METHODS FOR PREDICTING A CANCER PATIENT'S RESPONSE TO ANTIFOLATE CHEMOTHERAPY
The present invention provides methods for individualizing therapy for cancer treatment, and particularly for evaluating a patient's responsiveness to one or more antifolate therapeutic agents prior to treatment with such agents. Particularly, the invention provides an in vitro chemoresponse assay for predicting a patient's response to an antifolate agent, such as pemetrexed or methotrexate.
1 . A method for predicting the efficacy of an antifolate therapeutic agent for a cancer patient, comprising:
culturing malignant cells from a tumor specimen from the patient;
contacting the cultured cells with the antifolate therapeutic agent; and
evaluating the response of the malignant cells to the drug.
2 . The method of claim 1 , wherein the cancer is selected from lung, mesothelioma, breast, ovarian, colorectal, endometrial, thyroid, nasopharynx, prostate, head and neck, liver, kidney, pancreas, bladder, and brain.
3 . The method of claim 1 , wherein the cancer is lung cancer.
4 . The method of claim 3 , wherein the lung cancer is NSCLC or mesothelioma.
5 . The method of claim 1 , wherein the antifolate therapeutic agent is pemetrexed.
6 . The method of claim 1 , wherein the malignant cells are cultured from a plurality of tumor explants in a monolayer culture in growth media.
7 . The method of claim 6 , wherein the tumor explants are prepared by mechanical fragmentation of the patient's tumor specimen.
8 . The method of claim 7 , wherein the tumor specimen is minced.
9 . The method of claim 8 , wherein the explants each measure from about 0.25 to about 1.5 mm 3 .
10 . The method of claim 6 , where the monolayer cells are suspended in growth media, and the cells plated for chemosensitivity testing in treatment media.
11 . The method of claim 10 , wherein the growth media is removed prior to adding the treatment media.
12 . The method of claim 10 , wherein the treatment media is low folic acid media.
13 . The method of claim 12 , wherein the low folic acid media is RPMI.
14 . The method of claim 10 , wherein the cells are washed between the removal of growth media and the addition of treatment media.
15 . The method of claim 14 , wherein the cells are washed with PBS.
16 . The method of claim 10 , wherein dilutions of pemetrexed are added across a plurality of wells within the range of about 0.03 ng/ml to about 20 mg/ml, or about 1.0 nM to about 1.0 mM.
17 . The method of claim 1 , wherein the drug is contacted with the cells for about 3 days, and then cell viability quantified.
18 . The method of claim 17 , wherein cell viability is quantified by visible light, UV light, or fluorescent light.
19 . The method of claim 17 , wherein cells are stained with DAPI.
20 . The method of claim 1 , wherein the response of the drug to the chemotherapeutic agent is evaluated by preparing a dose response curve, and determining an adjusted AUC.
21 . The method of claim 20 , wherein the aAUC assigns weights relative to the degree of change in cytotoxicity between dose points.
22 . The method of claim 21 , wherein changes in cytotoxicity between dose points are quantified by a local slope, and the local slopes weighted along the dose-response curve to emphasize cytotoxic responses.
23 . The method of claim 22 , wherein aAUC is calculated by:
calculating a Cytotoxity Index (CI) for each dose;
calculating a local slope (S d ) at each dose point;
calculating a slope weight at each dose point, by W d=1 −S d ; and
calculating aAUC, where aAUC=Σ W d CI d , and where d represents each dose in a range.
24 . The method of claim 23 , wherein aAUC is calculated for a truncated dose response curve.
25 . The method of claim 23 , further comprising, assigning the tumor sample as being sensitive, resistant, or intermediate sensitive to the chemotherapeutic agent.