METHODS OF MEASURING ERBB SIGNALING PATHWAY ACTIVITY TO DIAGNOSE AND TREAT CANCER PATIENTS
Provided herein are methods for determining the functional status of a cellular pathway in a diseased cell sample obtained from an individual subject. These methods involve contacting a diseased cell sample obtained from the subject with a perturbing agent (e.g., an activating agent) known to perturb a specific cellular pathway when the pathway is functioning normally. A change in one or more physiological response parameters in the presence of the perturbing agent indicates that the cellular pathway targeted by the perturbing agent is functional in the individual subject. Methods of selecting a targeted therapeutic agent for an individual subject are also provided.
1 . A method of treating a human subject diagnosed with cancer whose cancer cells have non-overexpressed, non-amplified ErbB2 receptor, the method comprising administering to the subject a targeted therapeutic agent that affects an ErbB signaling pathway, wherein the subject has been selected for treatment using a method comprising:
preparing a sample of viable primary cancer cells obtained from the subject and culturing the sample in base media;
contacting (1) a first portion of the sample of viable primary cancer cells obtained from the subject with a perturbing agent, and (2) a second portion of the sample with a perturbing agent and a confirming agent, wherein the perturbing agent selectively affects an ErbB signaling pathway and the confirming agent selectively inhibits the effect of the perturbing agent on the same ErbB signaling pathway;
continuously measuring a physiological response parameter of the viable primary cancer cells in each portion of the sample;
determining a measurand by mathematical analysis of the continuous measurements of the physiological response parameter to identify the difference in value between the first portion of the sample and the second portion of the sample; and
comparing the measurand to a cut-off value derived from a normal reference interval of ErbB signaling pathway activity;
wherein a subject is selected for treatment with a targeted therapeutic agent that affects an ErbB signaling pathway when the measurand is greater than the cut-off value.
2 . The method of claim 1 , wherein the physiological response parameter is cell adhesion or attachment.
3 . The method of claim 1 , wherein the base media used to culture the sample is replaced with fresh base media no less than 12 hours but no more than 72 hours before they are contacted with the perturbing agent.
4 . The method of claim 1 , wherein the physiological response parameter is levels of cell metabolites.
5 . The method of claim 1 , wherein the physiological response parameter is cell enzyme levels related to mitochondrial function.
6 . The method of claim 1 , wherein the subject has non-overexpressed, non-amplified ErbB2 receptor breast cancer.
7 . The method of claim 1 , wherein the perturbing agent is selected from the group consisting of EGF, TGF-α, HB-EGF, amphiregulin, betacellulin, epigen, epiregulin, neuregulin-1, neuregulin-2, neuregulin-3, neuregulin-4, and combinations thereof.
8 . The method of claim 7 , which comprises:
contacting (1) a first portion of the sample of viable cancer cells obtained from the subject with neuregulin, and (2) a second portion of the sample with neuregulin and a confirming agent; and/or (3) contacting a third portion of the sample with an epidermal growth factor and (4) a fourth portion of the sample with an epidermal growth factor and a confirming agent; and
determining a measurand by mathematical analysis of the continuous measurements of the physiological response parameter to identify the difference in value between the first portion of the sample and the second portion of the sample and/or the difference in value between the third portion of the sample and the fourth portion of the sample.
9 . The method of claim 1 , wherein the targeted therapeutic agent is selected from the group consisting of trastuzumab, pertuzumab, lapatinib, afatinib, neratinib, ADXS-HER2, TAK-285, MM-302, MM-121, MM-111, REGN 1400, sapatinib, dacomitinib, poziomitinib, ASLAN001, LIM716, AV-203, Duligotuzumab, Lumretuzumab, Panitumumab, REGN955, MM-151, cetuximab, gefitinib, erlotinib, and combinations thereof.
10 . The method of claim 1 , wherein the targeted therapeutic agent binds to a site selected from the group consisting of domain II of an extracellular segment of HER2; domain IV of an extracellular segment of HER2; a cytoplasmic adenosine triphosphate-binding site of EGFR or HER2; a cysteine residue in an adenosine triphosphate binding site of EGFR or HER2; and combinations thereof.
11 . The method of claim 1 , wherein the subject is also treated with a therapeutic agent that does not affect an ErbB signaling pathway.
12 . The method of claim 1 , wherein the subject has non-overexpressed, non-amplified ErbB negative cancer selected from the group consisting of colon cancer, rectal cancer, endometrial cancer, gastric carcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, glioblastoma, hepatocellular carcinoma, small cell lung cancer, non-small cell lung cancer (NSCLC), melanoma, ovarian cancer, cervical cancer, pancreatic cancer, prostate carcinoma, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma and thyroid carcinoma or head and neck cancer.
13 . The method of claim 1 , wherein the confirming agent is 2C4 mouse monoclonal antibody.
14 . A method of treating a human subject diagnosed with non-overexpressed, non-amplified ErbB2 breast cancer, the method comprising administering to the subject lapatinib that affects an ErbB signaling pathway, wherein the subject has been selected for treatment using a method comprising:
preparing a sample of viable primary breast cancer cells obtained from the subject and culturing the sample in base media;
replacing the base media used to culture the sample with fresh base media no less than 12 hours but no more than 72 hours before the sample is contacted with a perturbing agent;
contacting (1) a first portion of the sample of viable primary breast cancer cells obtained from the subject with neuregulin as a perturbing agent, and (2) a second portion of the sample with neuregulin and a confirming agent, wherein the confirming agent selectively inhibits the same ErbB signaling pathway as neuregulin; and/or (3) contacting a third portion of the sample with an epidermal growth factor as a perturbing agent and (4) a fourth portion of the sample with an epidermal growth factor and a confirming agent, wherein the confirming agent selectively inhibits the same ErbB signaling pathway as epidermal growth factor;
continuously measuring cell adhesion or attachment of the viable primary breast cane cells contacted with the perturbing agent;
determining a measurand by mathematical analysis of the continuous measurements of the physiological response parameter to identify the difference in value between the first portion of the sample and the second portion of the sample and/or the difference in value between the third portion of the sample and the fourth portion of the sample;
comparing the measurand to a cut-off value derived from a normal reference interval for ErbB pathway activity; and
wherein a subject is selected for treatment with lapatinib when the measurand is greater than the cut-off value.
15 . The method of claim 14 , wherein the subject has a non-overexpressed, non-amplified ErbB2 and non-overexpressed, non-amplified EGFR breast cancer.
16 . The method of claim 14 , wherein the subject is also treated with a therapeutic agent that does not affect an ErbB signaling pathway.
17 . A method of treating a human subject diagnosed with non-overexpressed estrogen receptor breast cancer, the method comprising administering to the subject a targeted therapeutic agent that affects an estrogen-related signaling pathway, wherein the subject has been selected for treatment using a method comprising:
preparing a sample of viable primary breast cancer cells obtained from the subject and culturing the sample in a base media;
contacting (1) a first portion of the sample of viable primary breast cancer cells obtained from the subject with a perturbing agent known to upregulate or downregulate the estrogen-related signaling pathway, and (2) a second portion of the sample with the perturbing agent and a confirming agent known to inhibit the same estrogen-related signaling pathway activity;
continuously measuring a physiological response parameter of the viable primary breast cancer cells in each portion of the sample;
determining a measurand by mathematical analysis of the continuous measurements of the physiological response parameter to identify the difference in value between the first portion of the sample and the second portion of the sample; and
comparing the measurand to a cut-off value derived from a normal reference interval for estrogen receptor pathway activity;
wherein a subject is selected for treatment with a targeted therapeutic agent that affects an estrogen related signaling pathway when the measurand is greater than the cut-off value.
18 . The method of claim 17 , wherein the base media used to culture the sample is replaced with fresh base media no less than 12 hours but no more than 72 hours before they are contacted with the perturbing agent;
19 . The method of claim 17 , wherein the physiological response parameter is cell adhesion or attachment.
20 . The method of claim 17 , wherein the physiological response parameter is levels of cell metabolites.
21 . The method of claim 17 , wherein the physiological response parameter is cell enzyme levels related to mitochondrial function.
22 . The method of claim 17 , wherein the perturbing agent is selected from the group consisting of estradiol, estrone, raloxifene, estriol, genistein, DHEA, androstenedione, androstenediol, progesterone, hydroxyprogesterone, testosterone, sulfated forms thereof, and combinations thereof.
23 . The method of claim 17 , wherein the targeted therapeutic agent is selected from the group consisting of fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, palbociclib, abemaciclib, and combinations thereof.
24 . The method of claim 17 , wherein the targeted therapeutic agent binds to a site selected from the group consisting of a cytochrome P450 subunit of aromatase enzyme (CYP19A), activation function 1 (AFP-1) domain of an estrogen receptor, cyclin-dependent kinase 4 (cyclin D1), cyclin-dependent kinase 6 (cyclin D3), and combinations thereof.
25 . The method of claim 17 , wherein the subject is also treated with a therapeutic agent that does not affect an estrogen receptor-related signaling pathway.
26 . The method of claim 25 , wherein the subject is also treated with a therapy selected from the group consisting of chemotherapy, CDK4/CDK6 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, and combinations thereof.
27 . The method of claim 25 , wherein the subject is treated with a targeted therapeutic agent that affects an ErbB signaling pathway.
28 . The method of claim 27 , wherein the subject had previously received and failed to respond to an estrogen-related targeted therapy and wherein treatment with a targeted therapeutic agent that affects an ErbB signaling pathway enhances function of an estrogen-related targeted therapy.