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Patent Searching and Data

Document Type and Number:
WIPO Patent Application WO/1989/006162
Kind Code:
An improved method for predicting success of tumor therapies which relies on estimating the lowest effective concentration of a therapeutic agent that results in a majority of tumor cell kill in vitro is described. The method includes an assay and apparatus for simultaneously analyzing a series of dilutions of a therapeutic agent using 90 % cell kill as an index to effective cytotoxic concentration of the agent.

Application Number:
Publication Date:
July 13, 1989
Filing Date:
December 21, 1988
Export Citation:
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International Classes:
B01L3/14; B04B5/04; G01N1/28; G01N33/15; G01N33/50; (IPC1-7): B04B5/02; G01N33/573
Foreign References:
Other References:
WEISENTHAL et al., CANCER TREATMENT REP. 71 (7-8): 697-704 (1987).
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What is claimed is:
1. In a method for assaying tumor cells n vitro to determine their sensitivity to a therapeutic agent capable of killing tumor cells, using a dye excluded by living cells to stain dead cells after incubation with said agent, and counterstaining living cells, the improvement comprising: a. adding serial dilutions of a therapeutic agent capable of killing tumor cells to multiple samples containing heterogeneous populations of tumor and non tumor cells from a subject; b. adding an internal standard to each of said samples to verify that similar numbers of total cells are present when the samples are counted; c. adding said dye that is not taken up by living cells but is taken up by dead cells to each of said samples to stain cells killed by said agent; d. adding said dye to counterstain said cells not killed by said agent to facilitate determination of the number of living cells in each of said samples'; e. distinguishing the living tumor cells in each of said sample from living nontumor cells; f. counting the number of living tumor cells in each of said sample; and g. selecting the sample containing a majority of dead tumor cells compared to a control sample not exposed to said therapeutic agent, said sample corresponding to one of said dilutions of said therapeutic agent, whereby the lowest effective concentration of said therapeutic agent capable of killing a majority of tumor cells is determined.— .
2. The method of Claim 1 wherein said dye to counterstain is selected from the group consisting of hematoxylin/eosin, WrightGiemsa, periodic acid Shiff, and immunoperoxidase.—.
3. The method of Claim 1 wherein said agent is selected from the group consisting of melphalan, doxorubicin, vincristine, carmustine, etoposide, cisplatin, dexamethasone and cytarabine.
4. The method of Claim 3 wherein said majority is set at 90% of the tumor cells.
5. The method of Claim 3 wherein said range of concentrations includes five serial dilutions over a range of 16 fold total.
6. The method of Claim 5 wherein said dilutions include IX, 2X, 4X, 8X and 16X.
7. The method of Claim 1 wherein said range of concentrations is X, 10X, 100X, 1,000X and 10,000X.
8. The method of Claim 1 wherein the excluded dye is selected from the group consisting of Fast Green, Fast Green/Nigrosin and Nigrosin.
9. The method of Claim 7 wherein recovery of sufficient cells is verified by inclusion of an internal standard.
10. The method of Claim 9 wherein the internal standard is duck red blood cells.
11. A centrifuge tube holder and slide assembly for applying cellular material from multiple samples to discrete locations on a slide which comprises, when assembled: a. a centrifuge tube holder body having a base and a top, said body having a plurality of parallel elongated cylindrical centrifuge tube chambers arranged linearly for receiving a plurality of tubes containing a liquid sample of cellular material, each of said chambers having an aperture at said top corresponding to the location of said chambers; b. filter to absorb liquid from the samples, said filter having multiple circular openings spaced to be congruent with the apertures of the chambers placed at the top of said tube holder body; and c. a planar slide overlaying said filter for receiving cellular materials contained in the chambers in discrete regions on said slide.
12. The assembly of Claim 11 further including clamping means for securing said slide and filter onto said tube holder body.
13. The assembly of Claim 11 wherein the top of the tube holder body includes raised projections which are fitted into receivers in said filter spaced to correspond to the location of said projections for securing said filter onto the tube holder body.
14. The assembly of Claim 11, when assembled further including a plurality of centrifuge tubes placed in the chambers.
15. The assembly of Claim 14 wherein said centrifuge tubes taper to a diameter that is smaller at the bottom of the tube than the top.
16. The assembly of Claim 14 wherein said centrifuge tubes include a portion that extends above the top of the tube holder in a dimension so as to press against the filter.
17. The assembly of Claim 11 wherein the base of the tube holder includes means for permitting removal of the centrifuge tubes from the chambers by including pushing from the base.
18. The assembly of Claim 11 wherein the holder body is of a single piece constructed of a material selected from the group consisting of plastics, rubber and metals.

Field Of The Invention

The present invention relates to an improved in vitro method and apparatus to detect the resistance of tumor cells to chemotherapy. In particular, it relates to using .iri vitro tests to monitor the acquisition of resistance to chemotherapy in human tumors and methods to assess alternative therapeutic regimes.

Background of the Invention

The use of chemotherapy in treatment of human tumors is widespread, but frequently unsuccessful. One of the reasons for the lack of success in a large number of cases appears to be the acquisition of resistance, by some fraction of the tumor cell population, to the compounds selected for chemotherapy. In a large, proportion of individuals, initial successful remission is followed by relapse, presumably due to a mutation in a fraction of the tumor cells. Studies using established cell lines and animal tumors have permitted some understanding of the mechanisms of drug resistance in these cases (Chabner, B.A., et al., Proc. Am. Assoc. Cancer Research (1985) 26:390-391). However, it is unclear how mechanisms of drug resistance observed in these model systems relate to resistance occuring in individual cancer patients.

It is also known that human tumors are highly variable and that a drug regime effective in one patient may be totally ineffective in another. Efforts have been made to study the effects of drugs on individual human tumors, including solid tumors and hematologic

neoplasms using .in. vitro laboratory tests. Many of these assays are clonogenic in nature, i.e., they rely on the ability to culture and grow the neoplasms in vitro, which is often difficult.

An in vitro assay, the differential staining cyto- toxicity ("DiSC") assay, which permits the detection of - in vitro chemosensitivity of human cancer cells and which circumvents the necessity for tumor growth in culture has been reported ( eisenthal, L.M. , et al., Cancer Research 43:258-264, 749-757 (1983)). This assay, further described below as part of the method of the invention, relies on the ability of living cells to exclude certain dyes, whereas dead cells do not. In the assay, the number of individual cells capable (or not) of dye uptake is ascertained by counting populations using a microscope. Thus the assay provides a means for individually counting the number of tumor cells surviving in the presence or absence of drug when the biopsied tumor cells are subjected to the assay, and has been used to predict the chemosensitivity of human tumors to various drugs (Weisenthal, L.M., et al., Cancer 51:1490-1495 (1983)). A review of the development and relevance of this assay is set forth in Weisenthal, L.M., et al., in Recent Results in Cancer Research 94:161-173, Springer Verlag, Berlin-Heidelberg (1984).

Recently, a method for using the DiSC assay to dis¬ criminate between sensitive and resistant cell popula¬ tions in human tumors to detect and circumvent the acquisition of drug resistance during chemotherapy has been described (hereafter the "percent cell survival" DiSC assay method) (Weisenthal et al., Cancer Treatment Rep. 70:1283-1295 (1986)).

In the percent cell survival DiSC assay, for reliable results, the slides containing cells are counted by a skilled medical technologist with expertise in interpreting cytological preparations, particularly differential cell counting. This aspect of the procedure is time-consuming and tedious. In addition, there are problems of reproducibility associated with tumor specimens having appreciable cell clumping. A typical assay using 60 to 96 individual culture tubes requires 2 to 3 hours for slide preparation and up to 8 hours for slide reading. A rapid method for estimating the lowest effective concentration of chemotherapeutic agent at which most of the tumor cells in a sample are killed would improve the usefulness and practicability of the DiSC assay.

Devices are known for holding multiple centrifuge tubes containing liquid samples such as the devices described in U.S. Patent Nos. 4,389,374 and 4,462,964.

For cytotoxicity studies devices are known which permit a sample to be centrifuged in an individual sample chamber to deposit the cells in a single location on a slide, such as the sample chamber manufactured by Shandon Instruments, Inc., Sewickley, PA. These prior known devices require a transfer step in which the cells are treated with the cytotoxic agent in a sample tube then removed to the centrifuge sample chamber. It would thus be useful to provide an apparatus capable of centrifuging multiple samples simultaneously, and of depositing cellular material from the samples sequentially on a single slide for testing a range of cytotoxic agents, without requiring separate transfer of the samples into chambers for each centrifugation run.

Summarv of the Invention

The invention provides a method and apparatus for performing a differential staining cytoxicity assay to determine the sensitivity of tumor cells .in vitro to a therapeutic agent administered .in vitro by detecting the ability of cells to exclude a dye after incubation with the agent. The method includes assaying multiple samples of tumor cells from an individual subject using a range of concentrations of the therapeutic agent to provide an estimate of the lowest effective concentra¬ tion capable of killing most of the tumor cells. This method obviates the necessity for tedious cell counting, while retaining the advantages of the assay in identifying tumor cells in the presence of normal cells included in the sample. The ' apparatus facilitates processing of multiple samples of tumor cells covering the range of concentrations of the therapeutic agents selected, and includes a centrifuge tube holder containing a plurality of centrifuge tube chambers, a slide to receive the centrifuged cells from each of the inserted tubes, separated from the tubes by a filter punctured with circular openings matching the top circumferences of the tubes to absorb liquid during centrifugation, and to permit the passage of cellular material from the chambers to the slide that is placed over the filter on top of the centrifuge tube holder. A clamp is used to fasten the filter and slide on top of the tube holder after the tubes are inserted.

Brief Description of the Drawings

Figure 1 is an assembled isometric view of the apparatus and clamp for securing the apparatus together,

Figure 2 is an exploded isometric view of the apparatus at the point of assembly showing the centrifuge tube holder, centrifuge tubes, filter and glass slide.

Figure 3 is a staggered, assembled vertical section of the apparatus having the centrifuge tubes inserted.

Figure 4 is an enlarged view of a portion of Figure 3 showing the centrifuge tubes in and extending above the chambers that receive the centrifuge tubes.

Figure 5 is a photograph of a slide containing tumor cells treated with five dilutions of a chemothera¬ peutic drug using the apparatus of the invention.

Figure 6A-F are photographs of disks from a slide prepared using the apparatus of the invention, each disk containing tumor cells treated with a different concen¬ tration of the drug nitrogen mustard.

Figure 7 is a graph of the comparison of the percent cell survival method and the EMT 90 method.

Figure 8 is a graph of results of testing different chemotherapeutic agents on tumor cells using the percent cell survival method.

Figure 9 shows results of different chemotherapeutic agents on tumor cells determined by the EMT 90 method of the invention.

Figure 10 compares the response of tumor cells to the drug CDDP alone and with V/L obtained using the percent cell survival criterion and EMT/90 method.

Detailed Description of the Invention

The present invention employs the .in vitro DiSC assay procedure described in detail by Weisenthal et al., in Cancer Treatment Reports, 70(11) :1283-1295 (1986), incorporated by reference herein, modified as described below. The invention also comprises an apparatus for carrying out the centrifugation of multiple samples containing tumor cells constituting a range of concentrations of a therapeutic agent for use in the method of the invention. The method relies on the use of serial dilutions of the therapeutic compound to estimate the minimum effective concentration of drug at which 90% of the tumor cells in a population from a tumor sample are killed (i.e. 10% tumor cell survival). This endpoint concentration may be used to predict the concentration of therapeutic agent below which the tumor cells will be increasingly resistant to the agent. By comparing this endpoint between specimens, the relative drug sensitivity or resistance of the specimens may be determined. The higher the endpoint concentration the more resistant are the cells to the test drug; furthermore the concentration at which the drug is effective may be used to estimate dosage levels .in vivo. Comparative data of a number of drugs and protocols will also permit identification of those with the greatest likelihood of success. The method and apparatus may be used in conjunction with strategies to evaluate the continued effectiveness of the particular therapeutic agent, and/or to modify a therapeutic protocol in a human subject, as described by Weisenthal et al., Cancer Research, supra.

General Methods

In the previously described DiSC assay, bone marrow specimens, peripheral blood buffy coats (approximately 1 cm 3 ), or other appropriate tumor cell specimens are diluted with 6 to 10 volumes of complete RPMI-1640 medium containing 10 units/ml of heparin. This is underlayered with Ficoll-diatrizoate (Lymphocyte Separation Medium, Organon Teknika Corporation, Durham, NC) and centrifuged. The interface layer is washed and counted. Solid tumors are manually teased apart after incubation with collagenase/DNAse. Cells (3 to 8 X 10 4 ) are aliquoted into 0.8 ml capacity conical polystyrene or polypropylene tubes and brought to a volume of 0.1 ml with complete RPMI-1640 medium plus 10% heat-inactivated fetal calf serum. The appropriate concentrations, preferably five serial dilutions, of a drug solution are added to the cell suspensions and incubated for time periods according to the drug to be assayed. For example cells may be incubated either short term (e.g. 1 hour), longer term or continuously.

After several days or other desired time in culture, 0.01 ml of a suspension of acetaldehyde-fixed duck red blood cells (DRBC) containing 30,000 to 50,000 DRBC, is added to each tube. To this final volume, 0.2 ml Fast Green (solid tumors) or Fast Green Nigrosin (hematologic neoplasms) (1% Fast Green and 0.5% Nigrosin) in 0.15M NaCl is added to stain cells killed by the drug and the tubes vortexed. After 10 minutes the cell suspensions are again vortexed, and then transferred into a centrifuge apparatus, such as the apparatus described herein, to apply the cells onto a microscope slide in order of lowest to highest drug concentration over the dilution range.

The cell disks located on the slide after centri- fugation are counterstained with hematoxylin and eosin (H & E) (solid tumors) or are fixed with methanol for 20 seconds and counterstained with Wright-Giemsa (hema- tologic neoplasms) or with another stain such as PAS or immunoperoxidase to indicate living cells.

In the "percent cell survival" assay, mounting balsam (Histomount, National Diagnostics, Somerville, NJ) and coverslips are added. Slides are then counted at either 400X or 1000X on a standard light microscope by a trained hematology technologist. "Living" cells counterstain with H & E or Wright-Giemsa, while "dead" cells stain green with Fast Green and black or black- green with Fast Green Nigrosin. DRBC stain green and are easily identified as nucleated microelliptocytes. "Living" tumor cells and DRBC are counted by a technician capable of distinguishing tumor cells from non-tumor cells, and the living tumor cells/DRBC ratio is determined. The ratios from the serial dilutions of the drug-treated cultures are compared with the ratios from the control cultures (cultures containing drug vehicle, usually 0.9% NaCl) and expressed as a percent of control.

In the percent cell survival method, chemothera¬ peutic drugs are generally tested at only one or two concentrations owing to the considerable effort involved with the enumeration of individual cells .in the slide counting procedure. These concentrations are selected by obtaining preliminary experimental data and correlating .in vitro and .in vivo responses to the drug. In general, each assay tube required an individual cytocentrifuge slide on which 300 individual cells or more were counted to obtain a quantitative result. The

endpoint was the percent cell survival in drug treated cultures relative to control cultures.

In the method of the invention referred to as "EMT 90", a multiple dilution concentration range (preferably 5 dilutions) is tested. The assay endpoint is the lowest drug concentration (dilution) which results in essentially total tumor cell kill (cell kill estimated to exceed 90%).

In order to perform the assay according to the method of the invention, it is convenient to prepare the set of slides with all five concentrations simultaneously. Therefore, the centrifugation described above is done using the apparatus of the invention containing the reaction tubes for all five dilutions. The previous steps are conducted in plastic (e.g. poly¬ propylene) tubes which are designed to fit in the 5-tube carrier described below.

In more detail, to perform the assay of the invention, drugs in the selected range of concentrations (e.g. serial dilutions in the ranges of 1:2, 1:3, 1:5 or 1:10 depending on the therapeutic agent being tested) are added to the microtubes at time 0. The lowest con¬ centration (X) of therapeutic agent is determined individually for each drug for example using the clinical pharmacokinetic data available in the literature and from experiments to obtain preliminary test data. Times of exposure in the assay may be determined by performing preliminary experiments to determine which assay conditions most closely predict clinical outcomes. Thus the assay should be calibrated for different drugs with either a short-term, longer term or a continuous exposure. Continuous exposures are

simpler and the assay can be calibrated on the basis of a continuous exposure for most drugs.

Sample preparation depends on the nature of the tumor sample being taken, and follows standard guidelines for obtaining specimens for human studies. For example, depending on the nature of the tumor, specimens may include EDTA anticoagulated blood or marrow specimens and solid tumor biopsy specimens. Solid tumors are collected in complete tissue culture medium with antibiotics.

At the conclusion of the assay (generally 4 days), either Fast Green, Nigrosin, or Fast Green/Nigrosin stain is added to the culture tubes along with the acetaldehyde-fixed DRBC. The tubes are loaded into the apparatus of the invention described below, and spun at 500 to 1200 RPM on a cytospin centrifuge. The resulting slides are then counterstained with hematoxylin/eosin, Wright-Giemsa, or other specialized stain such as PAS, immunoperoxidase, or are processed for autoradiography.

Stained slides are then first read on a computerized Image Analyzer (Image Technology, Model 3000, Deer Park, New York). The Image Analyzer is initially set to count only the DRBC, to determine that all of the 5 disks on the slide contain approximately the same number of DRBC. If a particular disk has less than one-half the amount of DRBC as the- other disks, that particular assay dilution is deemed to be unreliable. If the disk has one-half or more the "standard" number of DRBC, then the surviving cell estimate for that particular disk is divided by the ratio of the actual number of DRBC for that disk to the actual number of DRBC in the "standard" control disks. (This is done for quality control purposes and to

normalize cell numbers in cases where there may be some loss of fluid during the cytocentrifuge process.)

After reading on the Image Analyzer, the slides are then screened by experienced technicians who are capable of recognizing tumor cells from non-tumor cells by detecting the living cells that have counterstained. The technician begins at the highest drug concentration, and proceeds to view the other slide cell disks in succession. The technician determines the first disk in which there appear to be greater than 10% surviving tumor cells relative to the control cultures. Thus, each disk is evaluated as a "yes" or "no" endpoint; "yes", if there are more than 10% surviving cells relative to the control cultures, and "no", if there are fewer than 10% surviving cells relative to the control cultures.

If five dilutions are used, there are six possible quantitative endpoints for the assay: If the lowest drug concentration used is "X" the possible drug concen¬ trations are: X, 2X, 4X, 8X, 16X, and greater than 16X. A "greater than 16X" result is arbitrarily called 32X. The lowest concentration capable of killing 90% of the tumor cells is designated "the EMT 90."

in a typical experiment, the first disk corresponding to the highest drug concentration (e.g. 16X) will contain predominantly dead tumor cells. The final disk containing the lowest drug concentration (e.g. IX) will contain predominantly living tumor cells. The first disk with tumor cell survival estimated to be less than 10% of the control culture will present the lowest drug concentration capable of 90% cell kill. The following disk corresponding to the next lowest drug

dilution will contain numerous living tumor cells, obviously more than 10% of the control cultures.

The reason for requiring a 90% tumor cell kill rather than 100% tumor cell kill is that there are occasionally situations in which one or two tumor cells are difficult to distinquish from normal cells; that is, either the tumor cells look relatively normal (e.g. resembling macrophages) or the normal cells may look abnormal so as to resemble tumor cells. In most assays, the vast majority of the cells can be accurately distinguished by an experienced reader as either being tumor cells or normal cells, but there may occasionally be a few cells which are difficult to distinguish.

Slides are preferably independently scored by two different readers. If the EMT 90 value agrees within one serial dilution, i.e. one unit of EMT 90 dilution X to 2X, between two observers, the results are averaged (e.g. to 1.5X). If the results disagree by more than one EMT 90 unit, then slides are reevaluated by the same observers and by additional observers until acceptable agreement is obtained within one EMT 90 unit and the corresponding results are then averaged and standard deviations are presented. Based on numerous tests, observations from experienced observers agree within one EMT 90 unit more than 90% of the time on first reading. Because the criterion of 90% tumor kill is so stringent, even substantial variation in the number of tumor cells/DRBC in each disk does not cause major differences in the EMT 90 value.

Centrifugation of the samples containing tumor cells treated with a range of dilutions of chemotherapeutic drug are preferably performed in the centrifugation apparatus shown in Figures 1-4 and

described herein. This apparatus allows multiple cell disks to be placed on a single slide, permitting a technician or automated reader device to rapidly scan an entire therapeutic agent concentration range from lowest to highest.

Referring to the drawings, in Figure 1 the assembled apparatus is shown fastened with a metal clamp. Centrifuge tube holder 10 is shown in Figure 2 having a base 12 and top 14 mounted in spaced generally parallel arrangement with the base 12. A ledge 16 is formed by an extension of the top 14 of the assembly over the body 18. Located in the top 14 are a plurality of apertures 20 connected to a plurality of chambers 22 in the body 18. The combination of the apertures 20 and chambers 22 is designed to receive a plurality of centrifuge tubes 24. The base 12 contains a plurality of apertures 26 connected to the chambers 22 for facilitating removal of the centrifuge tubes 24. The. top 14 also may contain raised projections 28 for holding a filter 30 as described below. The tube holder 10 is preferably made of a hard plastic such as acrylic (e.g. Lucite) or other material such as aluminum.

The centrifuge tubes 24 preferably taper to a diameter that is smaller at the bottom of the tube than at the top as shown in Figure 2 and include a cuff 32 at the top of the tube that extends above the aperture 20 and the top 14 of the assembly. The tubes may be constructed of a plastic such as polypropylene or polystyrene or similar material.

The tube holder 10 is designed to be used with a planar substrate such as a glass slide 34 for receiving centrifuged cellular material. The slide 34 may be manufactured so as to contain visible discrete regions,

(e.g. circular disks, to receive cellular material from samples contained in the centrifuge tubes 24). The filter 30 is inserted between the tube holder 10 and the slide 34 to absorb fluids from the samples in the centrifuge tubes 24 during operation. The filter 30 has apertures 38 that correspond to the apertures 20 in the top 14 of the tube holder 10, and openings 40 that receive the pegs 28 on the top 14 of the tube holder 10 to secure the filter onto the tube holder and to maintain alignment with the apertures 38 on the filter, and the cuffs 32 of the tubes. The typical order of assembly is the insertion of centrifuge tubes 24 containing a liquid sample into the chambers 22 in the tube holder 10, placement of the filter 30 onto the top 14 of the tube holder aligning the apertures 38 over the apertures 20 in the top 14 of the tube holder 10, followed by placing the slide 34 on top of the filter 30.

The volume of fluid sample used in the centrifuge tubes of the apparatus is preferably in a range of from 0.08 to 0.12 ml to prevent saturation of the filter paper with liquid.

As shown in Figures 1 and 3, the slide 34 and filter 30 are preferably secured onto the tube holder 10 by a clamp 42. The position of the centrifuge tubes 24 within the chambers 22 is shown in Figure 3, which presents two tubes 24 in section and a tube 44 in elevation. As shown in Figure 4, when assembled, the tops 32 of the centrifuge tubes 24 are pressed against the filter and slide so as to reduce the deposit of cellular material from each centrifuge tube 24 outside of the region on the slide corresponding to the aperture 20 in the tube holder 10.

Although a particular clamp 42 is shown in the Figures for the assembly of the present invention, it is envisioned that other types of clamps could be used embodying the same principles of attachment. Further, while a generally rectangular arrangement with five (5) chambers 22 has been shown for the assembly, it is contemplated that other configurations and numbers of chambers could be used without departing from the scope of the invention.

Once the desired number of centrifuge tubes 24 have been placed in the tube holder 10 and the filter 30 and slide 34 have been clamped in place, the entire assembly is designed to be placed in a centrifuge such as a Shandon cytocentrifuge (Shandon Southern Instruments, Inc., Sewickley, PA) which is subjected to centrifugal forces during a centrifugation run. A typical slide having five disks containing cells treated with five concentrations of a drug is depicted in Figure 5.

The EMT 90 values obtained can be used as an index of the efficacy of possible treatments for tumor cells biopsied on an individual basis. The EMT 90 values if obtained at various stages of drug therapy for the tumor can serve as an index for monitoring the acquisition of resistance to the treatment tested. The prospective efficacy of proposed changes in treatment protocol can also be evaluated.

At its simplest, the invention method can be used to compare sensitivity to various therapies, for example, those used in chemotherapy. Commonly used drugs include, for example, mechlorethamine, melphalan, carmustine, cytarabine, dexamethasone, doxorubicin, cisplatin, etoposide, vincristine and related antineoplastic agents as well as immune effectors such

as monoclonal antibodies, lymphokines, lymphokine- activated effector cells and interferons. The sensitivity of tumors to these materials can also be evaluated.

In addition, it may be possible to enhance the effects of these drugs using agents which have complementary functions, such as membrane-altering drugs. The potency of a chemotherapeutic agent is thus altered by coad inistration of, for example, cofactors and drugs used for other purposes. Whether such supplementation will potentiate a drug to which resistance has been acquired can be determined by comparing the EMT 90 for the drug with and without the supplement.

The following examples are presented to illustrate the method and apparatus of the invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the disclosure or the protection granted by Letters Patent granted hereon.


Illustration of the Method

To demonstrate the method, tumor cells were obtained by bone marrow aspiration on a patient having acute lymphoblastic leukemia (ALL) (Memorial Hospital Medical Center, Long Beach, CA) . The cells were isolated according to the methods described in Cancer Treat. Rep. 70:1283-95 (1986), incorporated by reference herein. The cells were placed in polypropylene microtubes (0.8 ml) and were exposed to fivefold dilutions (0.25 mg/ml or IX; 0.5 mg/ml or 2X; , 1.0 mg/ml or 4X; 2.0 mg/ml or 8X and 4.0 mg/ml or 16X) of the drug

nitrogen mustard (mechlorethamine hydrochloride, Merck, Sharp and Dohme, West Point, PA) for a continuous 4 day period of culture. Acetaldehyde-fixed DRBC (50,000) were then added to each tube along with 20 ml Fast Green (Sigma Chemical Co., St. Louis, MO.) and Nigrosin (Sigma Chemical Co., St. Louis, MO.). Culture tubes were then transferred to the 5 chamber acrylic centrifuge tube holder apparatus and the filter paper, planar slide and clamp were assembled as depicted in Figures 1 and 2. Following cytocentrifugation, at 500 rpm on a Shandon cytocentrifuge (Shandon Southern Instruments, Inc., Sewickley, PA) the slides were disassembled from the apparatus and were counterstained with Wright-Giemsa Stain (Sigma Chemical Co., St. Louis, MO).

Photomicrographs (original magnification 400X) of cells treated with the five concentrations of nitrogen mustard are shown in Figure 6A-F. (Figure 6A: control; Figure 6B: 0.25 mg/ml; Figure 6C: 0.50 ml/ml; Figure 6D: 1.0 mg/ml; Figure 6E: 2.0 mg/ml and Figure 6F: 4.0 mg/ml.) Each photograph depicts a representative portion of a disk. This figure demonstrates the ease with which an assay may be performed and a single slide visually scanned by a technician to determine the drug cutoff concentration for 90% tumor cell kill. As can be seen, almost all of the tumor cells in the first disk (IX concentration or 0.25 mg/ml; Figure 6B) are viable. At 8X (Figure 6E) there are fewer than 10% surviving tumor cells. At 16X (Figure 6F) essentially all of the tumor cells have disintegrated. Thus, the disk having 8X concentration of the nitrogen mustard is the lowest concentration of drug effecting 90% cell kill.


EMT 90 Determined Resistance of Tumor Cells

The method and apparatus of the invention were used to assay resistance of tumor cells from two patients (Memorial Hospital Medical Center, Long Beach, CA) with acute lymphoblastic leukemia to the drugs vincristine (Eli Lilly Co., Indianapolis, IN) and doxorubicin (Adria Laboratories, Columbus, OH). Cells were obtained from two different patients by means of bone marrow aspiration. Cells were isolated according to the method described in Cancer Treat. Rep. 70:1283-95, (1986), incorporated by reference herein. Cells were exposed to fivefold drug dilutions (as shown in the legend to the X axis in Figure 7), for a continuous 4 day period of culture. Following this period of drug exposure and culture in the specialized microtubes, ' 50,000 acetaldehyde-fixed DRBC were added to each tube along with Fast Green/Nigrosin. The culture tubes were transferred to the apparatus and centrifuged as described above in Example I.

Percent cell survival was determined for each drug concentration by counting 300 individual cells on each slide. Although 5 drug concentrations were individually counted for this comparison with the percent cell survival method, only 1 drug concentration would have normally been testedi 0.3 mg/ml for doxorubicin and 0.08 mg/ml for vincristine. Simultaneously, EMT 90 values were estimated by two different observers as described above. Figure 7 shows the relationship between the percent cell survival method and EMT 90 method in these two patients tested with the two drugs.


Modification of Drug Resistance

The method and apparatus of the invention were used to determine the ability of additional drugs to 5 circumvent acquired resistance of tumor cells and the results compared to those obtained in the same assay by calculating percent cell survival.

For the EMT 90 method, five different drugs were ( ^ tested: mephalan (LPAM) (Burroughs-Wellcome, Research Triangle Park, NC); vincristine (VCR) (Eli Lilly Co., Indianapolis, IN); carmustine (BCNU) (Bristol-Myers Laboratories, Syracuse, NY); nitrogen mustard (HN2) (Merck, Sharp and Dohme, West Point, PA); and etoposide ° (VP16) (Bristol-Myers Laboratories, Syracuse, NY). The drugs were tested at the concentrations shown in Table I. A resistance modulating drug, verapamil/lidocaine (V/L) was added at 0.5 mg/ml verapamil hydrochloride (Searle, Chicago, IL) and ^ 6.0 mg/ml lidocaine (Astra Pharmaceutical Products, Inc., Westboro, MA). For the percent cell survival method the following concentrations of drugs were used (concentrations were determined by training set data including results from correlation of .in vitro and .in 5 vivo applications of the drug): LPAM, 3.12(4X); VCR,

0.08(2X); BCNU, 4.0(4X); HN2, 1.0(4X) and VP16, 6.0(2X).








Cells were obtained from a single patient having acute lymphoblastic leukemia and were assayed as described above using a series of 5 dilutions of each drug and plated on slides in groups of five using the apparatus of Figures 1-4. The results were determined using the percent cell survival method and the EMT 90 assay as described in Example I.

As shown in Figure 8 using the percent cell survival method, the greatest cell kill (lowest percent cell survival) occurred with the drug VP16 alone and in

combination with V/L. And, for the dilution of VP16 selected, (6 mg/ml) significant potentiation of the effect of the chemotherapeutic drugs alone was not detected with the percent cell survival method.

Figure 9 summarizes the results for the response of tumor cells to five concentrations of five different drugs using the EMT 90 method. For LPAM, 90% cell kill occurred at 8X (6.25 mg/ml), for VCR 90% cell kill occurred at 4X (0.08 mg/ml), for VCR alone and IX (0.04 mg/ml) for VCR + V/L; for BCNU 90% cell kill occurred at 16X (16 mg/ml) for BCNU alone, and 8X

(8 mg/ml) for BCNU alone; 8X (2 mg/ml) for HN2 alone, and 4X (1.0 mg/ml) for HN2 alone; and 4X (18 mg/ml) for

VP16 alone and IX (2 mg/ml) for VP16 plus V/L.

As can be seen, in contrast to the results from the percent cell survival method for the drug concentrations selected, potentiation of cancer chemotherapeutic drug activity by the modulating agent V/L, defined as a decrease of two or greater units in the EMT 90 value, was detected for both VCR and VP16 in combination with

V/L. (A one unit variation or greater was determined to be significant since the difference between 16X and 8X is within the range of human observer variability, whereas the difference between 32X and 8X exceeds the range of observer variability.)


Evaluation of Resistance of Ovarian Cancer Cells Using EMT 90

A similar comparison of results using the percent cell survival and EMT 90 methods for treatment sensitivity was made using tumor cells from a 69-year- old female patient (Indio, California) having Stage IV

ovarian cancer. The patient had failed three previous therapeutic regimes using (1) cyclophosphamide, doxo¬ rubicin, cisplatin; (2) high dose intraperitoneal cisplatin; and (3) cisplatin plus cytarabine.

Indeed, the drug concentrations selected for the other four drugs (LPAM, VCR, BCNU and HN2) provided less than 90% tumor cell kill.

Tumor cells were obtained from a paracentesis of the patient's ovarian tumor ascites fluid and were incubated for four days with the following concentrations of cisplatin (Bristol-Myers Laboratories, Syracuse, NY), (CDDP) 0.75 mg/ml (IX); 1.5 mg/ml (2X); 3.0 mg/ml (4X) ; 6.0 mg/ml (8X) and 12 mg/ml (16X) alone and in combination with V/L (0.5 mg/ml verapamil and 6 mg/ml lidocaine) , and processed as described above in Example II. Slides containing cell disks corresponding to the five dilutions of CDDP (and CDDP + V/L) were scanned to determine the EMT 90 endpoint and results were plotted on a log scale as shown in Figure 10. Similarly, tumor cells were incubated with a single con¬ centration of CDDP (1.5 mg/ml) and CDDP + V/L, and were processed using the percent cell survival assay. Data for percent cell survival were plotted on a log scale on the same graph as for the EMT 90 results (Figure 10). As can be seen in Figure 10, the EMT 90 assay in this experiment provided similar results to those obtained for the percent cell survival assay. A 90% dramatic decrease in cell survival occurred for a four fold lower concentration of CDDP + V/L in the EMT 90 assay compared to CDDP alone. In the cell survival assay, an 85% decrease was measured at the same single concentration (1.5 mg/ml) of CDDP + V/L.

Therefore, the EMT 90 method provides an assay comparable to or more sensitive than the percent cell survival DiSC assay, and is also capable of indicating dramatic shifts in cell sensitivity with alternative therapeutic regimes.

The method and apparatus of the present invention relying on an effective drug concentration rather than percent cell survival, provide greater efficiency and reproducibility for assays to determine chemosensitivity using differential staining of tumor cells. The problems inherent in individual cell counting including tumor cell clumping are eliminated, and the speed of determining results are dramatically increased. For example, a trained technician can review 12 different assay slides (60 individual disks) and quantitate results at a rate of approximately 1 minute per slide or 12 minutes total with the EMT 90 method, compared to an average time of up to six (6) hours using the percent cell survival method previously described.

As will be apparent to those skilled in the art in which the invention is addressed, the present invention may be embodied in forms other than those specifically disclosed above, without departing from the spirit or essential characteristics of the invention. Particular embodiments of the present invention described above are therefore to be considered in all respects as illustrative and not restrictive. The scope of the present invention is as set forth in the appended claims rather than being limited to the examples contained in the foregoing description.

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