METHOD TO PREDICT WHETHER A TUMOR WILL REACT TO A CHEMOTHERAPEUTIC TREATMENT
Field of the Invention
The present invention relates to a method of personalized cancer therapy that employs a method of classifying a tumor to predict whether a patient will respond to a chemotherapeutic agent and a kit for use in the method.
Background to the Invention
The expression of various receptors has been correlated with the incidence of cancer. A particular example of this is the epidermal growth factor family of receptors, which is comprised of epidermal growth factor receptor (EGFR l)/ErbBl, ErbB2, ErbB3 and ErbB4 and is expressed and over-expressed in cancers of the lung, breast, colon and prostate, among others (Yarden and Sliwkowski, MoI Cell Biol 2, 127-37, 2001).
The over-expression of ErbBl in tumors has made it a popular target for the development of anti-cancer therapeutics, such as gefitinib ('Iressa'; ZD1839, AstraZeneca, Wilmington, DE). Gefitinib is a small molecule inhibitor of ErbBl that acts by reversibly competing with ATP for ErbBl 's tyrosine kinase domain (Wakeling et al., Cancer Res 62, 5749-54, 2002). In clinical application, 25 of 31 (81%) of tumors from patients experiencing partial responses or marked clinical improvement in response to gefitinib or erlotinib (OSI Pharmaceuticals and Roche) contained mutations in the tyrosine kinase domain (Lynch et al., N Engl J Med 350, 2129-39, 2004; Paez et al., Science 304, 1497-500, 2004; Pao et al., Proc Natl Acad Sci USA 101, 13306-11, 2004). Furthermore, no mutations were found in patients unresponsive to gefitinib (Lynch et al., supra; Paez et al., supra; Pao et al., supra). In spite of the clear correlation between ErbBl mutations and Iressa response, the link between ErbBl mutation and a mechanism of oncogenesis was hereunto unknown.
Administering an effective chemotherapeutic agent to cancer patients is critical. The determination of factors that influence, for example, response to a particular drug can be used to provide a patient with a personalised treatment regime. Such personalised treatment regimes offer the potential to maximize therapeutic benefit to the patient, whilst minimizing, for example, side effects that may be associated with alternative and less effective treatment regimes.
Therefore there is a need for methods that can predict a patients' response to a drug based on the results of a test diagnostic that indicates whether the patient is likely to respond to treatment with that drug.
Summary of the Invention
The present invention provides a simple assay to target patients who respond well to certain chemotherapeutic agents. The invention is based on the finding that the sustained presence of a cell surface receptor on a tumor can be correlated to increased responsiveness of that tumor to a chemotherapeutic agent. Accordingly, by examining for the sustained presence of a cell surface receptor on a patient's tumor it will be possible to determine if patients will have increased responsiveness to a chemotherapeutic agent of interest. The present invention thus permits the improved prognosis and quality of life of cancer patients by matching the treatments to individual patients and so making more effective use of the types of chemotherapeutic agents/drugs available.
In particular, the present invention permits the improved selection of a patient who is a candidate for treatment with an ErbBl receptor drug in order to predict an increased likelihood of response to the ErbBl receptor drug.
In one aspect, the invention includes a method of classifying a tumor. The method includes providing a tumor sample; contacting the sample with a cell surface receptor detecting agent; and determining the location of the detecting agent in the sample compared to a control, wherein a difference in location compared to the control is used to classify the tumor. The cell surface receptor can be ErbBl, insulin receptor (IR), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), colon carcinoma kinase (CCK), nerve growth factor receptor (NGFR), hepatocyte growth factor receptor (HGFR), ephrin receptor (EphR), tyrosine kinase receptor in endothial cells (TIE), receptor related to tyrosine kinase (RYK), discoidin domain receptor (DDR), rearranged during transfection (RET), receptor protein tyrosine kinase expressed in some epithelial cell types (ROS), leukocyte tyrosine kinase (LTK), receptor orphan (ROR), muscle-specific kinase (MUSK) or lemur (LMR). The tumor sample can be any appropriate tumor sample including a sample from a lung, breast, prostate, colon, ovary, pancreas, brain, oesophagus, endometrium, cervix, gastrointestinal tract or skin.
The detecting agent can be any agent that can be used to detect the cell surface receptor. In one example, the detecting agent is a labelled ligand of the receptor. The ligand can be an antibody, peptide or compound. The label can be a fluorescent label such as a green fluorescent protein, radioactive label, enzymatic label, and colorimetric label.
In another aspect, the invention includes a method for treating a tumor classified to have increased responsiveness to a cell surface receptor drug. The method includes providing a tumor sample from a test patient; contacting the sample with a detecting agent for a cell surface receptor; determining the location of the detecting agent in the sample compared to a control, wherein a difference in location compared to the control is used to classify the tumor as having an increased responsiveness to a cell surface receptor therapeutic agent; and administering to the patient having a tumor classified as having increased responsiveness a cell surface receptor drug.
In yet another aspect, the invention includes a method for predicting the likelihood that a patient who is a candidate for treatment with an epidermal growth factor receptor drug will respond to that treatment. The method includes providing a tumor sample whose activity is mediated alone, or in part, by ErbBl from a test patient; contacting the sample with an ErbBl receptor detecting agent; and comparing cell surface location of the detecting agent in the sample compared to a control, wherein a difference in location compared to the control is indicative that the patient has increased responsiveness to an ErbBl drug. In one example, an increased presence of the ErbBl detecting agent on the cell membrane compared to a control indicates that the test patient has increased responsiveness to an ErbBl drug such as gefitinib or an anti-ErbBl antibody such as trastuzumab or cetuximab.
In yet another aspect, the invention includes a kit for detecting if a patient has an enhanced ability to respond to an ErbBl inhibitor drug. The kit includes a detecting agent for an ErbBl located on the cell surface; and instructions for use.
In another aspect, the invention includes use of an ErbBl inhibitor in the manufacture of a medicament for the treatment of patients or patient populations having NSCLC identified according to the method described herein.
By "classifying" it is meant an ability to identify a subset of tumors that have enhanced ability to respond to a therapeutic agent.
Brief description of the drawings Fig. 1 depicts a histogram showing ligand-induced internalization in six cell lines A549, CaIu- 6, H292, NCI-H322, PC9 and SKBr3.
Detailed description of the invention
The present invention relates to a method of personalized cancer therapy that employs a method of classifying a tumor to predict a patients' responsiveness to a chemotherapeutic agent.
The invention is based on the finding that the sustained presence of a cell surface receptor on a tumor can be correlated to increased responsiveness of that tumor to a therapeutic agent. While not wishing to be bound by theory, it is believed that the sustained presence of a cell surface receptor is due to the receptors' reduced capacity to internalize. By using the method of the present invention, a subset of cancer patients can be identified that will have increased responsiveness to treatment with particular therapeutic agents.
The present invention is thus particularly suitable for use in predicting the response to therapeutic agents in cancers that mediate their activity alone, or in part, through a cell surface receptor such as ErbBl. Examples of cancers that are mediated alone, or in part, by ErbBl include advanced Non-small Cell Lung Cancer (NSCLC), for example adenocarcinoma. The method can be used to differenciate those patients that are responders and non-responders to an ErbB tyrosine kinase inhibitor. By Responders and non responders we mean objective tumor responses according to the Union International Contre Ie Cancer/World Health Organization (U ICC/WHO) criteria are categorised as follows: complete response (CR): no residual tumor in all evaluable lesions; partial response (PR): residual tumor with evidence of chemotherapy-induced 50% or greater decrease under baseline in the sum of all measurable lesions and no new lesions; stable disease (SD): residual tumor not qualified for CR; and progressive disease (PD): residual tumor with evidence of 25% or greater increase under baseline in the sum of all measurable lesions or appearance of new lesions. As defined herein non-responders are PD. The present invention is particularly effective for determining those patients that are CR or PR.
In a specific example, the present invention allows for the determination of a subset of lung cancer patients characterized by the sustained presence of the ErbBl located on the cell surface of a tumor. This subset of patients has been found to have a heightened response to an ErbB tyrosine kinase inhibitor, for example, gefϊtinib.
The present invention thus offers considerable advantages in the treatment of cancers by identifying individuals with cell surface receptors that exhibit a sustained presence of the receptor on the tumor surface and so determining which tumors would respond to a particular chemotherapeutic agent. Thus, the present method prevents patients from receiving ineffective or possibly "wrong" chemotherapeutic agents which can be detrimental to the physical and emotional well being of patients.
Diagnostic Assays
An exemplary method for detecting the presence of a cell surface receptor on a tumor sample involves obtaining a tumor sample from a test subject and contacting the tumor sample with an agent capable of detecting the cell surface receptor.
Tumor samples
Tumor samples can be taken from any patient where the tumor activity is mediated alone, or in part, through a cell surface receptor such as ErbBl . For example, the tumor can be a non- solid tumor such as leukaemia, multiple myeloma or lymphoma, or can be a solid tumor, for example bile duct, bone, bladder, brain/CNS, breast, colorectal, cervical, endometrial, gastric, head and neck, hepatic, lung, muscle, neuronal, oesophageal, ovarian, pancreatic, pleural/peritoneal rnembranes, prostate, renal, skin, testicular, thyroid, uterine and vulval tumors.
Appropriate tumor samples can be prepared as known in the art. For example, live tumor cells are obtained via a needle biopsy and then cultured in vitro according to standard procedures. Alternately, one could fix the tumor cells immediately following aspiration or remove the tumor (in whole or in part) and prepare a section for immunohistological staining. In vitro culturing of tumor cells will enable the measurement of internalization dynamics following stimulation, while immediately fixing samples will result in assaying the static localization of the receptor within the tumor.
Cell surface receptors
Cell surface receptors which are useful in the present invention are those that exhibit an increased time at the surface of the tumor compared to a control. The reason for this increased residency time can be, for example, that the receptor undergoes reduced ligand- induced internalization. Examples of receptors that are useful in the present invention include those receptors that have mutations in the receptor genes or proteins that result in an impaired ability to undergo ligand-induced internalization. Alternatively, an abnormality resulting in this effect would suffice; for example, mutations in the internalization apparatus or heightened interaction with a protein capable of restraining the receptor to the surface.
In one example, the ErbBl has been identified to have a number of mutations which have been shown to have reduced ligand-induced internalization. For example, ErbBl has been described to (i) have deletions in or around the kinase domain (Lynch et al., supra 2004; Paez et al., supra 2004); (ii) have single amino acid substitutions in or around the kinase domain (Lynch et al., supra 2004; Paez et al., supra 2004), and (iii) lack a portion of its extracellular domain (ErbBl vIII) (review: (Chen et al., Nature 328, 820-3, 1987)).
It is envisioned that the present method of classifying a tumor can be performed on any signalling cell surface receptor since internalization and down regulation is a characteristic of virtually all signalling receptors. An example of a cell surface receptor that would be useful in the present invention include receptor tyrosine kinases. Classes of these receptors are known (van der Geer et al., Annu Rev Cell Biol 10, 251-337, 1994; Wilks, Adv Cancer Res 60, 43-73, 1993) and include Class I receptor tyrosine kinases including the EGF family of receptor tyrosine kinases such as ErbBl, ErbB2, ErbB3 and ErbB4, Class II receptor tyrosine kinases including the insulin family of receptor tyrosine kinases such as the insulin and IGFI receptors and insulin-related receptor (IRR) and Class III receptor tyrosine kinases including the platelet-derived growth factor (PDGF) family of receptor tyrosine kinases such as the PDGFaa, PDGFbb and colony-stimulating factor 1 (CSFl) receptors.
A number of cell surface receptors as indicated in table 1 below have been identified to have a reduced ligand-induced internalization capacity as a consequence of mutations, deletions, truncations or rearrangements:
Table 1
Specific cases where changes in receptor trafficking are linked with disease.
Assay method
An exemplary method for detecting the sustained presence of a cell surface receptor on a rumor sample involves obtaining a tumor sample as described above from a test subject and contacting the tumor sample with an agent capable of detecting the cell surface receptor. An agent of interest includes any molecule such as a peptidomimetic, protein, peptide, nucleic acid, small molecule, an antibody or other drug candidate, that can bind the cell surface receptor. Agents that bind cell surface receptors are known in the art, for example, for ErbBl there arc numerous commercially available antibodies and ligands (including but not limited to EGF, TGFα, gefitnib, Amphiregulin, Heparin-binding EGF and Epiregulin)
To aid detection, agents can be labelled. Examples of labels include various enzymes, fluorescent materials, luminescent materials, and bioluminescent materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; an example of a luminescent material is luminol; and examples of bioluminescent materials include luciferase, luciferin, and aequorin.
The agents may be directly labelled with any readily detectable label such as a fluorescent, bioluminescent, phosphorescent, or radioactive label. In one example, the fluorescent label can be a green, blue, yellow or cyan fluorescent label. Alternatively, the agent can be indirectly labelled with a detectable substance, for example, indirectly labelled by reactivity with another reagent that is directly labelled. Examples of indirect labelling include detection
of a primary antibody using a fluorescently labelled secondary antibody or for example labelling with biotin such that it can be detected with fluorescently labelled streptavidin.
Labelling of an agent of interest can be carried out by chemical methods known in the art. A variety of coupling agents, including cross-linking agents, can be used for covalent conjugation. Examples of cross-linking agents include N,N'-dicyclohexylcarbodiimide (DCC; Pierce), N-succinimidyl-S-acetyl-thio- acetate (SATA), N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), ortho-phenylenedimaleimide (o-PDM), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (sulfo-SMCC). See, e.g., Karpovsky et al., J. Exp. Med. 160:1686, 1984; and Liu et al., Proc. Natl. Acad. Sci. USA 82:8648, 1985. Other methods include those described by Paulus, Behring Ins. Mitt., No. 78, 118-132, 1985; Brennan et al. Science 229:81-83, 1985, and Glennie et al., J. Immunol. 139:2367-2375, 1987. A large number of coupling agents, along with buffers, solvents, and methods of use, are described in the Pierce Chemical Co. catalog, pages T-155- T-200, 1994 (3747 N. Meridian Rd., Rockford 111., 61105, U.S.A.,; Pierce Europe B. V., P.O. Box 1512, 3260 BA Oud Beijerland, The Netherlands), which catalog is hereby incorporated by reference.
In one example, the agent is labelled with a green fluorescent protein (GFP). The GFP gene was originally cloned from the jellyfish Aequorea Victoria. It encodes a protein of 238 amino acids which absorbs blue light (major peak at 395 nm) and emits green light (major peak at 509 nm) (Prasher et al., Gene 15:229-223, 1992). GFP genes and functional proteins have been identified in a variety of organisms in the phyla hydrozoa, cnidaria, anthozoa and ctenophora. Both wild-type GFP and mutated GFP from Aequorea Victoria can be used as a label. The mutation of GFP (e.g., the substitution of certain amino acids in the GFP polypeptide) has been reported to yield GFP proteins with improved spectral properties. For example, mutating serine 65 to a threonine generates a GFP variant which has about sixfold greater brightness than wild-type GFP (Heim et al., Nature 372:663-664, 1995). The coding sequence for an enhanced GFP can be purchased commercially (Clontech, Palo Alto, Calif.)
Alternatively, a blue fluorescent protein (BFP) can also be used as a label. To obtain BFP, tyrosine 66 of GFP is mutated to a histidine. This mutated GFP protein fluoresces bright blue,
in contrast to the green of the wild-type protein.
The method of the invention can be used to determine if the labelled agent has a sustained presence on the surface of the tumor compared to a control. In one example, the sustained presence can be determined by determining the uptake of ligands and/or receptors. Uptake may be followed using labelled agents as described above. Uptake is either the disappearance from the cell surface and/or the appearance of labelled species within the cell.
The method of the invention can be applied to live samples of tumor tissue or can be applied to fixed samples.
The methods of the invention include comparing the location of the detecting agent in the sample to a control, wherein a difference in location compared to the control is used to classify the tumor. As noted above, the difference in location can be for example, determining if a labelled agent is present to a higher degree on the surface of the test tumor as compared to a control. Differences in location can be determined by, for example, visual inspection, for example, using microscopy to determine for a difference in binding of the labelled agent on the surface of the tumor compared to a control. In one example where the receptor is detected using a fluorescent label, control samples will typically show a punctuate staining indicative of internalization and a decrease in cell surface labelling, while tumor samples that are more sensitive to a therapeutic agent of interest will show continued presence of the labelled agent on the surface of the cell and a lack of any punctuate, internal staining. Early time points, for example, less than one hour, less than 45 minutes, less than 30 minutes, less than 15 minutes, etc, are the best indicators of this process, due to confounding effects of receptor recycling that may take place at longer times. Nonetheless, differences may still persist, and be measurable, at longer time points as well.
Alternately, differences in location can be determined using a radioactively-labelled detecting agent (Wiley and Cunningham, J Biol Chem 257, 4222-9, 1982). In this example, a 125I- labelled ligand (or 125I-labelled antibody + ligand) for the receptor of interest is incubated with the samples, for example, for around 10 minutes. The surface bound ligand is obtained by stripping the label from the surface with a mild acid strip solution (leaving the internalized ligand, in the cells, intact) and then quantitating via a gamma counter. The internalized ligand
is determined by solubilizing the cells and quantitating with a gamma counter. From this, one can calculate a ratio of internalized ligand to the total amount of bound ligand (surface + internal). Cells with a reduced capacity to undergo ligand-induced internalization will have a lower ratio than a control sample.
In another example, a difference in the kinetics of internalization of the tumor sample and the control can be determined. In this example, the rates of internalization of the detecting agent may be compared to either a control sample or a value or distribution of values from a representative 'normal' tissue sample. The kinetics of internalization can be determined by quantitating the change in the detecting agent on the surface and inside the cell. For example this is done with l25I-labelled ligands or antibodies that are added to cells at 37° for up to 10 min. At several time points, the amount of surface and internal receptors is quantified by standard procedures (Wiley and Cunningham, supra). The quantity of labelled internalized receptors can be plotted as a function of the integrated quantity of labelled surface receptors, yielding a linear relationship whose slope is the internalization rate constant (Wiley and Cunningham, supra). This rate constant characterizes the rate at which a receptor is internalized into the cell and can be compared to the range of values known for drug responsive and drug resistant samples.
The control sample can be a sample from a tumor that does not have a cell surface receptor having a reduced capacity for ligand-induced internalization, or the control can be a sample from a normal tissue sample. The control sample could consist of a negative control, that is, tissue tumor samples that are known to be resistant to the drug and a positive control, tissue tumor samples that are known to be sensitive to the drug.
Uses
Using the method described above, cancer patients having a tumor that will respond to a particular drug can be identified.
In particular, the present invention is particularly suitable for use in predicting the response to an ErbBl receptor drug in patients with a tumor that is dependent alone, or in part, on an EGF tyrosine kinase receptor. ErbBl receptor inhibitors are known in the art and include Iressa, erlotinib (OSI-774, CP-358774), PKI- 166, EKB-569, HKI-272 (WAY- 177820), lapatinib
(GW2016, GW-572016), canertinib (CI- 1033, PD183805), AEE788, XL647, BMS 5599626, GSK572016, AZD6474 or any of the compounds as disclosed in WO2004/006846 or WO03/082290. In an alternative embodiment the EGF receptor tyrosine kinase inhibitor is selected from an anti- ErbBl antibody such as cetuximab (C225), matuzumab (EMD-72000), panitumumab (ABX-EGF/ rHuMAb-EGFr), MRl - 1 , IMC- 11 F8 or EGFRL 11. The ErbB 1 tyrosine kinase inhibitors may be used as monotherapy or in combination with other drugs.
The present invention is particularly suitable for use in predicting the response in a lung cancer patient to gefitinib. In particular, the present invention offers considerable advantages in the treatment of tumors such as NSCLC, especially advanced NSCLC by determining which tumors would respond to gefitinib.
The present invention can be used to predict which patients should be administered particular therapeutic drugs. Methods of administration of such drugs are known in the art.
Kits
The invention also encompasses kits for detecting the presence of a cell surface receptor in a biological sample. For example, the kit can comprise a labelled agent capable of detecting a cell surface receptor in a tumor sample; a control sample; and instructions relating to how to detect the cell surface receptor.
The invention will be described in more detail and illustrated by the following examples that are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. Certain elements of the invention are also described in more detail below.
Examples
Example 1: To test the hypothesis that the ErbBl mutants may be defective in the ability to undergo ligand-induced internalization To test the hypothesis that the ErbBl mutants may be defective in the ability to undergo ligand-induced internalization we followed the internalization of EGF with 10 nM of fluorescently labelled EGF. This experiment was performed on H292 cells, which express wild- type (WT) ErbBl and PC-9 cells, which express a deletion mutant of ErbBl. Cells were
cultured in RPMI 1640 containing 10% FBS, antibiotics and L-glutamine (Invitrogen; cat# 21870-084). For ligand inductions cells were grown in 8 well chamber slides (Labtek; cat# 154941). Cells were plated overnight (O/N) in media containing serum. The media was then removed and cells washed 3 times with PBS. The cells were then incubated O/N in serum free media prior to addition of ligand.
ErbBl localization was assessed using an antibody raised against ErbBl (Biosource International, CA, USA; cat #44-796). Detection of antibody binding was carried out using a goat anti-mouse secondary antibody conjugated to Alexa-488 (Invitrogen/Molecular Probes; cat #A11001). Measurement of ligand induced ErbBl internalization was performed using immunostaining. For immunostaining, cells were washed in ice-cold PBS briefly and fixed in 0.5% formaldehyde for 5 min. Cells were then washed 3 times with PBS and incubated with primary antibody diluted 1/100 in 500 mg/ml of digitonin in PBS (Sigma-Aldrich; cat# R204382). After 1 hr, cells were washed with PBS and subsequently incubated for 1 hr with a goat anti-mouse secondary antibody conjugated to Alexa-488. Microscopic images were obtained using an inverted fluorescence microscope (Axiovert200M, Zeiss). Cells labelled with Alexa Fluor®-488 generated fluorescence were viewed by fluorescence microscopy using the appropriate filter sets to excite at 488 nm and measure emission at 530 nm.
For tracking EGF internalization Alexa Fluor®-488 EGF complex (Molecular Probes; cat #E-13345) was prepared as a 200 mg/mL stock solution in PBS, containing 1% BSA. Cells were incubated with serum free media containing 2 mg/ml of Alexa Fluor®-488 EGF complex. Cells were then washed with PBS and fixed with 0.5% formaldehyde. As a control, cells were incubated with 10 nM unlabeled EGF prior to addition of Alexa Fluor®-488 EGF. Binding of Alexa Fluor®-488 EGF was inhibited in these cases.
To test the hypothesis that the ErbBl mutants may be defective in the ability to undergo ligand-induced internalization we followed the internalization of EGF with 10 nM of fluorescently labelled EGF. To do this, ErbBl internalization was followed using an Alexa Fluor®-488 EGF complex. Cells were incubated with 2 μg/ml (10 nM) of the fluorescent EGF complex for 0, 5 or 10 min. Cells were then washed with PBS and fixed with 0.5% paraformaldehyde. Results are shown for H292 cells, which express WT ErbBl, and PC-9 cells, which express a deletion mutant of ErbBl. Following 10 minutes of incubation it is
clear that the WT ErbBl in the H292 cells has undergone significant internalization. By contrast, the mutant ErbBl in the PC-9 cells still remains on the surface, reflecting an impaired ability to undergo ligand-induced internalization.
As an additional confirmation that the mutant ErbBl have a reduced ability to internalize, we performed a similar experiment, following ErbBl rather than EGF. H292 or PC-9 cells were incubated with EGF for varying lengths of time and then stained for ErbBl. Briefly, H292 or PC-9 cells were incubated with either 10 nM EGF and stained with an anti-ErbBl antibody. Cells were washed, fixed, permeabilized and stained either immediately after addition of ligand (0 min) or following 10 min of incubation time, as indicated. As before, ErbBl undergoes significant internalization in the H292 cells as seen by the punctuate staining at 10 min. In PC-9 cells, ErbBl remains on the surface, showing little to no internalization. Similar results are obtained if one does the analogous experiment with TGFα, another ErbBl ligand.
Example 2: To test whether ligand-induced internalization is reduced where cells express a high level ofErbB2 relative to ErbBl
This example was to investigate whether ligand-induced internalization can be reduced where cells express a high level of ErbB2 relative to ErbB 1.
Six cell lines, A549, Calu-6, H292, NCI-H322, PC9 and SKBr3 cells, were assessed for their ability to internalize fluorescently labelled EGF ligand (labelled with Alexa488). The internalization experiment was performed as in Example 1. In the panel of six cell lines, three cell lines showed rapid internalization rates (A549, Calu-6, H292) and three cell lines showed delayed internalization kinetics (NCI-H322, PC9, and SKBr3). In the case of NCI-H322 and SKBr3 cell lines, both expressed high levels of ErbB2 and were relatively sensitive to Iressa (Fig. 1). Thus, re-inforcing that tumour cells that have delayed internalization kinetics (whether due to a mutation in EGFR or another mechanism) are likely to be sensitive to Iressa.
Example 3: Gefitinib and the treatment for non-small cell lung cancer.
This example is for the specific use of Gefitinib, or other small molecule tyrosine kinase inhibitors of the ErbB family, in the treatment for non-small cell lung cancer.
In order to determine if Gefinitib is likely to be effective in treating a non-small cell lung cancer patient a sample of tumor tissue is obtained via a needle biopsy. The sample is prepared for culture in vitro and grown in a minimal medium that is free of ErbB family ligands (particularly EGF and TGFα). Fluorescently labelled EGF (Alexa Fluor®-488 EGF, for example) is added to the cells at 37°C and incubated for 10 minutes. Following incubation with ligand, the cells are washed with PBS and fixed with formaldehyde. The internalization of Alexa Fluor®-488 EGF is assayed via microscopy and interpreted manually or via image analysis software. Internalized EGF appears as punctate spots within the cell, whereas surface bound EGF appears as a bright ring marking the cell border.