WO2007015926A2 - Systemes et procedes multiparametre de cytotoxicite a cytometrie de flux, compositions et kits d'evaluation de la susceptibilite de cellules cancereuses a une modalite de traitement - Google Patents

Systemes et procedes multiparametre de cytotoxicite a cytometrie de flux, compositions et kits d'evaluation de la susceptibilite de cellules cancereuses a une modalite de traitement Download PDF

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WO2007015926A2
WO2007015926A2 PCT/US2006/028159 US2006028159W WO2007015926A2 WO 2007015926 A2 WO2007015926 A2 WO 2007015926A2 US 2006028159 W US2006028159 W US 2006028159W WO 2007015926 A2 WO2007015926 A2 WO 2007015926A2
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cell
cells
cancer
cytotoxicity
cll
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WO2007015926A3 (fr
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James Z. Huang
Yanping Zhong
Antony Bakke
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Oregon Health And Science University
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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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the fields of oncology, cell biology, and flow cytometry. More specifically, provided herein are multiparameter flow cytometric cytotoxicity systems, methods, compositions, and kits for assessing, in a patient sample, whether one or more cancer cell(s) is susceptible to a chemotherapeutic or other cancer treatment modality.
  • cancers are typically comprised of multiple clonal cell populations. Cancer cells from one patient may differ in biology and, consequently, in response to a therapeutic regimen as compared to cancer cells from another patient afflicted with the same disease. In the United States, blood cancers account for 7% of all cancers for a total of
  • B-cell chronic lymphocytic leukemia (B-CLL) is the most common form of leukemia in North America and Europe.
  • the clinical course for CLL is quite variable. Montserrat, Hematol J. 5 Suppl. l:S2-9 (2004); Wendtner et al, Semin. Hematol. 41:224-233 (2004); and Leporrier, Hematol. J. 5 Suppl l:S10-19 (2004).
  • Some patients have an indolent clinical course while for other patients treatment is indicated. For those patients requiring treatment, however, clinical responses are quite variable; many patients show no response or only partial response.
  • a diagnosis of CLL leads to three possible treatment options: (1) "watch and wait", (2) chemotherapy, and (3) bone marrow transplantation.
  • Watch and wait is often chosen for non-aggressive types of cancer, largely because the available treatment regimens are more toxic than the disease itself.
  • Chemotherapy is chosen for patients whose cancer is growing and poses a significant risk to a patient's short-term health. Bone marrow transplantation is commonly performed upon recurrence of disease following a course of chemotherapy, typically due to out-growth of residual drug resistant cancer cells.
  • a limitation in current cancer treatment regimens lies in the vast number of choices of chemotherapy drugs and the high incidence of resistance to these drugs.
  • a patient is given 1 to 3 chemotherapy drugs for treatment.
  • An oncologist prescribes the drugs that work based upon the previous responses of patients with similar cancers.
  • Fludarabine is a first line drug for B-cell chronic lymphocytic leukemia (CLL).
  • the present invention addresses these and other related needs by providing, inter alia, multiparameter flow cytometric cytotoxicity systems, methods, compositions, and kits that are suitable for assessing, prior to the initiation of treatment, whether a patient sample has one or more cancer cell(s) that is susceptible to one or more candidate chemotherapeutic or other cancer treatment modality.
  • a tissue sample comprising one or more cancer cell(s) is collected from a patient and incubated in vitro with one or more treatment modality of interest.
  • Cancer cells and non cancer cells within the tissue sample are identified based upon the presence of cell population specific surface markers (cell- surface markers; typically antigens) using a label such as a protein ligand, antibody, and/or antibody fragment that specifically binds to a cell population surface-marker within the tissue sample.
  • Drug-induced cell death (cytotoxicity) in cancer cells as well as non-cancerous cells is subsequently detected and quantified without interference from healthy cells within the tissue sample. Cytotoxicity of one or more cancer cells within the cell population indicates that the drug or agent is effective in a treatment regimen against those one or more cancer cells. In contrast, absence of cytotoxicity of one or more cancer cells within the cell population indicates that the drug or agent is ineffective in a treatment regimen against those one or more cancer cell(s).
  • the present invention provides methods for assessing the suitability of a cytotoxic drug and/or treatment regimen comprising a cytotoxic drug for the treatment of a cancer patient.
  • the susceptibility to drug-induced toxicity of one or more normal cell in a patient sample is compared to the susceptibility to drug-induced toxicity of one or more cancer cell in the same patient sample.
  • the susceptibility of the cancer cell(s) is compared to the susceptibility of the normal cell(s) and the relative susceptibility is calculated and a corresponding in vitro therapeutic index is determined.
  • Such methods will find utility in assessing whether a given cytotoxic drug and/or treatment regimen is suitable for one or more individual cancer patient(s) wherein a high degree of susceptibility to a cytotoxic drug of one or more normal cell(s) as compared to one or more cancer cell(s) indicates that the cytotoxic drug is toxic the patient and consequently is not indicated for treatment of the patient whereas a low degree of susceptibility to a cytotoxic drug of one or more normal cell(s) as compared to one or more cancer cell(s) indicates that the cytotoxic drug is not toxic to the patient and consequently may be indicated for treatment of the patient without an expectation of in vivo toxicity.
  • the methods disclosed herein can be completed in two days or less, with early detection of cancer cell death, and are sensitive at a single cell level.
  • the assay is suitable for determining the best chemotherapeutic or other treatment modality for individual cancer patients.
  • the assay is suitable for all clinical samples including those with only a small amount of residual disease.
  • the samples can be either freshly harvested from the patient or cryopreserved.
  • Patient tissue samples that may be subjected to the multiparameter flow cytometric cytotoxicity systems and methods disclosed herein include, but are not limited to, blood, bone marrow, and biopsy samples of solid tumors.
  • Cancer cells within such patient samples that may be assayed for susceptibility to one or more chemotherapy or other treatment modality include, but are not limited to, cells associated with a wide variety of hematopoitic tancers including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous , leukemia (CML), hairy cell leukemia (HCL), non-Hodgkin's lymphoma (either T-cell, NK-cell, or B-cell lymphoma), and multiple myeloma.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogen
  • patient samples include biopsy samples from solid tumors such as soft tissue sarcomas and cancers of the brain, esophagus, skin, uterine cervix, bone, lung, liver, endometrium, bladder, breast, larynx, colon/rectum, stomach, ovary, pancreas, adrenal gland, and prostate. It will be understood that other patient samples may also be tested by the systems and methods presented herein.
  • solid tumors such as soft tissue sarcomas and cancers of the brain, esophagus, skin, uterine cervix, bone, lung, liver, endometrium, bladder, breast, larynx, colon/rectum, stomach, ovary, pancreas, adrenal gland, and prostate. It will be understood that other patient samples may also be tested by the systems and methods presented herein.
  • Candidate drugs or agents that may be tested for efficacy, individually or in combination, against one or more cancer cells from a patient sample include, without limitation, one or more known chemotherapy drug(s) and other small molecules as well as one or more therapeutic biomolecule such as, for example, an antibody or fragment thereof and/or a protein ligand or soluble receptor.
  • Suitable chemotherapy drugs that may be tested for efficacy in the present systems and methods include, but are not limited to, Altretamine, Arsenic Trioxide, Asparaginase, Bleomycin, Busulfan, Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide, Cytarabine, dacarbazine, Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Estramustine, Etoposide, Floxuridine, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan, Isotretinoin, Lomustine, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Paclitaxel, Pegaspargase, Pemetrexed
  • Suitable antibody therapeutics that may be tested for efficacy in the present systems and methods include, but are not limited to, Alemtuzumab, Bevacizumab, Cetuximab, Gemtuzumab ozogamicin, Trastuzumab, and Rituximab.
  • labels that specifically bind to a cancer cell bind to a cell-surface marker such that cancer cells can be distinguished from admixed non-cancer cells.
  • CeIl- surface markers include, for example, proteins, such as enzymes, hormones, oncofetal antigens, and blood-group antigens; and cell-surface displayed carbohydrates, including glycoproteins and glycolipids.
  • Labels that may be suitably employed for specifically labeling one or more cancer cells include protein ligands, antibodies, and fragments thereof.
  • Exemplary cell-surface protein markers include, but are not limited to, Alpha- fetal Protein (AFP), Human Chorionic Gonadotrophin (hCG), Calcitonin, CA27.29, CAl 5-3, CA19.9, CA125, CA72-4, Carcinoembryonic Antigen (CEA), CD22, CD3, CD33, CD13, CDw65, Lactoferrin (LF), CD68, CD15, CD15s, CD79a (MB-I), CD79b (B29), CD87 (uPA-R), and CDl 17 (c-kit).
  • AFP Alpha- fetal Protein
  • hCG Human Chorionic Gonadotrophin
  • Calcitonin CA27.29, CAl 5-3, CA19.9, CA125, CA72-4
  • CEA Carcinoembryonic Antigen
  • CD22 CD3, CD33, CD13, CDw65, Lactoferrin (LF)
  • LF Lactoferrin
  • CD68 CD15, CD15s, CD79a (MB
  • Exemplary cell-surface carbohydrate markers include, but are not limited to, N-linked and O-linked mucin-like glycoproteins; cell membrane-bound mucin-like carbohydrate structures such as Tn, sialyl-Tn, and T; and blood group-related carbohydrate structures such as Le(x), sialyl-Le(x), ABH, and Le(y).
  • An exemplary cell-surface glycolipid is CA19.9.
  • T-cell markers such as CDIa, CD2, CD3, CD4, CD5, CD7, CD8, TCR alpha-beta, and TCR gamma-delta
  • B-cell markers such as CDlO, CD19 CD20, CD21, CD22, CD23, CD24, CD79b, CD 103, Kappa, Lambda, IgG, IgM, IgD, IgA, and FMC-7
  • Myeloid/Monocytic-cell markers such as CD13, CD14, CD15, CD33, CDl Ib, CDl 17, and myeloperoxidase.
  • One or more other marker(s) may also be used such as, for example, CDl Ic, CD25, CD34, CD45, HLA-Dr, Tdt, CD16, CD30, CD38, CD41, CD42b, CD56, CD57, CD61, and glycophorin.
  • B-CLL exemplified herein, leukemic cells may be identified with a label comprising antibodies that specifically bind to CD5 and antibodies that specifically bind to CD19.
  • labels typically include one or more fluorescent dye such as, for example, allophycocyanin (APC), phycoerythrin (PE), and fluorescein- isothiocyanate (FITC).
  • Labels may also include a tag such as biotin.
  • the label may be a first antibody that does not otherwise include a fluorescent dye or other tag.
  • a second antibody may be employed wherein the second antibody is capable of specifically binding to the first antibody.
  • the second antibody typically includes one or more fluorescent dye or other detectable tag.
  • cytotoxicity may be measured by staining with annexin, anti-phosphatidylserine, and/or 7-AAD. Other assay systems may also be employed.
  • Figures IA- 1C present a gating strategy for measuring drug-induced cytotoxicity in B-CLL cells. Lymphocytes are gated based on forward and side scatter in region R
  • the surviving B-CLL cells are those viable cells that express both CD5 and
  • Figure 2 presents the variability of in vitro drug sensitivity profiles of 43 samples of B-CLL.
  • the drug concentrations for Fludarabine, Chlorambucil, Cladribine and Prednisolone are at 2.5 mg/ml, 2.5 mg/ml, 0.5 mg/ml, and 25 mg/ml, respectively.
  • Leukemic survival indices are displayed as a mean ⁇ SE of triplicate wells.
  • Figures 3A-3F present the correlation of in vitro drug sensitivity to Fludarabine, Chlorambucil, Cladribine, and Prednisolone.
  • a good correlation is observed between Fludarabine and Chlorambucil (Fig. 3A), between Fludarabine and Cladribine (Fig. 3B), and between Chlorambucil and Cladribine (Fig. 3C).
  • a poor correlation is seen between Prednisolone and Fludarabine (Fig. 3D), between Prednisolone and Chlorambucil (Fig. 3E), and between Prednisolone and Cladribine (Fig. 3F).
  • Figures 4A-4B present differences of in vitro drug sensitivity among different cytogenetic groups.
  • Figures 5A-5B present in vitro drug sensitivity of B-CLL cells from patients at different clinical stages of disease.
  • B-CLL cells from patients with early disease are more sensitive to Fludarabine than those from patients with advanced disease (Fig. 5A).
  • No significant difference is seen in B-CLL cells from patients with early and advanced stage exposed to Prednisolone (Fig. 5B).
  • the present invention provides multiparameter flow cytometric cytotoxicity systems, methods, compositions, and kits for assessing a clinical response to one or more drug and/or agent either alone or in combination.
  • a patient sample comprising one or more cancer cells is collected from a patient and contacted in vitro with one or more treatment modality. Cancer cells are specifically identified in the patient sample by contacting the cell population with a label that specifically binds to one or more cancer cell(s) but does not specifically bind normal cells within the cell population. Efficacy of the one or more candidate drug or agent is assessed by assaying for drug-induced cancer cell death (i.e. cytotoxicity) in the specifically labeled cancer cell population.
  • drug-induced cancer cell death i.e. cytotoxicity
  • Cytotoxicity of one or more cancer cells within the cell population indicates that the drug or agent is effective in a treatment regimen against those one or more cancer cells. In contrast, absence of cytotoxicity of one or more cancer cells within the cell population indicates that the drug or agent is ineffective in a treatment regimen against those one or more cancer cells.
  • the multiparameter flow cytometric cytotoxicity systems, methods, compositions, and kits of the present invention are exemplified herein by their application to the evaluation of the efficacy of four commonly used chemotherapeutic agents (i.e. Fludarabine, Chlorambucil, Cladribine, and Prednisolone) on chronic lymphocytic leukemia (CLL) cells from 43 patients.
  • CLL chronic lymphocytic leukemia
  • Specific drug-induced cytotoxicity of leukemic cells was measured after subjecting a patient sample to treatment with Fludarabine, Chlorambucil, Cladribine, and/or Prednisolone.
  • Leukemic cells were identified by staining with antibodies against CD5 and CD19. Cytotoxicity was assessed with Annexin V and 7- AAD.
  • Leukemic cell survival indices were calculated by expressing viable leukemic cells in test culture as percent of viable leukemic cells in control cultures.
  • the present methods can be advantageously completed in two days or less, with early detection of cell death, and are sensitive at a single cell level.
  • the results achieved by these methods yield drug sensitivity profiles that will find utility in guiding the selection of a drug having efficacy against a given disease.
  • the present methods also permit the identification of an appropriate drug for residual disease that exhibits resistance to an initial therapeutic regimen.
  • the present invention permits a determination of a drug modality having efficacy as a second line of chemotherapy to kill the remaining cancerous cells.
  • Patient samples that may be subjected to the multiparameter flow cytometric cytotoxicity methods disclosed herein include, but are not limited to, blood, bone marrow, and biopsy samples of solid tumors.
  • Cancer cells within such patient samples that may be assayed for susceptibility to one or more chemotherapy or other treatment modality include, but are not limited to, cells associated with a wide variety of hematopoitic cancers including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), non-Hodgkin's lymphoma (either T-cell, NK-cell, or B-cell lymphoma), and multiple myeloma.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • HCL
  • biopsy samples from solid tumors such as soft tissue sarcomas and cancers of the brain, eye, skin, liver, esophagus, uterine cervix, bone, lung, endometrium, bladder, breast, larynx, colon/rectum, stomach, ovary, pancreas, adrenal gland, and prostate.
  • solid tumors such as soft tissue sarcomas and cancers of the brain, eye, skin, liver, esophagus, uterine cervix, bone, lung, endometrium, bladder, breast, larynx, colon/rectum, stomach, ovary, pancreas, adrenal gland, and prostate.
  • Red blood cells in blood or bone marrow samples are, generally, removed by a lysis procedure or by ficoll gradient centrifugation, by standard methods.
  • Mononuclear cells typically containing cancer cells are either used fresh or cryopreserved.
  • An excisonal biopsy procedure is often performed to remove a cancer sample of an involved lymph node or other tissue.
  • the cells are disaggregated, single cells are prepared, and they are either used fresh or cryopreserved.
  • Candidate drugs or agents that may be tested for efficacy against one or more cancer cells from a patient sample include, without limitation, one or more known chemotherapy drugs and other small molecules as well as one or more therapeutic biomolecule such as, for example, an antibody or fragment thereof and/or a protein ligand or soluble receptor.
  • Suitable chemotherapy drugs that may be tested for efficacy in the present systems and methods include, but are not limited to, Altretamine, Arsenic Trioxide, Asparaginase, Bleomycin, Busulfan, Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide, Cytarabine, dacarbazine, Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Estramustine, Etoposide, Floxuridine, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan, Isotretinoin, Lomustine, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Paclitaxel, Pegaspargase, Pemetrexed
  • Suitable antibody therapeutics that may be tested for efficacy in the present systems and methods include, but are not limited to, Alemtuzumab, Bevacizumab, Cetuximab, Gemtuzumab ozogamicin, Trastuzumab, and Rituximab.
  • labels that specifically bind to a cancer cell bind to a cell-surface marker.
  • Cell-surface markers include, for example, proteins, such as enzymes, hormones, oncofetal antigens, and blood-group antigens; and cell-surface displayed carbohydrates, including glycoproteins and glycolipids.
  • Labels that may be suitably employed for specifically labeling one or more cancer cells include protein ligands, antibodies, and fragments thereof.
  • the term "specifically bind” as used in the context of the binding of a label to a cell-surface marker refers to binding to the cell-surface marker at a detectable level (within, for example, an ELISA assay) and absence of detectable binding with unrelated polypeptides under similar conditions.
  • Specific binding as used in this context, generally refers to the non-covalent interactions of the type that occur between an antibody and an antigen for which the antibody is specific.
  • the strength, or affinity of antibody-target antigen binding interactions can be expressed in terms of the dissociation constant (K d ) of the interaction, wherein a smaller K d represents a greater affinity.
  • K d dissociation constant
  • Specific binding properties of target-specific antibodies can be quantified using methods well known in the art.
  • One such method entails measuring the rates of target-specific antibody/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions.
  • both the "on rate constant” (K On ) and the “off rate constant” (K Off ) can be determined by calculation of the concentrations and the actual rates of association and dissociation.
  • the ratio of K off /K on enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant Kd. See, generally, Davies et al, Annual Rev. Biochem. 59:439-473 (1990).
  • Exemplary cell-surface cell-surface protein markers include, but are not limited to, Alpha-fetal Protein (AFP), Human Chorionic Gonadotrophin (hCG), Calcitonin, CA27.29, CA15-3, CA19.9, CA125, CA72-4, Carcinoembryonic Antigen (CEA), CD22, CD3, CD33, CD13, CDw65, Lactoferrin (LF), CD68, CD15, CD15s, CD79a (MB-I), CD79b (B29), CD87 (uPA-R), and CDl 17 (c-kit).
  • AFP Alpha-fetal Protein
  • hCG Human Chorionic Gonadotrophin
  • Calcitonin CA27.29
  • CA15-3 CA19.9
  • CA125 CA72-4
  • CEA Carcinoembryonic Antigen
  • CEA Carcinoembryonic Antigen
  • CD22 CD3, CD33
  • CD13 CDw65
  • Lactoferrin LF
  • CD68 CD15, CD15s, CD79a (
  • Exemplary cell-surface carbohydrate markers include, but are not limited to, N-linked and O-linked mucin-like glycoproteins; cell membrane-bound mucin-like carbohydrate structures such as Tn, sialyl-Tn, and T; and blood group-related carbohydrate structures such as Le(x), sialyl-Le(x), ABH, and Le(y).
  • An exemplary cell-surface glycolipid is CAl 9.9.
  • One or more other marker(s) may also be used such as, for example, CDl Ic, CD25, CD34, CD45, HLA-Dr, Tdt, CD16, CD30, CD38, CD41, CD42b, CD56, CD57, CD61, glycophorin.
  • Table 2, below, further identifies specific cell-surface antigens and their hematopoietic disease associations.
  • leukemic cells may be identified with a label comprising antibodies that specifically bind to CD5 and antibodies that specifically bind to CD 19.
  • labels typically include one or more fluorescent dye such as, for example, allophycocyanin (APC), phycoerythrin (PE), and fiuorescein- isothiocyanate (FITC).
  • Labels may also include a tag such as biotin.
  • the label may be a first antibody that does not otherwise include a fluorescent dye or other tag.
  • a second antibody may be employed wherein the second antibody is capable of specifically binding to the first antibody.
  • the second antibody typically includes one or more fluorescent dye or other detectable tag.
  • a wide variety of antibodies that specifically bind to the cell-surface antigens disclosed herein and that may be suitably employed in the systems and methods of the present invention are readily available from commercial sources.
  • Bioprocessing, Inc. (Scarborough, ME) provides antibodies to CaI 5-3, Ca27.29, breast tumor antigens, Cal25, ovarian tumor antigen, CaI 9-9, GI tumor antigen, CEA, carcinoembryonic antigen, AFP, alpha fetoprotein, Ca72.4, Tag-72, NSE, neuron specific enolase, Cyfra21-1, cytokeratin-19, PSA, prostate specific antigen using cell culture technologies; Diatec (Oslo, Norway) provides monoclonal antibodies including unconjugated antibodies and antibodies conjugated with FITC, PE, biotin, and APC against markers for leukemias and lymphomas; EMD Biosciences (San Diego, CA) and EXBIO Praha (Vestec, Czech Republic) provide monoclonal antibodies to human
  • Invitrogen/Molecular Probes (Eugene, OR) provides primary and secondary antibodies, including fluorescently tagged antibodies, for flow cytometry;
  • QED Biosciences, Inc. (San Diego, CA) provides antibodies directed to antibodies for cancer markers and apoptosis;
  • R&D Systems (Minneapolis, MN) provides antibodies against cytokine receptors;
  • Vector Laboratories (Burlingame, CA) provides a wide range of primary and secondary antibodies including primary antibodies for flow cytometry and against various tumor markers, hnmunostaining procedures for labeling cell-surface markers are readily available in the art.
  • the present invention provides multiparameter flow cytometry systems and methods to permit the rapid and simultaneous measurement of individual cells within the context of heterogenous cell populations.
  • Typical flow cytometers comprise three major systems: fluidic, optical, and electronic.
  • the fiuidics system pumps a suspension of cells through a nozzle, creating a stream with laminar flow properties.
  • the optical system focuses one or more laser beams on the stream and collects scattered and fluoresced light from cells as they pass through the laser beam.
  • the electronics measure and digitize these light emissions so that they can be analyzed on a desktop computer.
  • the terms “forward Scatter”, “FSC”, “FS” and “FALS” are used interchangeably to refer to the parameter that is a rough indicator of a cell's size.
  • the terms “side scatter”, “SSC”, and “SS” are used interchangeably to refer to the parameter that is a rough indicator of cellular granularity, membrane complexity, and number of organelles. Taken together, the two scatter parameters provide a morphological fingerprint of the cells passing through the flow cytometer.
  • Cell-surface markers such as proteins, carbohydrates, and lipids (see, above) are detected with one or more label, typically an antibody, that has been conjugated to a fluorescent molecule such as, for example, FITC, PE, Texas Red, and APC.
  • DNA content can be measured with propidium iodide (PI), 7-AAD, the Hoechst dyes, or DAPI. BrdU incorporation may be employed for measuring cell proliferation.
  • Apoptosis can be measured in a number of ways. The TUNEL technique identifies DNA strand breaks. Annexin V labeling detects changes in the plasma membrane asymmetry. Nuclear condensation and DNA loss are demonstrated by hypodiploid peaks in DNA content experiments. Uptake of Hoechst 33258 also increases with apoptotic changes.
  • cytotoxicity may be measured by staining with annexin, anti-phosphatidylserine, and/or 7- AAD.
  • CMC/ADCC cell-mediated cytotoxicity/antigen-dependent cytotoxicity
  • a cell tracking dye CFSE is utilized to label a patient cell population.
  • Cell death is measured by addition of 7AAD (live/dead).
  • 7AAD only enters membrane-compromised cells and binds to DNA.
  • Flow cytometry is utilized to gate on the target cells and measure 7AAD negative vs. 7AAD postitve cells.
  • Assay systems for measuring apoptosis may also be employed in the systems and methods presented herein.
  • a candidate therapeutic modality is incubated with a patient sample comprising one or more cancer
  • the cancer cells are stained, as described above, with a target cell-specific label such as, for example, a PE-conjugated mAb.
  • Annexin V-FITC which binds to cells expressing phosphatidylserine (an early marker of apoptosis) on the cell-surface, is subsequently added. Cancer cells are gated upon and quantified with respect to their annexin V positivity. The shift from annexin Vneg to annexin Vhi is a discrete event such that all cancer cells fall within discernible populations with respect to annexin V. This methodology permits the analysis of single cancer cells.
  • This Example discloses an in vitro multiparameter flow cytometry-based drug sensitivity and resistance assay for routine analysis of clinical samples containing a heterogeneous population of both leukemic and normal hematopoietic cells.
  • Red cells were removed from each patient sample by Ficoll gradient centrifugation by standard methods. Flow cytometric analysis was performed with fresh samples for diagnostic classification. The remaining cells were cryopreserved in liquid nitrogen with RPMI 1640 culture medium (GIBCO, Grand Island, NY) containing 20% heat-inactivated fetal bovine serum (FBS) (GIBCO, Grand Island, NY) and 10% DMSO (Sigma, St. Louis, MO).
  • RPMI 1640 culture medium Gib Island, NY
  • FBS heat-inactivated fetal bovine serum
  • DMSO Sigma, St. Louis, MO
  • Cryopreserved cells were thawed in a water bath at 37 0 C and transferred to RPMI 1640 tissue culture medium containing 2.5 mM MgCl 2 and 200 units/ml DNAase I. After incubation at room temperature for 10 minutes, cells were washed twice with phosphate buffered saline (PBS) and resuspended in RPMI 1640 supplemented with 20% FBS, 2 mM glutamine, 100 ⁇ g/ml streptomycin, and 100 ⁇ g/ml penicillin. Cell viability was determined by trypan blue dye exclusion. Cell concentration was adjusted to 1.5-3.0 x 10 6 viable cells/ml.
  • PBS phosphate buffered saline
  • each cell suspension was added to each well of a 96-well microtiter plate containing 50 ⁇ l of either Fludarabine (Sigma, St. Louis, MO), Chlorambucil (Sigma, St. Louis, MO), Cladribine (Sigma, St. Louis, MO), or Prednisolone (Sigma, St. Louis, MO), at 4x their respective empirically derived cut-off concentrations (EDCC), which produced the largest scatter of B-cell CLL cell survival seen among 12 representative samples with test protocols.
  • EDCC empirically derived cut-off concentrations
  • Each drug was tested in triplicate for each treatment group.
  • the culture plates were incubated at 37 0 C in humidified atmosphere containing 95% air and 5% CO 2 for 48 hours. At the end of the incubation period, the culture plates were centrifuged at 1500 rpm for 5 minutes and the supernatants discarded.
  • the cells in each well were resuspended with 100 ⁇ l of RPMI 1640 supplemented with 10% FBS.
  • 2.5 ⁇ l of CD19-APC (BD Biosciences, San Diego, CA) and 2 ⁇ l of CD5- PE (BD Biosciences, San Diego, CA) were added to each well. Plates were incubated on ice for 30 minutes in the dark. After one wash with 250 ⁇ l Ix binding buffer (BD Biosciences, San Diego, CA), 100 ⁇ l of Ix binding buffer was added.
  • the cells in each well were resuspended and transferred to 120 x 75 mm tubes. 2 ⁇ l of Annexin V-FITC (BD Biosciences, San Diego, CA) and 1 ⁇ l of 1 mg/ml 7-
  • AAD Sigma, St. Louis, MO
  • LCSI leukemic cell survival index
  • Bosanquet and Bell Blood 87:1962- 1971 (1996); Bosanquet and Bell, Leuk. Res. 20:143-153 (1996); and Bosanquet et al., Br. J. Cancer 76:511-518 (1997).
  • the presently described assay system is relatively robust and could be adapted to routine clinical laboratory, superior to other in vitro assays such as the MTT assay (Twentyman et al, Br. J. Haematol. 71:19-24 (1989)), the DISC assay (Bosanquet, Lancet 337:711-714 (1991)), and FCMA (Larsson et al, Int. J. Cancer 50:177-185 (1992)), requiring a relatively homogeneous population of leukemic cells in clinical samples typically seen in advanced stage only.
  • MTT assay wentyman et al, Br. J. Haematol. 71:19-24 (1989)
  • the DISC assay Bosanquet, Lancet 337:711-714 (1991)
  • FCMA Lisson et al, Int. J. Cancer 50:177-185 (1992)
  • CLL cells have morphological features indistinguishable from admixed normal lymphocytes that are almost always sensitive to chemotherapeutic drugs.
  • Our protocol is suitable to all routine clinical samples regardless of the percentages of B-CLL cells in the samples.
  • the present methods eliminate possible interference of spontaneous apoptosis that may overshadow the actual drug effect on the B-CLL cells seen in other assays. Therefore, the present assay system is suitable for both fresh and cryopreserved samples. This assay detects a remarkable variability in sensitivity to various drugs and their combinations in B-CLL cells, which is consistent with the reported results of other in vitro chemotherapy sensitivity assays. Morabito et al, Br. J. Haematol.
  • the difference of drug sensitivity among different cytogenetic groups of B-CLL provides additional validation for the reliability of in vitro drug effect as measured by our multiparameter flow cytometric cytotoxicity assay and supports the utility of personalization of therapy for B-CLL patients with different cytogenetic abnormalities and in vitro drug sensitivity profiles.
  • the methodology disclosed herein permit the assessment of drug sensitivity and resistance in drug discovery, clinical trials, and routine clinically practice and offers a cost effective approach to help clinicians identify optimal therapeutic regimens for biologically heterogeneous diseases.
  • Example 1 To correlate in vitro drug sensitivity/resistance results with cytogenetic abnormalities, each of the 43 cases described in Example 1 was further studied by interphase FISH in order to identify and characterize common cytogenetic abnormalities in B-CLL, including trisomy 12, deletion of p53, AMT, or 13q.
  • Interphase FISH was performed with fresh slides made from the fixed cell pellets of untreated cells.
  • Hybridization was performed using commercially available probes (Vysis, Downers Grove, IL) to detect genomic losses in 13q, ATM (I lq22.3), and p53 (17pl3.1), and to detect trisomy 12 (CEP12). 200 interphase nuclei were scored for hybridization signals for each probe. On the basis of the results in healthy controls, chromosomal losses and gains were interpreted as negative if they occurred in 5% or fewer of the nuclei.
  • an in vitro assay for drug sensitivity may have broad application in the clinical management.
  • this assay could be used to confirm the drug resistance to purine analogues or alkylating agents since occasional B-CLL with monoallelic loss or inactivation of the p53 gene may still have moderate Fludarabine sensitivity.
  • the assay may be used for identification of drugs that are potentially more effective than Fludarabine since B-CLL cells with Fludarabine resistance in vitro are not always resistant to Chlorambucil or Cladribine. See, Case 7 in Table 2. An alternative effective therapy should be sought early for these patients given that they almost invariably have an aggressive course.
  • in vitro drug sensitivity are also variable, suggesting additional biological factors may affect the drug effect in B-CLL cells.

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Abstract

L'invention concerne des systèmes et des méthodes multiparamètre de cytotoxicité à cytométrie de flux ainsi que des compositions et des kits permettant d'apprécier si une ou plusieurs cellules cancéreuses d'un échantillon prélevé sur un patient présentent une susceptibilité à un mode de chimiothérapie ou autre mode de traitement du cancer. La présente invention convient pour l'élaboration de stratégies thérapeutiques personnalisées permettant de choisir des modalités chimiothérapeutiques et autres à partir des profils de sensibilité à un médicament et de résistance de cellules cancéreuses de patients.
PCT/US2006/028159 2005-07-25 2006-07-21 Systemes et procedes multiparametre de cytotoxicite a cytometrie de flux, compositions et kits d'evaluation de la susceptibilite de cellules cancereuses a une modalite de traitement WO2007015926A2 (fr)

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WO2010135468A1 (fr) * 2009-05-19 2010-11-25 Vivia Biotech S.L. Procédés permettant de fournir des essais de médicaments personnalisés ex vivo pour des tumeurs hématologiques
CN102288767A (zh) * 2011-08-16 2011-12-21 内蒙古科慧生物科技有限责任公司 糖类抗原72-4(ca72-4)定量测定试剂盒及其检测方法
US8343733B2 (en) 2006-03-06 2013-01-01 Zetiq Technologies Ltd. Methods and compositions for identifying a cell phenotype
CN104407151A (zh) * 2014-11-19 2015-03-11 汕头大学医学院 Kindlin-2,Myosin-9和AnnexinⅡ三蛋白联合预测食管鳞癌患者预后试剂盒
US9182403B2 (en) 2009-05-19 2015-11-10 Zetiq Technologies Ltd. Kits for and methods of differential staining of cervical cancer cells and/or tissues
ES2674176A1 (es) * 2016-12-22 2018-06-27 Fundación Instituto De Investigación Marqués De Valdecilla Uso del Gen Prkaca para predecir la respuesta de un sujeto al tratamiento con un análogo de purina
CN116794313A (zh) * 2023-08-18 2023-09-22 江西赛基生物技术有限公司 基于流式细胞仪同时检测三项肿瘤标志物的试剂盒及方法

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US20050054018A1 (en) * 2002-07-17 2005-03-10 Li Chiang J. Activated checkpoint therapy and methods of use thereof

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US6180357B1 (en) * 1999-10-08 2001-01-30 Arius Research, Inc. Individualized patient-specific anti-cancer antibodies
US20050054018A1 (en) * 2002-07-17 2005-03-10 Li Chiang J. Activated checkpoint therapy and methods of use thereof

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US8343733B2 (en) 2006-03-06 2013-01-01 Zetiq Technologies Ltd. Methods and compositions for identifying a cell phenotype
US10018622B2 (en) 2006-03-06 2018-07-10 Zetiq Technologies Ltd. Methods and kits for differential staining of abnormal urinary system cells
US9057092B2 (en) 2006-03-06 2015-06-16 Zetiq Technologies Ltd. Methods and compositions for identifying a cell phenotype
JP2012527627A (ja) * 2009-05-19 2012-11-08 ビビア バイオテック ソシエダッド．リミターダ 血液学的腫瘍に対するエクスビボでの個別化医療試験を提供する方法
WO2010135468A1 (fr) * 2009-05-19 2010-11-25 Vivia Biotech S.L. Procédés permettant de fournir des essais de médicaments personnalisés ex vivo pour des tumeurs hématologiques
CN102460165A (zh) * 2009-05-19 2012-05-16 维维雅生物技术公司 用于为血液肿瘤提供离体个体化药物测试的方法
US9182403B2 (en) 2009-05-19 2015-11-10 Zetiq Technologies Ltd. Kits for and methods of differential staining of cervical cancer cells and/or tissues
AU2010249593B2 (en) * 2009-05-19 2016-03-17 Vivia Biotech S.L. Methods for providing personalized medicine tests ex vivo for hematological neoplasms
RU2636614C2 (ru) * 2009-05-19 2017-11-24 Вивия Байотек С.Л. Способы персонализированного медицинского тестирования ex vivo на гематологические новообразования
CN102288767A (zh) * 2011-08-16 2011-12-21 内蒙古科慧生物科技有限责任公司 糖类抗原72-4(ca72-4)定量测定试剂盒及其检测方法
CN104407151A (zh) * 2014-11-19 2015-03-11 汕头大学医学院 Kindlin-2,Myosin-9和AnnexinⅡ三蛋白联合预测食管鳞癌患者预后试剂盒
ES2674176A1 (es) * 2016-12-22 2018-06-27 Fundación Instituto De Investigación Marqués De Valdecilla Uso del Gen Prkaca para predecir la respuesta de un sujeto al tratamiento con un análogo de purina
CN116794313A (zh) * 2023-08-18 2023-09-22 江西赛基生物技术有限公司 基于流式细胞仪同时检测三项肿瘤标志物的试剂盒及方法
CN116794313B (zh) * 2023-08-18 2023-11-03 江西赛基生物技术有限公司 基于流式细胞仪同时检测三项肿瘤标志物的试剂盒及方法

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