical density of the wells at a wavelength of approximately 600 nm using a spectrophotometer,
f. determining a kinetic units (KU) value for each well from the optical density and time measurements, and
g. correlating the KU value with an ability of the at least one drug candidate or the at least one drug combination to induce apoptosis in the cancer cells if the KU value is positive, or an inability of the at least one drug candidate or at least one drug combination to induce apoptosis in the cancer cells if the KU value is not positive;
wherein an increase in the KU value of the drug combination compared to the KU value of the drug candidate alone is indicative of an increase in effectiveness.
2. The method of claim 1, wherein the drug candidate is an individual anti-cancer drug.
3. The method of claim 1, wherein the drug candidate is a combination of anti-cancer drugs.
4. The method as in any one of claims 1-3, wherein the tumor site tested is from the
primary tumor site.
5. The method as in any one of claims 1-3, wherein the tumor site tested is from a metastatic tumor site.
6. The method as in any one of claims 1-3, wherein the viable cancer cells are from a blood cancer.
7. The method of claim 6, wherein the blood cancer is selected from the group consisting of acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.
8. The method as in any one of claims 1-3, wherein the viable cancer cells are from a solid tumor.
9. The method of claim 8, wherein the solid tumor is selected from the group consisting of breast cancer, lung cancer, ovarian cancer, gastric cancer, and uterine cancer.
10. A method of identifying a drug candidate that increases the effectiveness of another drug candidate in an individual patient comprising:
a. plating a single-cell suspension of viable cancer cells obtained from a tumor site in said subject in at least three wells of a plate suitable to be read by a spectrophotometer,
b. adding a drug candidate to one well in an amount sufficient to achieve a target drug candidate concentration,
c. adding a different drug candidate to a different well in an amount sufficient to achieve a target drug candidate concentration,
d. adding the drug candidate of step (b) and the drug candidate of step (c) to form a drug combination to a different well in an amount sufficient to achieve a target drug candidate concentration,
e. measuring, at selected time intervals for a selected duration of time, the optical density of the wells at a wavelength of approximately 600 nm using a spectrophotometer,
f. determining a kinetic units (KU) value from each well from the optical density and time measurements, and
g. correlating the KU value with an ability of the at least one drug candidate to induce apoptosis in the cancer cells if the KU value is positive, or an inability of the at least one drug candidate to induce apoptosis in the cancer cells if the KU value is not positive;
wherein an increase in the KU value of the drug combination compared to the KU value of the drug candidate alone is indicative of an increase in effectiveness.
1 1. The method of claim 10, wherein the drug candidate is an individual anti-cancer drug.
12. The method of claim 10, wherein the drug candidate is a combination of anti-cancer drugs.
13. The method as in any one of claims 10-12, wherein the tumor site tested is from the primary tumor site.
14. The method as in any one of claims 10-12, wherein the tumor site tested is from a metastatic tumor site.
15. The method as in any one of claims 10-12, wherein the viable cancer cells are from a blood cancer.
16. The method of claim 15, wherein the blood cancer is selected from the group consisting of acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.
17. The method as in any one of claims 10-12, wherein the viable cancer cells are from a solid tumor.
18. The method of claim 17, wherein the solid tumor is selected from the group consisting of breast cancer, lung cancer, ovarian cancer, gastric cancer, and uterine cancer.
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