WO2015127234A1

WO2015127234A1 - Use of ibrutinib to treat egfr mutant cancer

Info

Publication number: WO2015127234A1
Application number: PCT/US2015/016859
Authority: WO
Inventors: Bingliang Fang; Jack A. Roth; Michael Wang; Li Wang; Shuhong Wu; John V. HEYMACH; Stephen G. Swisher; Wayne L. HOFSTETTER
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 2014-02-20
Filing date: 2015-02-20
Publication date: 2015-08-27

Abstract

Provided herein are methods of using irreversible BTK inhibitors, such as, for example, ibrutinib (PCI-32765), to treat EGFR mutant cancers, HER2 amplified cancers, and/or HER2 overexpressing cancers.

Description

DESCRIPTION

USE OF IBRUTINIB TO TREAT EGFR MUTANT CANCER

[0001] This application claims the benefit of United States Provisional Patent Application No. 61/942,255, filed February 20, 2014, incorporated herein by reference in its entirety.

[0002] The invention was made with government support under Grant No. R01 CA124951-01A2 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

[0003] The present invention relates generally to the field of oncology. More particularly, it concerns methods of treating EGFR inhibitor-resistant cancer patients with ibrutinib.

2. Description of Related Art

[0004] EGFR mutations are frequently detected in lung adenocarcinoma patients, especially those who have no smoking history (Pao et al, 2004; Ding et al, 2008). The high susceptibilities of EGFR mutant lung cancer cells to gefitinib and erlotinib (Pao et al, 2004; Lynch et al, 2004; Mukohara et al, 2005) have made these two agents the first choice for treatment of EGFR mutant cancers. Unfortunately, despite dramatic responses of EGFR mutant lung cancer patients to gefitinib or erlotinib, acquired resistance occurs at a median of 10-13 month after the treatment initiation (Zhou et al, 2011; Lee et al, 2013). While a variety of mechanisms have been identified for the acquired resistance, including a second T790M mutation at exon 20 of the EGFR gene (Pao et al, 2005; Kobayashi et al, 2005), amplification of MET gene (Bean et al, 2007; Cappuzzo et al., 2009; Engelman et al, 2007), mutations of KRas gene (Linardou et al, 2008) and activation of AXL or c-Src kinases (Byers et al, 2013; Zhang et al, 2012; Stabile et al, 2013), the most common cause of the resistance in clinics is the T790M mutation in the EGFR, which is found in about 50% of those patients (Pao et al, 2005; Bean et al, 2007; Kosaka et al, 2006). Effort has been made to develop EGFR kinase inhibitors that are effective for EGFR T790M mutants (Zhou et al, 2009; Kwak et al, 2005; Chang et al, 2012), including development and approval of afatinib for clinical application (Sequist et al, 2013 a) and clinical trials on some novel anti-EGFR agents (Sequist et al, 2013b). Nevertheless, the need remains for effective therapeutics for treatment of EGFR mutant cancers.

SUMMARY OF THE INVENTION

[0005] In some embodiments, there are provided methods of treating a patient having cancer comprising administering a therapeutically effective amount of an irreversible BTK inhibitor, such as any of those disclosed in U.S. Pat. No. 7,514,444, which is incorporated herein by reference in its entirety, to a patient determined to have a cancer that is resistant to erlotinib and/or gefitinib. In some aspects, the cancer comprises (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression. In one aspect, the irreversible BTK inhibitor is ibrutinib. In various aspects, the EGFR mutation comprises a T790M substitution, a L858R substitution, a deletion in exon 19, a G719x substitution, and/or a L861Q substitution.

[0006] In some aspects, the cancer is metastatic, recurrent, or multi-drug resistant. In some aspects, the cancer is colorectal, breast, prostate, lung, or pancreatic cancer. In one aspect, the cancer is non-small cell lung cancer.

[0007] In certain aspects, the method further comprises administering at least a second anticancer therapy to the subject. Such second anticancer therapy may be a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, or cytokine therapy. The chemotherapy may be an NF-KB/STAT-3 inhibitor (e.g., auranofin), an AXL inhibitor (e.g., SGI-7079, cabozantinib), an ALK/MET inhibitor (e.g., crizotinib), a RAF/VEGFR inhibitor (e.g., sorafenib), an AKT inhibitor (e.g., MK2206), a Src inhibitor (e.g., dasatinib), a JAK/STAT-3 inhibitor (e.g., ruxolitinib), or a WNT/β- catenin inhibitor (e.g., LGK974135). [0008] In some embodiments, there are provided methods of treating a patient having cancer comprising administering a therapeutically effective amount of ibrutinib in combination with an AXL inhibitor (e.g., SGI-7079, cabozantinib) to a patient determined to have a cancer that comprises an EGFR mutation (e.g., T790M, exon 19 deletion, L858R, G719x, and/or L861Q) and AXL protein overexpression and/or E-cadherin protein underexpression. Overexpression can be defined as an expression level in the cancer sample that is elevated relative to a control sample (e.g., a non-tumor sample obtaining from the patient or a sample obtained from a healthy patient). Underexpression can be defined as an expression level in the cancer sample that is decreased relative to a control sample.

[0009] In some aspects, the patient is a human. In some aspects, the patient is a non- human mammal. In some aspects, the patient is treated at least a second time. In some aspects, the patient is treated over a period of 1 week to 6 months. In some aspects, the patient has previously undergone at least one round of anti-cancer therapy.

[0010] In some embodiments, there are provided methods of treating a patient having cancer comprising: (a) selecting a patient determined to comprise a cancer comprising (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression; and (b) administering a therapeutically effective amount of an irreversible BTK inhibitor, such as any of those disclosed in U.S. Pat. No. 7,514,444, to the patient. In one aspect, the irreversible BTK inhibitor is ibrutinib.

[0011] In some aspects, selecting a patient comprises obtaining a sample of the cancer and determining whether the cancer comprises (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression. In one aspect, the method comprises providing a report of the determining. The report may be a written or electronic report. In some aspects, the report is provided to the patient, a health care payer, a physician, an insurance agent, or an electronic system.

[0012] In certain aspects, the EGFR mutation comprises a T790M substitution. In certain aspects, the amino acid present at position 790 of the EGFR protein is determined by mass spectrometry, western blot, ELISA, or sequencing a nucleic acid comprising at least a portion of the protein coding sequence of the EGFR protein. In certain aspects, the EGFR mutation further comprises a L858R EGFR mutation, a deletion in exon 19, a G719x substitution, and/or a L861Q substitution. In certain aspects, the amino acid present at position 858, 861, and/or 719 of the EGFR protein is determined by mass spectrometry, western blot, ELISA, or sequencing a nucleic acid comprising at least a portion of the protein coding sequence of the EGFR protein. In certain aspects, a deletion in exon 19 of EGFR is determined by mass spectrometry, western blot, ELISA, or sequencing a nucleic acid comprising at least a portion of the protein coding sequence of the EGFR protein. In some aspects, the EGFR mutation comprises a substitution at C797. [0013] In certain aspects, HER2 gene amplification is determined by FISH or quantitative PCR of genomic DNA. In certain aspects, HER2 protein overexpression is determined by western blot, ELISA, mass spectrometry. Overexpression can be defined as an expression level in the cancer sample that is elevated relative to a control sample (e.g., a non- tumor sample obtaining from the patient or a sample obtained from a healthy patient).

[0014] In some aspects, selecting a patient comprises obtaining results from a test that determines whether the cancer comprises (a) an EGFR mutation; (b) HER2 gene amplification; or (c) HER2 protein overexpression; or taking a patient history that reveals that results. [0015] In some aspects, the cancer is metastatic, recurrent, or multi-drug resistant. In some aspects, the cancer is colorectal, breast, prostate, lung, or pancreatic cancer. In some aspects, the cancer is non-small cell lung cancer.

[0016] In certain aspects, the method further comprises administering at least a second anticancer therapy to the subject. Such second anticancer therapy may be a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, or cytokine therapy. The chemotherapy may be an NF-KB/STAT-3 inhibitor (e.g., auranofin), an AXL inhibitor (e.g., SGI-7079, cabozantinib), an ALK/MET inhibitor (e.g., crizotinib), a RAF/VEGFR inhibitor (e.g., sorafenib), an AKT inhibitor (e.g., MK2206), a Src inhibitor (e.g., dasatinib), a JAK/STAT-3 inhibitor (e.g., ruxolitinib), or a W T/β- catenin inhibitor (e.g., LGK974135).

[0017] In some aspects, the patient is treated at least a second time. In other aspects, the patient is treated over a period of 1 week to 6 months. In certain aspects, the patient is a human. In other aspect, the patient is a non-human mammal.

[0018] In one embodiment, there are provided methods of selecting a drug therapy for a cancer patient comprising: (a) obtaining a sample of the cancer; (b) determining the presence of (i) a mutation in the EGFR protein expressed in the cancer; (ii) amplification of the HER2 gene in the cancer; or (iii) overexpression of the HER2 protein in the cancer; and (c) selecting an irreversible BTK inhibitor, such as any of those disclosed in U.S. Pat. No. 7,514,444, if (i) a mutation is determined to be present in the EGFR protein expressed in the cancer; (ii) the HER2 is determined to be amplified in the cancer; or (iii) the HER2 protein is determined to be overexpressed in the cancer. In one aspect, the irreversible BTK inhibitor is ibrutinib. In one aspect, the method comprises administering a therapeutically effective amount of ibrutinib to the patient. In some aspects, the mutation in the EGFR protein is a T790M and/or L858R substitution.

[0019] In one embodiment, there are provided compositions comprising ibrutinib for use in the treatment of a cancer in a subject, wherein the cancer has been determined to comprise (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression. In some aspects, the EGFR mutation comprises a T790M substitution, a L858R substitution, a deletion in exon 19 of EGFR, a G719x substitution, or a L861Q substitution. In some aspects, the cancer is colorectal, breast, prostate, lung (e.g. non-small cell lung cancer), or pancreatic cancer.

[0020] In certain aspects, the composition comprises at least a second anticancer therapy. Such second anticancer therapy may be a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, or cytokine therapy. The chemotherapy may be an NF-KB/STAT-3 inhibitor (e.g., auranofin), an AXL inhibitor (e.g., SGI-7079, cabozantinib), an ALK/MET inhibitor (e.g., crizotinib), a RAF/VEGFR inhibitor (e.g., sorafenib), an AKT inhibitor (e.g., MK2206), a Src inhibitor (e.g., dasatinib), a JAK/STAT-3 inhibitor (e.g., ruxolitinib), or a W T/p-catenin inhibitor (e.g., LGK974135).

[0021] In some aspects, there is provided the use of ibrutinib in the manufacture of a medicament for the treatment of a cancer, wherein the cancer has been determined to comprise (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression.

[0022] In variations on any of the above embodiments, ibrutinib is administered systemically. In some embodiments, ibrutinib and a second agent are administered by distinct routes. In some embodiments, ibrutinib or the second agent are administered orally, intraarterially or intravenously. In some embodiments, ibrutinib is administered after the second agent. In some embodiments, ibrutinib is administered before and after the second agent. In some embodiments, ibrutinib is administered concurrently with the second agent. In some embodiments, ibrutinib is administered at least a second time. In some embodiments, the second agent is administered at least a second time. [0023] As used herein, "essentially free," in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0024] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0025] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.

[0026] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0027] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0029] FIGS. 1A-D. Antitumor activities of ibrutinib in non-small cell lung cancer ( SCLC) cell lines and EGFR T790M mutant tumors. FIG. 1A. Calculated 50% inhibitory concentration [IC5₀] on a logarithmic scale for 39 NSCLC cell lines by dose-response cell viability assay after ibrutinib treatment for 72 h. FIG. IB. Dose-response curves of erlotinib, afatinib and ibrutinib for the HI 975 cell line, which has a T790M mutation. The data are means with standard deviations for two assays performed in quadruplicate. The viability of control cells treated with dimethyl sulfoxide was assigned a value of 100. FIG. 1C. In vivo growth of HI 795 tumors. The mice were treated as indicated. The values are means ± SD of data from 5 mice per group. * indicates P < 0.05 when compared with the control group, using a two-sided Student's t-test. FIG. ID. Kaplan Meier Survival Curve of the animals shown in FIG. 1C.

[0030] FIGS. 2A-D. Western blot analysis of epidermal growth factor receptor (EGFR) phosphorylation (p-EGFR) and cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3 (Casp-3). FIG. 2A. H1975 and H3255 cells were treated with erlotinib and ibrutinib with the dose as indicated. Phospho-Y1068 and total EGFR were determined at 24 h after treatment. FIG. 2B. HCC827, H292 and A549 cells were treated with erlotinib or ibrutinib at the doses as indicated. Cell lysates were harvested for protein phosphorylation analyses at 24 h. Note, for A549 cells, 10 ng/ml EGF was added to the cells to activate EGFR 15 min before harvesting cells. FIG. 2C. HCC827 cells were treated with 0.5 μΜ erlotinib or 0.5 μΜ ibrutinib and tested for EGFR phosphorylation at different time points as indicated. FIG. 2D. HCC827 cells were treated with ibrutinib and tested for EGFR phosphorylation and caspase-3 and PARP 1 cleavage at 48 h. [0031] FIG. 3. Bruton tyrosine kinase (BTK) expression in lung cancer cell lines.

The expression of BTK in lung cancer cell lines was tested by Western blot analysis. Mantle cell lymphoma cell line Jeko-1 was used as a positive control, β-actin was used as a loading control. No BTK expression was detected in the lung cancer cell lines susceptible to ibrutinib.

[0032] FIGS. 4A-C. Activity in acquired erlotinib-resistant cell lines with EGFR mutations. The responses of parental PC9, HCC827, and HCC4006 cell lines are shown in FIGS. 8A-F. FIG. 4A. Allele frequencies of T790M mutations in PC9 and PC9ER determined by allele-specific PCR. The values represent the mean + SD of three assays. FIG. 4B. Dose response of erlotinib and ibrutinib in PC9ER cells. The data are means with standard deviations for two assays, performed in quadruplicate. FIG. 4C. IC5₀ values of erlotinib, afatinib, ibrutinib, and CO-1686 in HCC4006ER and HCC827ER. Both were negative for T790M mutations. [0033] FIGS. 5A-C. Molecular biomarkers associated with responses to the anti- EGFR agents erlotinib, afatinib, and ibrutinib. FIG. 5A. Gene mutations associated with response. Colors represent fold change in IC5₀ values. FIGS. 5B-C. Protein (B) and mRNA (C) levels associated with response and identified at FDR of 20% and 5%, respectively. Colors represent correlation coefficients. Green, sensitive; red, resistant.

[0034] FIGS. 6A-C. Combination effects of auranofin and ibrutinib on HI 975 and H1650 cell lines. FIGS. 6A-B. Dose response to ibrutinib in the absence and presence of 0.25 μΜ auranofin. The data are the means with standard deviations for two assays, performed in quadruplicate. FIG. 6C. Ibrutinib's IC5₀ values in the presence or absence of auranofin.

[0035] FIGS. 7A-B. FIG. 7A. AXL expression is significantly higher in mesenchymal NSCLC cell lines. FIG. 7B. Mesenchymal cell lines are relatively more sensitive to the AXL inhibitor, SGI-7079, whereas epithelial cell lines are relatively more sensitive to erlotinib.

[0036] FIGS. 8A-F. HCC827 and HCC4006 NSCLC cells are sensitive to erlotinib, whereas EGFR TKI resistant variants (ER 1-6) are resistant (FIGS. 8A&D). The majority of resistant variants have acquired a mesenchymal phenotype as indicated by loss of E-cadherin and gain of AXL expression (FIGS. 8B&E). Parental HCC827 and HCC4006 cells are sensitive to ibrutinib, while EGFR TKI resistant variants are not (FIGS. 8C&F).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0037] Current methods for the treatment of EGFR mutant cancer use reversible EGFR inhibitors (e.g., erlotinib and gefitinib). A second mutation of EGER in T790M often causes resistance to erlotinib and gefitinib. Ibrutinib (PCI-32765) is an irreversible inhibitor for Bruton tyrosine kinase (BTK) that has been evaluated in humans for treatment of lymphoma and myeloma. Ibrutinib remains effective for cancer cells with the T790M mutation that are resistant to erlotinib.

[0038] Provided herein are methods of using irreversible BTK inhibitors, such as, for example, ibrutinib (PCI-32765) to treat EGFR mutant cancers. By testing a panel of non- small cell lung cancer cell (NSCLC) lines, ibrutinib was found to selectively inhibit the growth of EGFR mutant NSCLC cells, including cancer cells that harbor a T790M mutation and are resistant to conventional erlotinib. Thus, ibrutinib is a candidate drug for treatment of EGFR mutant cancers, including erlotinib- or gefitinib-resistant tumors.

I. Ibrutinib

[0039] Ibrutinib (PCI-32765) selectively and irreversibly inhibits Bruton tyrosine kinase (BTK) (Honigberg et al, 2010; Pan et al., 2007), which is specifically required for the B-cell antigen receptor signaling pathway (Davis et al, 2010). Previous studies revealed that ibrutinib specifically inhibited the proliferation of B-cell lymphoma with active B-cell antigen receptor signaling (Davis et al, 2010) and multiple myeloma cells expressing BTK (Tai et al, 2012). Oral administration of ibrutinib led to promising in vivo activity against spontaneous B-cell non-Hodgkin lymphoma in dogs and experimental rheumatoid arthritis in mice (Honigberg et al, 2010; Pan et al, 2007). Ibrutinib also inhibited growth of chronic lymphocytic leukemia and multiple myeloma cells inoculated into immune defective mice (Tai et al, 2012; Ponader et al, 2012). Ibrutinib was approved in November 2013 by the FDA for treatment of mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) due to its remarkable single-agent efficacy in relapsed or refractory disease (objective response rate of 60%-70% and a complete response of 16%-20%) (Advani et al., 2013; Byrd et al, 2013; Wang et al., 2013). Moreover, clinical trials of ibrutinib in lymphoma or CLL patients revealed that it has an excellent safety profile at wide dose ranges (420-840 mg/day). Side effects appear less common and less severe in patients treated with ibrutinib compared to patients treated with other EGFR inhibitors (Sequist et al, 2013a; Eilers et al, 2010; Lee et al, 2012; Petrelli et al, 2012; Advani et al., 2013; Brown et al, 2013; Byrd et al., 2013; Wang et al, 2013). Long-term therapy with ibrutinib was associated with modest toxicity, and most adverse events were grade 1 or 2 (Byrd et al, 2013; Wang et al, 2013; O'Brien et al, 2014). Ibrutinib is in clinical trials for treatment of lymphoma and myeloma with increased BTK activity or expression.

[0040] Most EGFR-mutant NSCLC cells, including the T790M mutant cell line HI 975, are highly susceptible to ibrutinib, with 50% growth inhibition at <0.2 μΜ, which is well within clinically achievable concentrations used for the treatment of lymphoma (Byrd et al, 2013; Advani et al, 2013). Moreover, ibrutinib was recently reported to be well tolerated when used in combination with other targeted therapeutics (Burger et al, 2014; Younes et al, 2014). Inhibitors targeting bypass signaling pathways, including the NF-KB/STAT-3 inhibitor auranofin (Bernhard, 1982; Chaffman et al, 1984; Larsen et al, 1984; van Riel et al, 1984), as well as the AXL inhibitor SGI-7079 that targets the EMT pathway (Byers et al, 2013), can dramatically sensitize lung cancer cells to EGFR inhibitors, including ibrutinib. Auranofin has been used to treat rheumatoid arthritis (Bernhard, 1982; Chaffman et al, 1984; Larsen et al, 1984; van Riel et al, 1984) for more than 30 years and the AXL inhibitor cabozantinib (Smith et al, 2013; Bowles et al, 2011) has been approved for treatment of thyroid cancer (Viola et al, 2013). Thus, repurposing ibrutinib for NSCLC therapy and enhancing its efficacy through combination with other approved drugs has the potential to rapidly and significantly impact the clinical treatment of EGFR-mutant NSCLC patients.

[0041] In a previous study of ibrutinib' s activity against a panel of kinases, ibrutinib inhibited wild-type EGFR with an IC5₀ value of 5.6 nM (Honigberg et al, 2010). However, its effects on mutant EGFR and in EGFR-mutant lung cancer cells, which do not express BTK, the known target of ibrutinib, have not been previously reported. The present finding that ibrutinib selectively inhibits EGFR-mutant NSCLC cells, including those with T790M mutant EGFR, by directly inhibiting EGFR phosphorylation (Gao et al, 2014) is highly innovative (Haura and Rix, 2014).

[0042] However, ibrutinib 's activity relationship with mutant EGFR is unknown. There are multiple locations in the EGFR gene where driver mutations can occur, and these correlate with differential sensitivity to EGFR inhibition. The most common mutations, so- called "typical" mutations, are deletions in exon 19 and L858R point mutations in exon 21. Together these account for -85% of the EGFR mutations seen in lung cancer, and they correlate with high response rates to erlotinib, gefitnib, and afatinib (Fukuoka et al, 201 1 ; Sequist et al, 2013; Rosell et al, 2012). There are other less common mutations that also seem sensitive to TKIs, such as G719x and L861Q (Lynch et al, 2004). Some atypical mutations correlate with resistance to EGFR inhibitors, such as exon 20 insertions (Arcila et al, 2013; Wu et al., 2008). Clinical trials suggest that patients with typical mutations have better outcomes than those with atypical mutations (Sequist et al, 2013). Moreover, it is not clear whether mutations in the covalent binding site (C797) for the second and third generation of anti-EGFR therapeutics (Ward et al, 2013; Cross et al, 2014; Tjin et al, 2014) will affect the activity of ibrutinib or other novel EGFRi. The activity of ibrutinib against wild-type and mutated forms of HER-2, another EGFR family member, will also be determined. The molecular insights into the actions of ibrutinib-mediated EGFR inhibition and the possible roles of atypical, including C797 mutations, will facilitate the design of combinational strategies to overcome resistance to EGFRi therapy.

[0043] To this end, a clinical trial will be performed testing ibrutinib in EGFR-mutant NSCLC patients, which will allow for investigation of possible novel, clinically relevant mechanisms associated with responses to ibrutinib. Novel therapeutic combinations targeting multiple mechanisms of EGFRi resistance, including inhibition of bypass signaling pathways (NF-KB and STAT-3) with the FDA-approved drug auranofin and the AXL inhibitor cabozantinib (Smith et al, 2013; Bowles et al, 2011) will also be tested. Thus, the direct effect of ibrutinib on EGFR and HER-2 and clinically relevant mutations in preclinical models will be determined. In addition, the T790M independent mechanisms of resistance to anti-EGFR therapies (i.e., bypass signaling pathways and EMT) will be characterized, while developing effective combination therapies to target them.

II. EGFR Tyrosine Kinase Inhibitor Resistance

[0044] Epidermal growth factor receptor (EGFR) is a known oncogenic driver in lung tumorigenesis and has been extensively investigated as a therapeutic target in lung cancer. Activating EGFR mutations are detected in about 10%- 17% of lung adenocarcinoma patients in the United States and Europe (Rosell et al, 2009; Pao et al, 2004; Ding et al, 2008; Marchetti et al, 2005; Gahr et al, 2013) and in about 30%-65% of lung cancer patients in Asia (Choi et al, 2013; Li et al, 2011; Gao et al, 2010; Tanaka et al, 2010). These mutations are more commonly seen in women and non-smokers (Rosell et al., 2009; Pao et al, 2004; Ding et al, 2008; Marchetti et al, 2005; Gahr et al, 2013). The inducible expression of human lung cancer-related mutant EGFR genes in transgenic mice causes the development of lung adenocarcinoma, whereas stopping the inducible expression of mutant EGFR leads to tumor regression (Ji et al, 2006; Politi et al, 2006), demonstrating that activating EGFR is required and sufficient for lung cancer tumorigenesis and malignancy maintenance. The finding that EGFR-mutant lung cancer cells are highly susceptible to the reversible EGFR inhibitors gefitinib (Pao et al, 2004; Lynch et al, 2004; Paez et al, 2004) and erlotinib (Pao et al, 2004; Shepherd et al, 2005) has made these two agents the first choice for therapy in patients whose tumors harbor EGFR mutations. Both gefitinib and erlotinib have been reported to significantly improve progression-free survival in patients with EGFR-mutant lung cancer (Cappuzzo et al, 2010; Fukuoka et al, 2011; Thatcher et al., 2005). [0045] Unfortunately, despite initial dramatic responses to gefitinib and erlotinib in patients with EGFR-mutant lung cancer, most patients invariably develop acquired resistance to these agents within 10-13 months after treatment initiation (Zhou et al, 201 1 ; Maemondo et al., 2010). Among the various mechanisms of resistance to anti-EGFR therapy, one of the most commonly observed in the clinic is the T790M mutation in exon 20 of the EGFR gene (Pao et al, 2005; Kobayashi et al, 2005; Yun et al, 2008; Engelman et al, 2006), which is found in about 50% of erlotinib- or gefitinib-resistant tumors (Bean et al, 2007; Pao et al, 2005; Kosaka et al, 2006). T790M-independent mechanisms of resistance include "bypass" signaling via activation of alternative pathways. These tumors often upregulate alternative receptors (e.g., HER2 [Takezawa et al., 2012], MET [Bean et al, 2007; Cappuzzo et al, 2009; Engelman et al, 2007]) and downstream pathways (e.g., IL-6R/STAT-3 [Kim et al, 2012; Sen et al, 2012], NF-κΒ [Bivona et al, 2011], PI3K/AKT via PTEN loss [Sos et al, 2009]) to sustain survival signals. Another major mechanism of resistance is upregulation of epithelial-to-mesenchymal transition (EMT) (Byers et al, 2013; Zhang et al, 2012; Giles et al, 2013; Meyer et al, 2013; Cufi et al, 2013; Shien et al., 2013), which is significantly associated with resistance to EGFR inhibitors in non-small cell lung cancer (NSCLC) cell lines and clinical specimens of patients treated in the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) study (Kim et al, 2011). EGFRi resistant NSCLC tumors also have enhanced expression of classical EMT markers and the EMT-associated receptor tyrosine kinase AXL is a potential therapeutic target for overcoming anti-EGFR therapy associated with this mesenchymal phenotype (Byers et al, 2013). The resistance to EGFR inhibitors caused by activation of AXL and EMT has also been reported by others (Zhang et al, 2012; Giles et al, 2013; Meyer et al, 2013; Cufi et al, 2013; Shien et al., 2013), suggesting that it is likely a third major subgroup of resistance in T790M negative tumors.

[0046] In an effort to target the largest subset of resistance mechanisms, substantial efforts have been made to develop EGFR kinase inhibitors that are effective against T790M mutations in EGFR (Zhou et al, 2009; Kwak et al., 2005; Chang et al, 2012; Lee et al, 2013). Several second-generation EGFR inhibitors, most of which are irreversible and inhibit two or more receptors in the EGFR family, have been evaluated in clinical trials (Miller et al, 2012; Ramalingam et al, 2012; Hirsch et al, 2013; Yu and Riely, 2013). Among these second-generation EGFR inhibitors, only afatinib, an irreversible dual EGFR/HER2 inhibitor that exhibits some activity against T790M mutant tumors preclinically (Li et al, 2008), has been approved by the U.S. Food and Drug Administration (FDA) (July 2013) for the treatment of metastatic NSCLC with EGFR mutation. However, because of the toxicity profile of afatinib with significant acneiform rash and diarrhea, as well as the lack of improvement in overall survival in patients with previously treated disease (Langer, 2013; Sequist et al, 2013), afatinib is only approved for frontline treatment of EGFR mutant NSCLC, not for treatment of previously treated tumors. A phase lb trial for combination therapy with cisplatin/paclitaxel or cisplatin/5-fluorouracil revealed that the maximum tolerated dose for afatinib was reduced to 20-30 mg/day (Vermorken et al, 2013), suggesting that combining afatinib with other therapeutic agents may not be feasible. Moreover, T790M- negative cancer cells remain resistant to third-generation EGFR inhibitors. Therefore, developing new therapeutic strategies or agents to overcome resistance to conventional EGFR inhibitors is urgently required.

[0047] Ibrutinib, an irreversible BTK inhibitor recently approved by the FDA for the treatment of mantle cell lymphoma and chronic lymphocytic leukemia, can function as an EGFRi and selectively inhibit growth and induce apoptosis in EGFR-mutant NSCLC cells in vitro and in vivo, including erlotinib-resistant cells that harbor a T790M mutation (Gao et al, 2014). A phase I/II clinical trial will test ibrutinib in EGFR-mutant NSCLC. Moreover, ibrutinib inhibits HER2 signaling in NSCLC cells, indicating that it may have clinical application in resistant tumors with HER2 overexpression. Targeting other mechanisms of resistance, including bypass mechanisms and EMT pathways, can dramatically sensitize lung cancer cells to EGFRi, including ibrutinib. For example, inhibitors of STAT-3, NF-κΒ, and AXL can dramatically sensitize NSCLC cells to EGFRi. Auranofin (Bernhard, 1982; Chaffman et al., 1984; Larsen et al., 1984; van Riel et al, 1984), an inhibitor of thioredoxin reductase (TXNRD) (Lima and Rodriguez, 2011 ; Madeira et al, 2012; Schuh et al., 2012), STAT-3 (Kim et al, 2007; Nakaya et al, 201 1), and NF-κΒ (Nakaya et al, 201 1), has been used to treat rheumatoid arthritis (Bernhard, 1982; Chaffman et al, 1984; Larsen et al, 1984; van Riel et al, 1984) since the 1980s. AXL inhibitor cabozantinib is also approved for thyroid cancer. Combination therapy using these agents may provide an opportunity to overcome resistance by drug repurposing and targeting multiple mechanisms of EGFRi resistance. III. Detection of Mutations, Amplification, and Expression

[0048] Certain embodiments concern detecting, either in vivo or in a sample, a mutation in the EGFR gene. Other embodiments concern detecting amplification of the HER2 gene. Other embodiments concern detecting a change in expression level of HER2, E- cadherin, and/or AXL gene products (e.g., mR A or protein).

A. Nucleic Acid Detection

[0049] In some embodiments, assessing the presence of a mutation, can involve detecting or quantifying a coding nucleic acid, such as an R A or DNA encoding a gene product (e.g., EGFR). For example, in some aspects all or a portion of cancer cell genome is sequenced to detect the presence of a mutation (e.g., a substitution, deletion, inversion or insertion). In some aspects, polymerase chain reaction (PCR) may be used to amplify all or part of an EGFR sequence.

[0050] In further embodiments, a mutation in a gene coding sequence can be detected by determining the sequence of all or part of a coding RNA. For instance, reverse transcription PCR can be employed to determine the sequence of an EGFR coding sequence. In still further aspects quantitative PCR or RT PCR can be employed to determine whether cells comprise a reduced amount of an E-cadherin coding sequence or an increased amount of an AXL or a HER2 coding sequence.

[0051] In some embodiments, PCR products described above may be subjected to sequence analysis to identify specific kinds of variations using standard sequence analysis techniques. Within certain methods, exhaustive analysis of genes is carried out by sequence analysis using primer sets designed for optimal sequencing. The present embodiments provide methods by which any or all of these types of analyses may be used. Using the known sequence of the human genome (or cDNAs derived therefrom), oligonucleotide primers may be designed to permit the amplification of sequences throughout the EGFR gene and surrounding sequence. Likewise, in some cases, DNA sequencing may be used to detect and/or quantify EGFR, AXL, HER2 and/or E-cadherin coding nucleic acids. Methods for such sequence include, but are not limited to, reversible terminator methods (e.g., used by Illumina® and Helicos® Biosciences), pyrosequencing (e.g., 454 sequencing from Roche) and sequencing by ligation (e.g., Life Technologies™ SOLiD™ sequencing). [0052] In PCR , the number of molecules of the amplified target DNA increase by a factor approaching two with every cycle of the reaction until some reagent becomes limiting. Thereafter, the rate of amplification becomes increasingly diminished until there is no increase in the amplified target between cycles. If a graph is plotted in which the cycle number is on the X axis and the log of the concentration of the amplified target DNA is on the Y axis, a curved line of characteristic shape is formed by connecting the plotted points. Beginning with the first cycle, the slope of the line is positive and constant. This is said to be the linear portion of the curve. After a reagent becomes limiting, the slope of the line begins to decrease and eventually becomes zero. At this point the concentration of the amplified target DNA becomes asymptotic to some fixed value. This is said to be the plateau portion of the curve.

[0053] The concentration of the target DNA in the linear portion of the PCR amplification is directly proportional to the starting concentration of the target before the reaction began. By determining the concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. Thus, these methods can be employed to detect EGFR sequences that are deleted in a cell, to detect a reduced overall expression of E-cadherin coding sequence, or to detect an increased overall expression of AXL or HER2 coding sequences. [0054] In some embodiments, nucleic acids are detected or quantified following gel separation and staining with ethidium bromide and visualization under UV light. In some embodiments, if the nucleic acid results from a synthesis or amplification (e.g., by PCR) using integral radio- or fluorometrically-labeled nucleotides, the products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.

[0055] In some embodiments, visualization is achieved indirectly. Following separation of nucleic acids, a labeled nucleic acid is brought into contact with the target sequence. The probe is conjugated to a chromophore or a radiolabel. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety. One example of the foregoing is described in U.S. Pat. No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present embodiments.

[0056] Mutations in a gene can further be assessed by hybridization techniques. Northern and Southern blotting techniques are, for instance, well known to those of skill in the art. Northern and Southern blotting involves the use of RNA or DNA, respectively, as a target. Briefly, a probe is used to target an RNA or DNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter. Subsequently, the blotted target is incubated with a probe (such as a labeled probe) under conditions that promote denaturation and rehybridization. Because the probe is designed to base pair with the target, the probe will binding a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished. Similarly, a labeled probe can be used for in situ hybidization to detect the presence of heterozygous mutations EGFR or the presence of increased copies of the HER2 gene. For example, fluorescence in situ hybridization (FISH) may be employed.

B. Protein Detection

[0057] In some aspects, methods of the embodiments concern detection of the expression or activity of HER2, AXL, or E-cadherin proteins. For example, immunodetection methods for binding, purifying, removing, quantifying and/or otherwise generally detecting HER2, AXL, or E-cadherin proteins can be employed. Antibodies may be employed to detect and/or quantify HER2, AXL, or E-cadherin in a subject or sample, e.g., a tumor biopsy. Some immunodetection methods include, for example, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, mass spectrometry, and Western blot.

1. ELISAs

[0058] As detailed above, immunoassays, in their most simple and/or direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. [0059] In some embodiments, antigen-specific antibodies are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the protein antigen, such as a clinical sample, is added to the wells. After binding and/or washing to remove non-specifically bound immune complexes, the bound protein antigen may be detected. Detection is generally achieved by the addition of another antigen-specific antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection and quantification may also be achieved by the addition of a second antigen-specific antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[0060] In some embodiments, the samples suspected of containing the protein antigen are immobilized onto the well surface and/or then contacted with the antigen-specific antibodies of the embodiments. After binding and/or washing to remove non-specifically bound immune complexes, the bound antibodies are detected and quantified. Where the initial antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.

[0061] In some embodiments, the antigen proteins, polypeptides and/or peptides are immobilized. In some embodiments, ELISA involves the use of antibody competition in the detection. In this ELISA, labeled antibodies against an antigen are added to the wells, allowed to bind, and/or detected by means of their label. The amount of antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against the antigen before and/or during incubation with coated wells. The presence of an antigen in the sample acts to reduce the amount of antibody against wild type or mutant protein available for binding to the well and thus reduces the ultimate signal. This is also appropriate for detecting antibodies against an antigen in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies. [0062] Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are steps are well known to a skilled artisan.

2. Immunohistochemistry

[0063] Antigen-specific antibodies may also be used in conjunction with both fresh- frozen and/or formalin- fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and/or is well known to those of skill in the art (Brown et ah, 1990; Abbondanzo et ah, 1990; Allred ei a/., 1990). [0064] Briefly, frozen-sections (e.g., vascular tissue sections) may be prepared by rehydrating 50 ng of frozen "pulverized" tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in 70°C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections.

[0065] Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections.

3. Mass Spectrometry

[0066] By exploiting the intrinsic properties of mass and charge, mass spectrometry (MS) can resolved and confidently identified a wide variety of complex compounds, including proteins. Traditional quantitative MS has used electrospray ionization (ESI) followed by tandem MS (MS/MS) while newer quantitative methods are being developed using matrix assisted laser desorption/ionization (MALDI) followed by time of flight (TOF) MS. One can generate mass spectrometry profiles without regard for the identity of specific proteins. Alternatively, mass spectrometry may be used to look for the levels of proteins particularly. [0067] ESI is a convenient ionization technique that is used to produce gaseous ions from highly polar, mostly nonvolatile biomolecules, including lipids. The sample is injected as a liquid at low flow rates (1-10 μΙ7ηιίη) through a capillary tube to which a strong electric field is applied. The field generates additional charges to the liquid at the end of the capillary and produces a fine spray of highly charged droplets that are electrostatically attracted to the mass spectrometer inlet. The evaporation of the solvent from the surface of a droplet as it travels through the desolvation chamber increases its charge density substantially. When this increase exceeds the Rayleigh stability limit, ions are ejected and ready for MS analysis.

[0068] A typical conventional ESI source consists of a metal capillary of typically 0.1-0.3 mm in diameter, with a tip held approximately 0.5 to 5 cm (but more usually 1 to 3 cm) away from an electrically grounded circular interface having at its center the sampling orifice. A potential difference of between 1 to 5 kV (but more typically 2 to 3 kV) is applied to the capillary by power supply to generate a high electrostatic field (10⁶ to 10⁷ V/m) at the capillary tip. A sample liquid carrying the analyte to be analyzed by the mass spectrometer is delivered to tip through an internal passage from a suitable source (such as from a chromatograph or directly from a sample solution via a liquid flow controller). By applying pressure to the sample in the capillary, the liquid leaves the capillary tip as small, highly electrically-charged droplets, and further undergoes desolvation and breakdown to form single or multicharged gas phase ions in the form of an ion beam. The ions are then collected by the grounded (or negatively charged) interface plate and led through an orifice into an analyzer of the mass spectrometer. During this operation, the voltage applied to the capillary is held constant. Aspects of construction of ESI sources are described, for example, in U.S. Patents 5,838,002; 5,788, 166; 5,757,994; RE 35,413; and 5,986,258.

[0069] In ESI tandem mass spectroscopy (ESI/MS/MS), one is able to simultaneously analyze both precursor ions and product ions, thereby monitoring a single precursor product reaction and producing (through selective reaction monitoring (SRM)) a signal only when the desired precursor ion is present. When the internal standard is a stable isotope-labeled version of the analyte, this is known as quantification by the stable isotope dilution method. This approach has been used to accurately measure pharmaceuticals and bioactive. Newer methods are performed on widely available MALDI-TOF instruments, which can resolve a wider mass range and have been used to quantify metabolites, peptides, and proteins. Larger molecules such as peptides can be quantified using unlabeled homologous peptides as long as their chemistry is similar to the analyte peptide. Protein quantification has been achieved by quantifying tryptic peptides. Complex mixtures such as crude extracts can be analyzed, but in some instances sample clean up is required.

[0070] Secondary ion mass spectroscopy, or SIMS, is an analytical method that uses ionized particles emitted from a surface for mass spectroscopy at a sensitivity of detection of a few parts per billion. The sample surface is bombarded by primary energetic particles, such as electrons, ions (e.g., O, Cs), neutrals or even photons, forcing atomic and molecular particles to be ejected from the surface, a process called sputtering. Since some of these sputtered particles carry a charge, a mass spectrometer can be used to measure their mass and charge. Continued sputtering permits measuring of the exposed elements as material is removed. This in turn permits one to construct elemental depth profiles. Although the majority of secondary ionized particles are electrons, it is the secondary ions which are detected and analysis by the mass spectrometer in this method.

[0071] Laser desorption mass spectroscopy (LD-MS) involves the use of a pulsed laser, which induces desorption of sample material from a sample site - effectively, this means vaporization of sample off of the sample substrate. This method is usually only used in conjunction with a mass spectrometer, and can be performed simultaneously with ionization if one uses the right laser radiation wavelength.

[0072] When coupled with Time-of-Flight (TOF) measurement, LD-MS is referred to as LDLPMS (Laser Desorption Laser Photoionization Mass Spectroscopy). The LDLPMS method of analysis gives instantaneous volatilization of the sample, and this form of sample fragmentation permits rapid analysis without any wet extraction chemistry. The LDLPMS instrumentation provides a profile of the species present while the retention time is low and the sample size is small. In LDLPMS, an impactor strip is loaded into a vacuum chamber. The pulsed laser is fired upon a certain spot of the sample site, and species present are desorbed and ionized by the laser radiation. This ionization also causes the molecules to break up into smaller fragment-ions. The positive or negative ions made are then accelerated into the flight tube, being detected at the end by a microchannel plate detector. Signal intensity, or peak height, is measured as a function of travel time. The applied voltage and charge of the particular ion determines the kinetic energy, and separation of fragments is due to different size causing different velocity. Each ion mass will thus have a different flight- time to the detector. [0073] One can either form positive ions or negative ions for analysis. Positive ions are made from regular direct photoionization, but negative ion formation requires a higher powered laser and a secondary process to gain electrons. Most of the molecules that come off the sample site are neutrals, and thus can attract electrons based on their electron affinity. The negative ion formation process is less efficient than forming just positive ions. The sample constituents will also affect the outlook of a negative ion spectra.

[0074] Other advantages with the LDLPMS method include the possibility of constructing the system to give a quiet baseline of the spectra because one can prevent coevolved neutrals from entering the flight tube by operating the instrument in a linear mode. Also, in environmental analysis, the salts in the air and as deposits will not interfere with the laser desorption and ionization. This instrumentation also is very sensitive, known to detect trace levels in natural samples without any prior extraction preparations.

[0075] Since its inception and commercial availability, the versatility of MALDI- TOF-MS has been demonstrated convincingly by its extensive use for qualitative analysis. For example, MALDI-TOF-MS has been employed for the characterization of synthetic polymers, peptide and protein analysis, DNA and oligonucleotide sequencing, and the characterization of recombinant proteins. Recently, applications of MALDI-TOF-MS have been extended to include the direct analysis of biological tissues and single cell organisms with the aim of characterizing endogenous peptide and protein constituents. [0076] The properties that make MALDI-TOF-MS a popular qualitative tool— its ability to analyze molecules across an extensive mass range, high sensitivity, minimal sample preparation and rapid analysis times— also make it a potentially useful quantitative tool. MALDI-TOF-MS also enables non-volatile and thermally labile molecules to be analyzed with relative ease. It is therefore prudent to explore the potential of MALDI-TOF-MS for quantitative analysis in clinical settings, for toxicological screenings, as well as for environmental analysis. In addition, the application of MALDI-TOF-MS to the quantification of peptides and proteins is particularly relevant.

IV. Treatment of Disease

[0077] "Treatment" and "treating" refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, a treatment may include administration of a pharmaceutically effective amount of ibrutinib to a cancer patient in need thereof.

[0078] "Subject" and "patient" refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human. [0079] The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0080] In certain aspects, the administration of ibrutinib may be used for the treatment of diseases, including cancers that are resistant to erlotinib and/or gefitinib. The invention specifically discloses treatment methods using ibrutinib in combination with various secondary agents.

[0081] Tumors for which the present treatment methods are useful include any malignant cell type, such as those found in a solid tumor or a hematological tumor. Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma. [0082] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; B-cell lymphoma; low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

A. Pharmaceutical Preparations

[0083] Where clinical application of a therapeutic composition containing ibrutinib is undertaken, it will generally be beneficial to prepare a pharmaceutical or therapeutic composition appropriate for the intended application. In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. [0084] The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. The preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.

[0085] As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0086] The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the effect desired. The actual dosage amount of a composition of the present embodiments administered to a patient or subject can be determined by physical and physiological factors, such as body weight, the age, health, and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, idiopathy of the patient, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, a dose may also comprise from about 1 μg/kg/body weight to about 1000 mg/kg/body weight (this such range includes intervening doses) or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 μg/kg/body weight to about 100 mg/kg/body weight, about 5 μg/kg/body weight to about 500 mg/kg/body weight, etc. , can be administered. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0087] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes. One preferred method of administration is hepatic artery infusion. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0088] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0089] The compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0090] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

B. Combination Treatments

[0091] In certain embodiments, the compositions and methods of the present embodiments involve ibrutinib in combination with a second or additional therapy.

[0092] The methods and compositions, including combination therapies, enhance the therapeutic or protective effect, and/or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and/or the inhibition of cellular hyperproliferation. This process may involve contacting the cells with both ibrutinib and a second therapy. A tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) comprising one or more of the agents (i.e., ibrutinib or a second anti-cancer agent), or by contacting the tissue, tumor, and/or cell with two or more distinct compositions or formulations, wherein one composition provides 1) ibrutinib, 2) a second anti-cancer agent, or 3) both ibrutinib and an anti-cancer agent. Also, it is contemplated that such a combination therapy can be used in conjunction with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

[0093] The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing, for example, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

[0094] Ibrutinib may be administered before, during, after, or in various combinations relative to an anti-cancer treatment. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where ibrutinib is provided to a patient separately from an anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with ibrutinib and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

[0095] In certain embodiments, a course of treatment will last 1-90 days or more (this such range includes intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered. This time period may last 1-7 days, and/or 1-5 weeks, and/or 1-12 months or more (this such range includes intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary. [0096] Various combinations may be employed. For the example below ibrutinib therapy is "A" and a second anti-cancer therapy is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

[0097] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy. i. Chemotherapy

[0098] A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

[0099] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DFMO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine,plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[00100] Specific, non-limiting classes of chemotherapeutics that are contemplated for use herein include NF-KB/STAT-3 inhibitors (e.g., auranofin, compounds disclosed in PCT Publn. No. WO2012/142615), AXL inhibitors (e.g., SGI-7079, cabozantinib, BMS 777607, BGB324, ASP2215, compounds disclosed in PCT Publn. Nos. WO2007/030680, WO2008/045978, WO2008/080134, WO2008/083353, WO2008/083354, WO2008/083356, WO2008/083357, WO2008/083367, WO2009/054864, WO2009/137429, WO2010/083465, and U.S. Patent No. 8,658,669), ALK/MET inhibitors (e.g., crizotinib, compounds disclosed in PCT Publn. No. OW2006/021884, ceritinib, alectinib, brigatinib, entrectinib, PF-06463922, TSR-01 1, CEP-37440, X-396), RAF/VEGFR inhibitors (e.g., sorafenib, vemurafenib, AZ628), AKT inhibitors (e.g., MK2206, perifosine, GSK-2141795), Src inhibitors (e.g., dasatinib, SU6656), JAK/STAT-3 inhibitors (e.g., ruxolitinib, tofacitinib, baricitinib, CYT387, GLPG0634, GSK2586184, lestaurtinib, pacritinib, TG101348), and WNT/p-catenin inhibitors (e.g., LGK974135, cardionogen 1, FH 535, IWP 4, TAK715). ii. Radiotherapy

[00101] Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. iii. Immunotherapy

[00102] The skilled artisan will understand that immunotherapies may be used in combination or in conjunction with methods of the embodiments. In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (Rituxan®) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[00103] In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and pl55. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand. [00104] Examples of immunotherapies currently under investigation or in use are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739, 169; Hui and Hashimoto, 1998; Christodoulides et al, 1998); cytokine therapy, e.g., interferons α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al, 1998; Davidson et al, 1998; Hellstrand et al, 1998); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al, 1998; Austin- Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-pl 85 (Hollander, 2012; Hanibuchi et al, 1998; U.S. Patent 5,824,31 1). It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein. iv. Surgery

[00105] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs' surgery).

[00106] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 months. These treatments may be of varying dosages as well. v. Other Agents

[00107] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.

V. Examples

[00108] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Materials and Methods

[00109] Cell lines. The human non-small cell lung cancer (NSCLC) cell lines were maintained in the inventors' laboratories. Authentication for each cell line was performed by DNA fingerprint analysis as described (Byers et al, 2013). All cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin in a 37°C incubator with 95% humidity and 5% C0₂.

[00110] Chemicals and antibodies. Ibrutinib, erlotinib, and afatinib were obtained from Selleck Chemicals (Houston, TX). Monoclonal antibodies (mouse anti- human) for Western blot analysis were obtained from Cell Signaling Technology (Beverly, MA), except for β-actin, which was acquired from Sigma (St Louis, MO). Secondary antibodies were purchased from Li-Cor Corp (Lincoln, NE).

[00111] Cell viability and molecular biological assays. Quadruplicate cell viability assays were performed using the sulforhodamine B assay. Western blot analysis for protein expression and phosphorylation, and flow cytometric analysis for apoptotic cells were performed as previously described (Liu et al., 2012). All experiments were performed at least twice.

[00112] Animal experiments. Animal experiments were carried out in accordance with Guidelines for the Care and Use of Laboratory Animals (NIH publication number 85-23) and the institutional guidelines of M.D. Anderson Cancer Center. Subcutaneous tumors were established in 6- to 8-week-old female nude mice (Charles River Laboratories Inc., Wilmington, MD) by inoculation of 2 x 10⁶ H1975 cells into the dorsal flank of each mouse. After the tumors grew to 3-5 mm in diameter, the mice were grouped randomly into four groups (5/group) and treated with oral administration of 1) ibrutinib (25 mg/kg/day); 2) erlotinib (50 mg/kg/day); and 3) solvent (5% DMSO, 2% Tween®-80 and 0.2% 2-hydroxypropyl- -cyclodextrin). Tumor volumes were calculated by using the formula a x b² x 0.5, where a and b represented the larger and smaller diameters, respectively. Mice were killed by CO₂ asphyxiation when the tumors had grown to 15 mm in diameter or had ulceration. [00113] Statistical analysis. Each experiment or assay was performed at least twice, and representative examples are shown. Data are reported as means ± standard deviations (SD). Statistical significance of the differences between treated samples was determined by using the two-tailed Student's t-test and one way ANOVA analysis. Differences were considered statistically significant at P < 0.05. The mean survival time and accumulative survival curve were determined by Kaplan-Meier estimation. The mean survival times were compared by log-rank test. All statistical analyses were performed by using IBM SPSS Statistics software, version 19 (IBM, Armonk, NY).

Example 1 - Selective antitumor activity of ibrutinib in EGFR-mutant non-small cell lung cancer cells [00114] To identify new potential agents for the treatment of lung cancer and to test whether ibrutinib can be used for treatment of solid tumors, the antitumor activities of ibrutinib were evaluated in a panel of lung cancer cell lines using a cell viability assay (Liu et al, 2012) following 3 days of treatment with 0.03-31 μΜ ibrutinib. For the 39 non-small cell lung cancer (NSCLC) cell lines tested, the 50% inhibitory concentration [IC5₀] values of ibrutinib ranged from 0.002 to 31 μΜ. Among these cell lines, 36 had IC5₀ values between 2 and 31 μΜ. For the remaining three cell lines, HCC827, H1975, and H292, the IC5₀ values were between 0.002 and 0.195 μΜ (FIG. 1A), which were within the clinically achievable concentrations of ibrutinib in the doses used for treatment of lymphoma (Advani et al, 2013; Byrd et al, 2013). HCC827 and H1975 cells are known to harbor epidermal growth factor receptor (EGFR) mutations, whereas the H292 cell line has wild-type EGFR. Subsequent analysis showed that EGFR was constitutively active in H292 cells and that H292 cells were also susceptible to the EGFR inhibitor erlotinib (Shepherd et al, 2005). These results suggest that ibrutinib is specific for EGFR-mutant or -constitutively active NSCLC cells.

[00115] Erlotinib and ibrutinib antitumor activities were compared in nine other NSCLC cell lines, seven of which have mutations or deletions in the EGFR gene. As shown in FIG. IB and Table 1, ibrutinib induced an antitumor spectrum similar to that of erlotinib in these cell lines. One exception is the HI 975 cell line, which harbors a T790M mutation in EGFR, and was resistant to erlotinib but sensitive to ibrutinib. HI 650 cells, which harbor the EGFR mutations and PTEN loss (Sos et al, 2009), were resistant to erlotinib, ibrutinib, and afatinib (IC5₀ = 2.63 μΜ), a second generation of EGFR inhibitors that are approved for treatment of EGFR mutant lung cancer (Sequist et al, 2013). The dose- response of ibrutinib was compared to that of erlotinib and afatinib in HI 975 cells and it was found that ibrutinib induced antitumor activity similar to that of afatinib (FIG. IB).

Table 1. IC5₀ (μΜ) of erlotinib and ibutinib in NSCLC cell lines

Del, deletion; Ex, exon; WT, wild-type. *Note, H2170 has HER2 amplification.

Example 2 - Ibrutinib effectively suppresses the in vivo growth of T790M mutant tumors [00116] To test whether ibrutinib could elicit in vivo antitumor activity in

EGFR-mutant tumors, xenograft tumors were established from HI 975 cells in nude mice. When the tumors reached 4-5 mm in diameters, the animals were treated daily with ibrutinib (25 mg/kg), erlotinib (50 mg/kg), or solvent. The results showed that treatment with ibrutinib, but not erlotinib, significantly suppressed HI 975 tumor growth and prolonged survival of the tumor-bearing animals (FIGS. 1C-D). While the mean survival times for solvent- and erlotinib-treated animals were both 17.8 days (95% confidence interval [CI] = 14.3 - 21.3), the mean survival time for ibrutinib treated animals was 29.8 days (95% CI = 26.0 - 33.6, P = 0.008, log-rank test), demonstrating the in vivo efficacy of ibrutinib in erlotinib-resistant EGFR-mutant cancer. Example 3 - Ibrutinib inhibits EGFR activation in EGFR-mutant NSCLC, including

NSCLC with T790M mutations

[00117] Whether antitumor activity of ibrutinib in NSCLC cells was mediated by inhibition of BTK or by direct effect on EGFR was determined. The expression of BTK was not detectable in any cell line tested (FIG. 3), indicating that ibrutinib-induced antitumor activity in these cells is not mediated by BTK. In contrast, treatment of HI 975 and H3255 cells with erlotinib and ibrutinib led to a similar dose-dependent inhibition of phosphor- EGFR at the Y1068 site in H3255 cells. However, only ibrutinib inhibited pY1068 in H1975 cells (FIG. 2A). The basal EGFR phosphorylation at Yl 173 was only detectable in HCC827. Like erlotinib, ibrutinib induced dose-dependent inhibition of EGFR Y1173 phosphorylation in HCC827 cells, and constitutive Y1068 phosphorylation in H292 cells, although at relatively higher doses compared to that observed in EGFR mutant cells. Similar results were observed for EGF-stimulated Y1068 phosphorylation in A549 cells (FIG. 2B), suggesting that at a higher dose, ibrutinib was able to suppress wild-type EGFR activity, consistent with other studies about ibrutinib's effect on EGFR (Honigberg et al., 2010; Pan et al, 2007). These results suggest that ibrutinib can function as an EGFR inhibitor in NSCLC cells, even though it does not have the 4-anilinoquinazoline core structure of most other EGFR inhibitors. Ibrutinib-induced inhibition of EGFR phosphorylation occurred as early as 30 min after treatment (FIG. 2C).

[00118] Whether ibrutinib induced growth suppression or apoptosis in HCC827 cells was also tested. Flow cytometric analysis on apoptotic cells and Western blot analyses of poly(ADP-ribose) polymerase (PARP1) and caspase-3 cleavage showed that ibrutinib induced a dose-dependent increase of apoptotic cells (42% of apoptotic cells at 72 h after treatment with 1 μΜ ibrutinib versus <10% of apoptotic cells in the control group) and cleavage of PARP 1 and caspase-3 in HCC827 cells (FIG. 2D), demonstrating that apoptosis is the major mode of action in EGFR mutant NSCLC, such as HCC827 cells.

[00119] To perform direct biochemical analyses of EGFR inhibition, EGFR assay kits (Promega or Invitrogen) will be used to determine the direct effect of ibrutinib on wild-type and mutant EGFR. Afatinib or CO- 1686 (Selleck Chemicals, Houston, TX) will be used as a positive control. Varying doses of ibrutinib and positive controls will be tested for their effects on recombinant wild-type and mutant (L858R, T790M, and T790M/L858R) EGFR proteins (BPS Bioscience, San Diego, CA). IC5₀ values will be calculated on the basis of the dose-response curves. The results will allow for a determination of whether ibrutinib has a differential effect on wild-type and mutant EGFR.

[00120] Although T790M mutations are commonly seen in patients resistant to anti-EGFR therapies, few T790M mutant cell lines are available. Mutant EGFR can transform both Ba/F3 cells and immortalized human bronchial epithelial cells, rendering them sensitive to anti-EGFR therapy (Li et al., 2008; Greulich et al., 2005; Jiang et al, 2005; Yuza et al, 2007). Therefore, retroviral plasmids expressing wild-type, mutant, and double-mutant EGFR (including G719S/T790M, L858R/T790M), atypical mutations (e.g., L861Q), and various deletions in exon 19 with and without T790M mutations, have been obtained (Li et al, 2008; Greulich et al., 2005; Yuza et al, 2007). These wild-type and mutant EGFR retroviral vectors will be used to stably transfect Ba/F3 (Jiang et al, 2005; Shimamura et al, 2006) and human bronchial epithelial cells (Greulich et al, 2005; Yuza et al, 2007). Transfected cells will be used to compare the activity of ibrutinib and newer generation EGFRi (CO-1686 and AZD9291). Dose-dependent EGFR inhibition, cell viability, and apoptosis induction will be determined in parallel for all three agents in these cell lines. Proteomic profiling (RPPA) will be also performed on all untreated and treated transfected cells to determine baseline predictive markers of response and compare signaling changes between mutation groups. These results will provide additional evidence regarding signaling pathways modulated by ibrutinib in different EGFR-double mutant backgrounds compared with that of 3rd-generation EGFR inhibitors.

[00121] The effects of ibrutinib, CO-1686, and AZD9291 on EGF-stimulated

EGFR activation will also be determined in A549 and H460 cells. For this purpose, A549 and H460 cells will be treated with various doses of ibrutinib, CO-1686, and AZD9291 for 6 h. EGF (10-100 ng/ml) will be added to the cultures for 15 min. The cell lysate will then be analyzed for EGFR phosphorylation. The results will allow comparison of the effects of ibrutinib with the newer EGFRi on EGF-stimulated EGFR activation of wild-type EGFR, as previously reported (Gao et al, 2014; Xu et al, 2010; Naumov et al, 2009). Example 4 - Structural basis of ibrutinib's effects on EGFR in cultured cells

[00122] Since ibrutinib is reported to form a covalent complex with BTK through Cys481 and a conserved cysteine is found in EGFR at Cys797 (Pan et al, 2007) and in HER2 at Cys775, it is likely that ibrutinib binds to EGFR through Cys797. This cysteine residue is also the covalent binding site for all second- and third-generation EGFR inhibitors (Ward et al., 2013), including afatinib (Li et al., 2008), AZD9291 (Cross et al, 2014), and CO-1686 (Tjin et al, 2014). Recent studies revealed that mutation of this cysteine to serine in BTK (C481S) resulted in resistance to ibrutinib in both MCL and CLL patients (Woyach et al, 2014; Chiron et al, 2014). Therefore, if C797 in EGFR is critical for ibrutinib's anti- EGFR activity, a mutation in this cysteine is expected to cause resistance to ibrutinib in EGFR mutant NSCLC cells. [00123] Due to limited information on resistance to second- and third- generation anti-EGFR agents, resistance caused by EGFR C797 mutations has not yet been reported, and cells with this mutation are not available. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated target-specific genome editing, a novel technology that can efficiently induce mutagenesis in target genes in cultured cells and animals (Shalem et al, 2014; Wang et al, 2013; Zhou et al, 2014), will be used to generate EGFR C797 mutations in HCC827 and HI 975 cells. Alternatively, in vitro mutagenesis will be performed on retroviral plasmids that encode mutant EGFRs to generate a C797S mutation in the original plasmid backbones. The C797S mutant EGFR will then be stably transfected into HCC827 and HI 975 cells. The C797 mutant cell lines will allow a determination as to whether putative mutations in this site will cause resistance to ibrutinib and the novel anti- EGFR therapeutics under development. The cell lines may also be used to develop strategies to overcome putative resistance to second- and third-generation anti-EGFR therapeutics.

[00124] Cell viability assays will be performed to determine whether C797 mutations in EGFR affect the activity of ibrutinib, afatinib, CO- 1686, and AZD9291 in these cell lines. EGFR C797 mutations are expected to cause resistance to afatinib, AZD9297, and CO-1686 because these agents are known to inhibit EGFR through covalent binding to C797. If ibrutinib also inhibits EGFR through C797, its activity is expected to be affected by C797 mutations. Otherwise, C797 mutations may not have a drastic impact on ibrutinib's anti- EGFR activity, although they may have an impact on activities of other C797-binding anti- EGFR agents. The results will allow a determination of the possible impact of a C797 mutation on novel anti-EGFR therapeutics.

Example 5 - Ibrutinib in cells with acquired resistance to erlotinib and combinatorial approaches to augment anti-EGFR efficacy [00125] Nearly 50% of NSCLC cases with acquired resistance to EGFR TKIs develop resistance through T790M-independent mechanisms that involve reactivation of the key intracellular signals or activation of bypass pathways. Increased activation of alternate pathways results in maintenance of PI3K/AKT signaling despite inhibition of EGFR. Alternative or bypass pathways known to be associated with EGFR inhibitor resistance include amplification of MET (Bean et al., 2007; Cappuzzo et al, 2009; Engelman et al., 2007), loss of PTEN (Sos et al, 2009), and activation of the NF-κΒ (Bivona et al, 201 1) or IL-6R/STAT-3 pathway (Kim et al, 2012; Sen et al, 2012). A third key mechanism of resistance is acquisition of a mesenchymal phenotype (Byers et al, 2013; Zhang et al., 2012; Giles et al, 2013; Meyer et al, 2013; Cufi et al, 2013; Shien et al, 2013). EMT is associated with a loss of cell adhesion proteins, including E-cadherin, and increased invasiveness. Moreover, EMT is associated with increased AXL expression (Byers et al, 2013), which is a potential therapeutic target in overcoming EMT-associated resistance to EGFR TKIs. Studies in NSCLC cell lines and clinical specimens have demonstrated that EMT and AXL activation are significantly associated with resistance to anti-EGFR therapy (Byers et al, 2013).

[00126] HI 650 cells, which harbor the EGFR mutation together with a loss of

PTEN (Sos et al, 2009), were found to be resistant to ibrutinib, erlotinib, and afatinib. The acquired erlotinib-resistant cell lines HCC4006ER and HCC827ER, which are T790M negative, were found to be resistant to ibrutinib, afatinib, and CO- 1686. STAT-3/NF-KB inhibitor, auranofin, and AXL inhibitor, SGI-7079, dramatically augmented the therapeutic efficacy of EGFR inhibitors, including ibrutinib, in erlotinib-resistant NSCLC cells.

[00127] Erlotinib-resistant clones were generated from three EGFR mutant NSCLC cell lines (PC9, HCC827, and HCC4006) by repeated exposure and selection against erlotinib. T790M was only detectable in erlotinib-resistant PC9 (PC9ER) cells (FIG. 4A) by allele-specific PCR (Broude et al, 2001), but not in the resistant HCC4006 (HCC4006ER) and HCC827 (HCC827ER) cells. PC9ER cells remained susceptible to ibrutinib (IC₅₀ = 0.18 μΜ) (FIG. 4B), while HCC4006ER and HCC827ER were pan-resistant to ibrutinib, afatinib, and CO- 1686 (FIG. 4C). These results were consistent with data showing that some resistant tumors (e.g., those undergoing EMT [Byers et al, 2013; Zhang et al, 2012; Giles et al, 2013; Meyer et al, 2013; Cufi et al., 2013; Shien et al, 2013]) likely activate independent signaling and survival mechanisms, and are therefore likely to require combination regimens.

[00128] Resistance in HI 650 NSCLC cells has been reported to be associated with PTEN loss (Sos et al, 2009). A comparison of molecular profiling with other EGFR mutant cell lines revealed that HI 650 cells expressed relatively low levels of PRKCH, AP 1G2, INADL, FOX03, and EGFR-pY992, but with elevated expression of TNFR2, IKBIP, and p27 levels. These molecules have been shown to be significantly associated with response to anti-EGFR therapeutics (FIG. 7). Whether these molecules contribute to EGFR resistance is not clear. Overexpression and knockdown approaches will be used to determine whether modulating the expression levels of those molecules restores the sensitivity of HI 650 to ibrutinib, erlotinib, and CO- 1686. Western blot and real-time PCR analyses will be used to compare the expression of these molecules in HCC827, HCC827ER, HCC4006, and HCC4006ER cell lines. Finally, gene mutations in the exons of 200 cancer-related genes will be determined by exome sequencing, which includes most of the 138 cancer driver genes affected by mutations (Vogelstein et al, 2013), and protein profiling by RPPA in parental and resistant HCC827ER and HCC4006ER cells. New mutations or significant protein changes detected in HCC827ER or HCC4006ER cells will be further tested for their causal relationship with anti-EGFR resistance, as described above for HI 650 cells.

[00129] It is possible that resistance to ibrutinib may develop after multiple cycles of drug treatment. For the in vivo studies, some tumors may not completely regress and may regrow after treatment stops. If those tumors do not respond to another cycle of treatment, it will indicate the presence of in vivo resistance. For those tumors, tumor cells in culture will be obtained and their response compared to the parental tumor cells. Tumor tissue will also be collected and gene expression and proteomic analysis performed. PCR analysis will be conducted to validate the absence of the T790M mutation. RPPA analysis will be conducted to validate bypass pathways identified in vitro (MET, STAT-3/NF-KB, AXL) and to identify additional "targetable" bypass pathways. It is possible, however, that re-cultured tumor cells will respond to ibrutinib treatment, despite in vivo resistance. In this case, whether tumor microenvironment factors may be involved in the resistance will be determined, as previously reported (Piao et al., 2012). [00130] Ibrutinib is highly active against T790M mutant and HER2-amplified cell lines, and inhibitors of both AXL and NF-KB/STAT-3 can dramatically sensitize lung cancer cells to EGFR inhibitors, including ibrutinib. Thus, it is hypothesized that ibrutinib alone will be effective for treatment of acquired resistance caused by T790M mutation and HER2 amplifications, whereas combination therapy will be required to overcome the resistance caused by alterations in cooperative or parallel pathways. It will be determined whether MET inhibitor crizotinib, the AXL inhibitor cabozantinib, and/or auranofin sensitize HCC4006ER and HCC827ER cells to ibrutinib or the novel anti-EGFR agents CO- 1686 and AZD9291. If the mechanistic characterization of resistance in T790M negative cells leads to novel pathways that contribute to resistance, it will be determined whether ibrutinib, CO- 1686, and AZD9291 can be used in combination with pathway-targeting drugs to treat T790M-negative tumors. Potential therapeutic agents include ALK/MET inhibitor crizotinib, RAF/VEGFR inhibitor sorafenib, AKT inhibitor MK2206, Src inhibitor dasatinib, JAK/STAT-3 inhibitor ruxolitinib, and W T/p-catenin inhibitor LGK974135. All these agents will be obtained from Selleck Chemicals. Single agent activity of these compounds will be determined in HI 650, HCC4006ER, HCC827ER, and HI 975 cell lines using a dose- dependent cell viability assay, and the IC₅₀ values will be calculated. The combination activity will also be determined using a dose-dependent cell viability assay with a ratio of the two agents on the basis of their IC₅₀ values. The combined effects of the two agents will then be analyzed by calculating the combination index with CalcuSyn software (Biosoft, Ferguson, MO), as previously described (Huang et al, 2002; Meng et al, 2010). The combination of ibrutinib, CO- 1686, and AZD9291 with crizotinib, cabozantinib, auranofin, or inhibitors for pathways associated with resistance to anti-EGFR therapy, may overcome resistance. Combinations found to restore sensitivity of resistant cells in vitro will be validated in vivo.

[00131] An unbiased RPPA assay will be used to determine the downstream signaling effects of ibrutinib and the optimal combinatorial therapies identified. In brief, three cell lines (e.g., H1975, H1650 and HCC827ER) will be treated with solvent, single agents, or combinations at their optimal concentrations (ICso-ICso). Cells will be harvested at 0.5, 1, 2, 4, 8, and 24 h after treatment, and lysates analyzed by RPPA. The time-dependent molecular changes identified will be further analyzed using the Ingenuity Pathway Analysis server to identify possible pathways involved in the pharmacological interaction of the combination therapy. The possible contributions of the candidate targets in enhanced efficacy in combination therapies will be further characterized using specific inhibitors and enforced gene overexpression or knockdown, as previously described (Wei et al, 2009; Lu et al, 2013; Wei et al., 2010). The results will allow a determination as to the possible molecular changes associated with the treatment response of combination therapy; these may be useful as biomarkers for monitoring treatment responses. The excellent safety profile observed in clinical trials of ibrutinib in combination therapy (Burger et al, 2014; Younes et al, 2014) suggests that ibrutinib is a good candidate for combinatorial therapies in anti-EGFR therapy.

[00132] To translate the optimized combination therapy to future clinical trials, the possible treatment-related toxicity of the combination therapy will be determined in Balb/c or C3H mice. The animals will be treated with doses in combination therapy. Mice will be observed daily, and their weights will be recorded every 2-3 days for 60 days. Five mice from each group will be euthanized on day 3 after the last treatment and at the end of the experiment (day 60). Complete pathological analyses will be performed for all animals, including moribund animals, at necropsy. The following tissues (and organs) will be collected and fixed in cold, buffered, neutral 10% formalin for histopathological examination: adrenal gland, bone (femur), bone marrow (femur), brain, colon, intestines, esophagus, eyes, gall bladder, gonads, heart, kidneys, liver, lungs, lymph nodes, pancreas, skin, spleen, spinal cord, stomach, and urinary bladder. Hematology profiles (such as blood cell counts for erythrocytes, platelets, reticulocytes, total and differential leukocytes, and nucleated red blood cells and hemoglobin concentration) and clinical chemistry profiles (such as blood urea nitrogen, creatinine, serum aspartate aminotransferase, serum alanine aminotransferase, alkaline phosphatase, total bilirubin, total protein, and albumin) will be determined. Blood samples and changes in histopathological features will be examined microscopically as described previously (Gu et al, 2000). The paired t-test, one-way ANOVA, or Wilcoxon signed-rank test will be used to compare mouse weights and the testing parameters before treatment and at various time points after treatment. The results will allow a determination as to whether the combination therapy causes toxicity and organs susceptible to the combination therapy. If necessary, pharmacokinetic studies will be performed as recently reported (Wu et al, 2014). The information will be useful for patient monitoring in future clinical trials.

[00133] Possible treatment-related toxicity for effective combination therapy will be further evaluated in Balb/c or C3H mice. The animals will be treated with doses in combination therapy. The mice will be observed daily, and their weights will be recorded every 2-3 days for 60 days. Five mice from each group will be euthanized on day 3 after the last treatment and at the end of the experiment (day 60). Moribund animals will be killed immediately. Complete histopathological and clinical pathological analyses will be performed for all animals, including moribund animals, at necropsy. The following tissues (and organs) will be collected and fixed in cold, buffered, neutral 10% formalin for histopathological examination: adrenal gland, bone (femur), bone marrow (femur), brain, colon, intestines, esophagus, eyes, gall bladder, gonads, heart, kidneys, liver, lungs, lymph nodes, pancreas, skin, spleen, spinal cord, stomach, and urinary bladder. Hematology profiles (such as blood cell counts for erythrocytes, platelets, reticulocytes, total and differential leukocytes, and nucleated red blood cells and hemoglobin concentration) and clinical chemistry profiles (such as blood urea nitrogen, creatinine, serum aspartate aminotransferase, serum alanine aminotransferase, alkaline phosphatase, total bilirubin, total protein, and albumin) will be determined. Blood samples and changes in histopathological features will be examined microscopically as described previously (Gu et al., 2000). The paired t-test, oneway ANOVA, or Wilcoxon signed-rank test will be used to compare mouse weights and the testing parameters before treatment and at various time points after treatment. The results will allow a determination as to whether the combination therapy causes toxicities and what organs are susceptible to the combination therapy. This information will be useful for patient monitoring.

Example 6 - Targeting EGFR inhibitor resistance via bypass signaling pathways: HER2 amplification

[00134] The activities of erlotinib, ibrutinib, and afatinib were compared in 86 NSCLC cell lines. Of those 86 cell lines, gene mutation data were available in 76 cell lines, Illumina® mRNA expression profiling on 11544 unique genes were available in 67 lines, and proteomic data [determined by reverse phase protein array (RPPA)] for 193 proteins were available in 55 lines (He et al, 2012; Wei et al, 2009; Akbani et al, 2014). From this initial screen, additional NSCLC cell lines that are highly susceptible to ibrutinib or afatinib were identified. In particular, the lung squamous cancer cell line H2170, which is EGFR wild-type, but has copy number amplifications of both EGFR (4.4 copies/cell) and HER2 (135 copies/cell), was highly sensitive to both ibrutinib and afatinib (IC₅₀ = 0.011 μΜ for both agents), consistent with reports that afatinib and ibrutinib are active against HER (Kosaka et al, 2006; Li et al, 2008; Grabinski et al, 2014). These results are also consistent with a recent report on ibrutinib 's anticancer activity in ERBB2-positive breast cancer cells (Grabinski and Ewald, 2014). HER2 overexpression is detected in 20% (Berghoff et al, 2013; Nakamura et al, 2005; Heinmoller et al, 2003) of lung cancers, and HER2 amplifications or mutations are detected in ~3% (Stephens et al, 2004; Mazieres et al, 2013; Arcila et al, 2012; Yoshizawa et al., 2014). Amplification of HER2 is thought to account for -10% of NSCLC with acquired resistance to EGFRi and represent a distinct resistance mechanism, independent of T790M mutation (Takezawa et al, 2012). These data indicate that ibrutinib may also be used to treat HER2 amplified lung cancer or to overcome acquired resistance to anti-EGFR therapy caused by HER2 amplification.

[00135] Moreover, two-tailed t-test, Fisher exact test, Spearman rank test, and Pearson correlation test were performed to determine the correlation between treatment response to anti-EGFR agents and gene mutations, mRNA levels, and protein levels in above- mentioned 86 NSCLC cell lines. To adjust for multiple tests, a beta-uniform mixture model (Pounds et al., 2003) was used to estimate the false discovery rate (FDR). These analyses indicate that EGFR mutations are significantly associated with the sensitivity of all three drugs (P = 0.001) (FIG. 5A). A gene expression analysis revealed that 15 mRNA (FDR at 5%) and nine proteins or phospho-proteins (FDR at 20%) were differentially associated with response to the three anti-EGFR agents (FIGS. 5B-C). Furthermore, these analyses revealed that high levels of HER2 protein expression were significantly correlated with sensitivity to ibrutinib (r = 0.4, P = 0.002).

[00136] In addition to upregulating HER2, EGFRi resistant NSCLC also upregulates multiple bypass signaling pathways including PI3K/AKT (via loss of PTEN [Sos et al, 2009; She et al, 2005]), NF-κΒ (Bivona et al., 2011 ; Tanaka et al, 201 1; Sakuma et al, 2012), and STAT-3 (Kim et al, 2012; Chiu et al, 201 1 ; Harada et al, 2012). In this scenario, it may be possible to combine ibrutinib with other targeted therapies to achieve clinical benefit. Therefore, whether anti-inflammatory drug auranofin, which inhibits STAT-3 (Kim et al, 2007; Nakaya et al, 2011) and NF-κΒ (Nakaya et al, 2011) activity, and is FDA approved for the treatment of rheumatoid arthritis (Bernhard, 1982; Chaffman et al, 1984; Larsen et al, 1984; van Riel et al, 1984), can enhance ibrutinib's activity in EGFR mutant NSCLC cells was investigated. Activation of both STAT-3 (Kim et al., 2012; Chiu et al, 2011 ; Harada et al, 2012) and NF-κΒ (Bivona et al, 2011 ; Tanaka et al, 2011 ; Sakuma et al, 2012) pathways is known to promote resistance to anti-EGFR therapy. Specifically, treatment of cancer cells with EGFR inhibitors such as afatinib and dacomitinib can activate the IL-6/JAK/STAT-3 signaling pathway, which in turn induces resistance to these agents (Kim et al, 2012). Inhibition of the STAT-3 pathway has been shown to overcome resistance to EGFRi therapy in lung cancer (Chiu et al, 2011 ; Harada et al, 2012), head and neck cancer (Sen et al, 2012), pancreatic cancer (Nagaraj et al, 2011), and glioma (Lo et al, 2008). Furthermore, inhibition of IL-6/JAK/STAT-3 signaling or NF-κΒ activity with siRNA or small-molecule inhibitors dramatically sensitizes cancer cells to treatment with EGFR inhibitors (Kim et al, 2012; Sen et al, 2012; Bivona et al, 2011 ; Sakuma et al., 2012; Chiu et al, 201 1). To investigate whether ibrutinib's activity can be enhanced by combination therapy with auranofin, ibrutinib dose responses were examined in HI 975 and HI 650 cell lines in the presence or absence of auranofin. While low concentrations of auranofin (0.25 μΜ) alone demonstrate mild activity, its presence dramatically sensitized HI 975 and HI 650 cells to ibrutinib (IC₅₀ value reduced 50-100 fold) (FIG. 6), demonstrating the feasibility of enhancing ibrutinib's activity by combination therapy. Recently, clinical trials demonstrated that ibrutinib is well tolerated when used in combination with other targeted therapeutics (Burger et al, 2014; Younes et al, 2014). Therefore, targeting bypass signaling pathways, including HER2, STAT-3 and NF-κΒ, through combinatorial approaches may enhance anti- EGFR therapy. [00137] RPPA will be performed to determine time-dependent changes in molecular targets of multiple cancer related pathways in H2170 cells after treatment with ibrutinib. The activity of ibrutinib and the HER2 inhibitor lapatinib in other HER2 amplified lung cancer cell lines, H650 (7.2 copies/cell), H1092 (6.2 copies/cell), and HCC954 (6.6 copies/cell), will be determined. Based on preliminary results in H2170 cells and recent report on ibrutinib anti-HER2 activity in breast cancer cell lines (Grabinski and Ewald, 2014), ibrutinib may be used for treatment of HER2 amplified lung cancers. Wild-type and mutant HER2 plasmids (Li et al, 2004; Wang et al, 2004) will be used to stably transfect EGFR mutant NSCLC cell lines PC9, HCC827, and HCC4006. The transfected cells will be used to compare the activity of ibrutinib, afatinib, CO- 1686, and AZD9291. The results will allow a determination as to whether ibrutinib can be used to overcome the resistance caused by HER2 amplification/mutations.

[00138] The effects of ibrutinib on in vivo growth of NSCLC cells with HER2 amplification/overexpression will be tested. Briefly, in vivo activity of ibrutinib will be determined by treating tumors derived from cell lines described above in nude mice daily with either ibrutinib or solvent, when tumors reach 50-100 mm³ in volume. A subset of tumors (5/group) from all experiments (vehicle- and ibrutinib-treated xenografts) will be snap frozen and protein lysates collected and analyzed by RPPA to compare ibrutinib-induced signaling changes and determine proteomic markers of response to ibrutinib in HER2 mutant NSCLC. Example 7 - Targeting EGFR inhibitor resistance by targeting EMT

[00139] Molecular profiling was performed on NSCLC cell lines and clinical specimens from patients treated in the BATTLE trial (Kim et al, 201 1) and it was demonstrated that mesenchymal cells showed significantly greater resistance to EGFR inhibitors, compared to more epithelial cells. Moreover, mesenchymal NSCLC also expressed increased levels of the receptor tyrosine kinase AXL and showed a trend toward greater sensitivity to the AXL inhibitor SGI-7079, whereas the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing AXL (FIG. 7) and in a xenograft model of mesenchymal NSCLC (Byers et al, 2013). Furthermore, using NSCLC cell lines with acquired resistance to erlotinib (described in Example 4), the expression of AXL, as well as other classical EMT markers (e.g., E-cadherin) was investigated by Western blotting. In erlotinib-resistant HCC4006 (HCC4006ER) and HCC827 (HCC827ER) cells, AXL protein expression was elevated, while expression of the epithelial marker, E-cadherin, was lost in most resistant clones (FIG. 8). These data indicate that EMT/AXL expression may be a mechanism of resistance to EGFRi, including ibrutinib. To overcome EMT-mediated EGFRi resistance, ibrutinib may be used in combination with the AXL inhibitor cabozantanib, which is approved for use in thyroid cancer.

Example 8 - Patient-derived xenografts (PDXs) from lung cancer patients

[00140] The effects of ibrutinib will be tested in multiple NSCLC mouse models, including 1) xenografts established from cell lines, 2) genetically engineered mouse models (GEMMS), and 3) PDXs. For xenograft experiments, in vivo activity of ibrutinib will be determined by treating tumors derived from cell lines (HI 975 and others as described above) in nude mice daily with either ibrutinib (25-50 mg/kg) or solvent when tumors reach 50-100 mm³ in volume.

[00141] Recent studies show that PDXs exhibit similar response rates for several therapeutic agents compared to those observed clinically (Sivanand et al, 2012; Rubio-Viqueira et al, 2006; Richtner et al, 2008). PDXs can also be used to effectively select targeted therapy treatment regimens for cancer patients bearing specific mutations (Hidalgo et al, 201 1; Morelli et al, 2012), suggesting that PDXs are clinically relevant tumor models for efficacy studies. Twenty-three PDXs were established from lung cancer surgical specimens over the past two years and gene mutations were determined in these PDXs and their paired primary tumors by ultra-deep exome sequencing (average coverage of about 600 fold) for 202 cancer-related genes, including 138 cancer driver genes often affected by mutation (Vogelstein et al, 2013). Among these PDXs, two had double atypical mutations in the kinase domain of EGFR, one with S81 1C and D855N mutations and the other with V674F and P959L mutations. These mutations are predicted to be oncogenic drivers based on predictions made using SIFT (Kumar et al, 2009), Polyphen (Adzhubei et al, 2010), Condel (Gonzalez-Perez et al, 2011), and Mutation Assessor (Reva et al, 201 1) software. These two PDXs with EGFR mutations, in addition to two commercially available lung cancer PDXs with mutations in EGFR and acquired resistance to erlotinib from the Jackson Laboratory (Bar Harbor, Maine) (one with an EGFR L858R mutation (TM00199) and the other with a T790M mutation (TM00204), will be used to determine in vivo activity of the anti-EGFR therapies. Tumors will be harvested from the first (Fl) or second (F2) passage in animals and will be sectioned into 2-3 mm³ each; they will then be replanted into 40-50 mice for in vivo efficacy studies.

[00142] The effects of ibrutinib will be tested on mouse models (GEMMs) that harbor the EGFR L858R and L858R/T790M mutations (Politi et al, 2006; Regales et al, 2009). Analysis of tumors from EGFR-mutant GEMMs bearing the L858R allele has been previously reported (Xu et al, 2010). Bi-transgenic mice expressing TetO- EGFRL858R/T790M and tetracycline trans activator will be fed doxycycline from the time of weaning. Adenocarcinoma develops at about 4 weeks after receiving doxycycline (Politi et al, 2006; Regales et al, 2009). Treatment will start 4-5 weeks after receiving doxycycline. Mice will be sacrificed at 3-6 weeks after treatment and tumor burden will be determined by analysis of lung histopathology.

[00143] In vivo activity in xenografts established from EGFR-mutant human lung cancer cell lines. Subcutaneous tumor models established from two EGFR mutant NSCLC cell lines (H1975, H1650, PC9ER, HCC827ER, or HCC4006ER) will be used for this study. Tumors will be established by inoculating 1-5 x 10⁶ cells in the rear flank sites of nude mice. Mice will be randomly assigned to groups (10 mice/group) before treatment begins, which will be when tumors reach 3-5 mm in diameter. The drugs will be administered in a manner similar to how they are used in the clinic. For example, ibrutinib and auranofin will be given via oral gavage. Animals treated with single-drug therapy, and solvent alone will be used as controls. [00144] For all studies, mice will be randomly divided into groups before treatment starts, when tumors reach 50-100 mm³ in volume. Ibrutinib will be given via oral gavage daily at doses of 25-50 mg/kg. Animals treated with solvent will be used as controls. A subset of tumors from all experiments (vehicle and ibrutinib treated xenografts, GEMMs, and PDXs) will be snap frozen and protein lysates collected and analyzed by RPPA to compare ibrutinib-induced signaling changes between mutation subsets and determine proteomic markers of response to ibrutinib. [00145] Mice will be monitored for growth of subcutaneous tumors and body weight changes every 2-3 days for up to 16 weeks. Tumors will be measured with calipers to determine the largest and smallest diameters. The tumor volume (V) will be calculated according to the formula V = ab²/2, in which a is the largest and b is the smallest diameter (Carlsson et al., 1983). The results will be subjected to an analysis of variance (A OVA) using SPSS software (Gu et al, 2000). A P-value of < 05 will be considered significant. For GEMMs, the tumor burden will be determined by analysis of lung histopathology at the end of experiment (3-6 weeks after the treatment).

[00146] Mice will be killed when tumors reach 1500-2000 mm³ in volume; the survival duration of the tumor-bearing animals will be recorded. At the end of the study (16 weeks after the last treatment), any mice that remain alive will be killed and their tumors and various organs (lungs, heart, stomach, liver, kidneys, brain, gonads, and spleen) will be harvested for histopathologic evaluation (Gu et al, 2000). Survival data will be analyzed using a Kaplan-Meier survival analysis with SPSS software. The results will show whether the treatments improve survival in mice bearing xenografts from cell lines or PDXs of lung cancer.

[00147] Molecular changes associated with treatment responses. Proteomic changes that occur in the effective treatment group (five mice per group) will be determined in tumors derived from cell lines and PDXs. Mice will be euthanized 24 h after the three sequential treatments. Tumors and various organs (lungs, heart, stomach, liver, kidneys, brain, gonads, and spleen) will be harvested, snap frozen, and lysates analyzed by RPPA. Proteomic data quality will be assessed as previously described (Gao et al, 2014; Haura and Rix, 2014). Differences in marker levels between treatment arms and time points will be determined by ANOVA. Results will be confirmed by immunohistochemistry for relevant proteins (e.g., EGFR, p-EGFR, HER2, MET, etc.), AXL/EMT pathway, and other key proteins or phospho-proteins identified by RPPA. Analyses of in vivo proteomic changes in tumors will allow a determination of whether those molecular changes can be used as markers for monitoring treatment responses in clinical evaluations. Example 9 - Efficacy of ibrutinib in non-small cell lung cancer patients with EGFR- mutant tumors

[00148] Based on preclinical data, a clinical trial has been developed to study the efficacy of ibrutinib in patients with NSCLC ("A Phase I/II Study of Ibrutinib in Previously Treated EGFR Mutant Non-Small Cell Lung Cancer"). Patients eligible for this trial will have EGFR mutant NSCLC that has progressed on treatment with frontline TKIs (e.g., erlotinib, gefitinib, or afatinib).

[00149] Biopsies will be obtained prior to treatment with ibrutinib to study resistance mechanisms to frontline EGFR inhibition. Patients will then receive treatment with ibrutinib. There will be a dose escalation phase, with initial patients dosed at 560 mg daily (the approved dose for MCL) and dose escalation to 840 mg daily if treatment is well tolerated. Patients will continue treatment until progressive disease, intolerable side effects, or withdrawal of consent. There will also be biopsies performed at progression, to determine mechanisms of resistance to ibrutinib. [00150] Up to 38 patients will be enrolled on this trial— up to 18 on the dose escalation phase, and a total of 20 at the recommended phase II dose. The primary endpoint for the expansion cohort will be response rate by RECIST 1.1 ; secondary endpoints will include progression free survival and overall survival. Although the small numbers of patients will limit the power of this analysis, response to therapy will be correlated with the mechanism of acquired resistance to frontline TKI. Studies on MCL and CLL (Woyach et al, 2014; Chiron et al, 2014) revealed that analysis on specimens from 5-6 patients was able to identify clinically relevant mechanisms of resistance to ibrutinib. It is hypothesized that ibrutinib will be most effective in patients with secondary resistance mechanisms, such as T790M mutation and in patients who have upregulated bypass signaling pathways (e.g., HER2 amplification), but will be less effective in patients with STAT-3/NF-KB activation or AXL overexpression.

[00151] Fresh frozen core needle biopsies (2-3) from patients prior to ibrutinib treatment (day 0) and at disease progression will be obtained and analyzed by RPPA and whole-exome/whole-transciptome sequencing. Changes in protein expression from Day 0 to progression for markers in the EGFR/HER2, MET, STAT-3/NF-KB, and AXL/EMT pathways will be assessed by ?-test. Baseline marker expression and the degree of marker change following treatment will be correlated with clinical response for each RPPA marker. The core biopsy specimens stored in RNAlater® (Life Technologies) will also be analyzed for whole-exome/whole-transciptome sequencing to determine mutational/expressional differences between before treatment and resistant or recurrent tumor specimens after treatment. Remaining tissue from the cores will be preserved for IHC validation of top markers and for future analysis of mR A and/or DNA.

[00152] Analysis on clinical specimens are expected to lead to identification of some novel mutations in EGFR or related pathways or altered gene expressions that occur either only or with higher frequencies in resistant tumors. Molecular characterizations will be used to determine whether those mutations are causally associated with resistance.

[00153] It is expected that lung cancer with EGFR mutations, including the second T790M mutations and/or HER2 amplifications, will be sensitive to the ibrutinib treatment in preclinical and clinical studies, and that mutations in the covalent binding site for newer EGFRi (C797) will cause resistance to those agents, including ibrutinib. However, it is possible that cells or tumors that initially respond to ibrutinib may develop resistance after repeated exposure or treatment cycles, as observed for other EGFRi (Politi et al, 2010). For example, in vivo tumors may regrow after treatment stops. Should this occur, the tumor- bearing animals will be treated again to determine whether the tumors still respond to the treatment. If the tumors do not respond, it will indicate that in vivo resistance may have been induced. Those tumors will be harvested to characterize possible mechanisms of resistance.

* * *

[00154] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

Advani et al, Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) has significant activity in patients with relapsed/refractory B-cell malignancies. J Clin Oncol, 31(l):88-94, 2013.

Adzhubei et al, A method and server for predicting damaging missense mutations. Nature

Methods, 7:248-249, 2010.

Akbani et al, A pan-cancer proteomic perspective on The Cancer Genome Atlas. Nature

Communications, 5:3887, 2014.

American Veterinary Medical Association. AVMA Guidelines on Euthanasia. On the world wide web at avma.org/issues/animal_welfare/euthanasia.pdf, 2007.

Arcila et al, Prevalence, clinicopathologic associations, and molecular spectrum of ERBB2

(HER2) tyrosine kinase mutations in lung adenocarcinomas. Clinical Cancer

Research, 18:4910-4918, 2012.

Arcila et al, EGFR exon 20 insertion mutations in lung adenocarcinomas: prevalence, molecular heterogeneity, and clinicopathologic characteristics. Molecular Cancer

Therapeutics, 12:220-229, 2013.

Bean et al, MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci

USA, 104(52):20932-20937, 2007.

Berghoff et al, Frequent overexpression of ErbB— receptor family members in brain metastases of non-small cell lung cancer patients. Apmis, 121: 1144-1152, 2013.

Bernhard, Auranofin therapy in rheumatoid arthritis. Journal of Laboratory & Clinical

Medicine, 100: 167-177, 1982.

Bivona et al, FAS and NF-kB signalling modulate dependence of lung cancers on mutant EGFR. Nature, 471 :523-526, 2011.

Bowles et al., Multi-targeted tyrosine kinase inhibitors in clinical development: focus on XL-

184 (cabozantinib). Drugs of Today, 47:857-868, 2011.

Broude et al, Multiplex allele-specific target amplification based on PCR suppression. Proc.

Natl. Acad. Sci. USA, 98:206-211, 2001. Brown, Ibrutinib (PCI-32765), the First BTK (Bruton's Tyrosine Kinase) Inhibitor in Clinical

Trials. Current Hematologic Malignancy Reports, 8: 1-6, 2013.

Burger et al, Safety and activity of ibrutinib plus rituximab for patients with high-risk chronic lymphocytic leukaemia: a single-arm, phase 2 study. Lancet Oncology, 15: 1090-1099, 2014.

Byers et al, Reciprocal regulation of c-Src and STAT3 in non-small cell lung cancer.

Clinical Cancer Research, 15:6852-6861, 2009.

Byers et al, Proteomic profiling identifies dysregulated pathways in small cell lung cancer and novel therapeutic targets including PARP1. Cancer Discovery, 2:798-811, 2012. Byers et al, An epithelial-mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance. Clin Cancer Res., 19(l):279-290, 2013.

Byrd et al, Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J

Med., 369(l):32-42, 2013.

Cappuzzo et al, MET increased gene copy number and primary resistance to gefitinib therapy in non-small-cell lung cancer patients. Ann Oncol, 20(2):298-304, 2009. Cappuzzo et al, Erlotinib as maintenance treatment in advanced non-small-cell lung cancer: a multicentre, randomised, placebo-controlled phase 3 study. Lancet Oncology, 11:521-

529, 2010.

Carlsson et al, Estimation of liver tumor volume using different formulas - an experimental study in rats. Journal of Cancer Research & Clinical Oncology, 105:20-23, 1983. Chaffman et al, A preliminary review of its pharmacological properties and therapeutic use in rheumatoid arthritis. Drugs, 27:378-424, 1984.

Chang et al., Design, synthesis, and biological evaluation of novel conformationally constrained inhibitors targeting epidermal growth factor receptor threonine790 -> methionine790 mutant. J Med Chem., 55(6):2711-2723, 2012.

Chiron et al, Cell-Cycle Reprogramming for PI3K Inhibition Overrides a Relapse-Specific

C481S BTK Mutation Revealed by Longitudinal Functional Genomics in Mantle Cell

Lymphoma. Cancer Discovery, 4: 1022-1035, 2014.

Choi et al, EGFR mutation testing in patients with advanced non-small cell lung cancer: a comprehensive evaluation of real-world practice in an East Asian tertiary hospital.

PLoS ONE, 8:e56011, 2013.

Chiu et al, Suppression of Stat3 activity sensitizes gefitinib-resistant non small cell lung cancer cells. Biochemical Pharmacology, 81 : 1263-1270, 2011. Corbett et al., In vivo methods for screening and preclinical testing. In: Anticancer Drug

Development Guide: preclinical screening, clinical trials, and approval, 2 ed.;

Teicher, B. A.; Andrews, P. A. Eds.; Humana Press: New Jersey, pp. 99-124, 2004. Cross et al, AZD9291, an Irreversible EGFR TKI, Overcomes T790M-Mediated Resistance to EGFR Inhibitors in Lung Cancer. Cancer Discovery, 4: 1046-1061, 2014.

Cufi et al., Silibinin suppresses EMT-driven erlotinib resistance by reversing the high miR-

21/low miR-200c signature in vivo. Science Reporter, 3:2459, 2013.

Davis et al, Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.

Nature, 463(7277):88-92, 2010.

Ding et al, Somatic mutations affect key pathways in lung adenocarcinoma. Nature,

455(7216): 1069-1075, 2008.

Eilers et al, Dermatologic infections in cancer patients treated with epidermal growth factor receptor inhibitor therapy. J Natl Cancer Inst., 102(l):47-53, 2010.

Engelman et al, Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer. Journal of Clinical Investigation,

116:2695-2706, 2006.

Engelman et al, MET amplification leads to gefitinib resistance in lung cancer by activating

ERBB3 signaling. Science, 316(5827): 1039-1043, 2007.

Fichtner et al, Establishment of patient-derived non-small cell lung cancer xenografts as models for the identification of predictive biomarkers. Clinical Cancer Research,

14:6456-6468, 2008.

Fukuoka et al, Biomarker analyses and final overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS).

Journal of Clinical Oncology, 29:2866-2874, 2011.

Gahr et al, EGFR mutational status in a large series of Caucasian European NSCLC patients: data from daily practice. British Journal of Cancer, 109: 1821-1828, 2013.

Gao et al, Spectrum of LKB1, EGFR, and KRAS mutations in Chinese lung adenocarcinomas. Journal of Thoracic Oncology, 5: 1130-1135, 2010.

Gao et al, Selective antitumor activity of ibrutinib in EGFR-mutant non-small cell lung cancer cells. Journal of the National Cancer Institute, 106(9):dju204, 2014.

Giles et al, Axl mediates acquired resistance of head and neck cancer cells to the epidermal growth factor receptor inhibitor erlotinib. Molecular Cancer Therapeutics, 12:2541-

2558, 2013. Gonzalez-Perez and Lopez-Bigas, Improving the assessment of the outcome of nonsynonymous SNVs with a consensus deleteriousness score, Condel. American Journal of Human Genetics, 88:440-449, 2011.

Grabinski and Ewald, Ibrutinib (Imbruvica) potently inhibits ErbB receptor phosphorylation and cell viability of ErbB2-positive breast cancer cells. Invest New Drugs, 32: 1096-

1104, 2014.

Greulich et al, Oncogenic transformation by inhibitor-sensitive and -resistant EGFR mutants.

PLoS Medicine, 2:e313, 2005.

Gu et al, Tumor-specific transgene expression from the human telomerase reverse transcriptase promoter enables targeting of the therapeutic effects of the Bax gene to cancers. Cancer Res., 60:5359-5364, 2000.

Harada et al, JAK2-related pathway induces acquired erlotinib resistance in lung cancer cells harboring an epidermal growth factor receptor-activating mutation. Cancer Science,

103: 1795-1802, 2012.

Haura and Rix, Deploying ibrutinib to lung cancer: another step in the quest towards drug repurposing. Journal of the National Cancer Institute, 106(9):dju250, 2014.

He et al, Aberrant expression of proteins involved in signal transduction and DNA repair pathways in lung cancer and their association with clinical parameters. PLoS ONE, 7:e31087, 2007.

Heinmoller et al, HER2 status in non-small cell lung cancer: results from patient screening for enrollment to a phase II study of herceptin. Clinical Cancer Research, 9:5238- 5243, 2003.

Hidalgo et al, A pilot clinical study of treatment guided by personalized tumorgrafts in patients with advanced cancer. Molecular Cancer Therapeutics, 10: 1311-1316, 2011. Hirsch et al, Epidermal growth factor receptor inhibition in lung cancer: status 2012. Journal of Thoracic Oncology, 8:373-384, 2013.

Honigberg et al, The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc Natl

Acad Sci USA, 107(29): 13075-13080, 2010.

Huang et al, Combined TRAIL and Bax gene therapy prolonged survival in mice with ovarian cancer xenograft. Gene Ther., 9: 1379-1386, 2002.

Ji et al, The impact of human EGFR kinase domain mutations on lung tumorigenesis and in vivo sensitivity to EGFR-targeted therapies. Cancer Cell, 9:485-495, 2006. Jiang et al, Epidermal growth factor-independent transformation of Ba/F3 cells with cancer- derived epidermal growth factor receptor mutants induces gefitinib-sensitive cell cycle progression. Cancer Res., 65:8968-8974, 2005.

Kim et al, Auranofin blocks interleukin-6 signalling by inhibiting phosphorylation of JAK1 and STAT3. Immunology, 122:607-614, 2007.

Kim et al., The BATTLE trial: personalizing therapy for lung cancer. Cancer Discovery, 1:44-53, 2011.

Kim et al, Activation of IL-6R/JAK1/STAT3 Signaling Induces De Novo Resistance to

Irreversible EGFR Inhibitors in Non-Small Cell Lung Cancer with T790M Resistance Mutation. Molecular Cancer Therapeutics, 11 :2254-2264, 2012.

Kobayashi et al, EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N

Engl J Med., 352(8):786-792, 2005.

Kosaka et al, Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib. Clin Cancer Res., 12(19):5764-5769, 2006.

Kumar et al, Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nature Protocols, 4: 1073-1081, 2009.

Kwak et al, Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc Natl Acad Sci USA, 102(21):7665-7670, 2005.

Langer, Epidermal growth factor receptor inhibition in mutation-positive non-small-cell lung cancer: is afatinib better or simply newer? Journal of Clinical Oncology, 31 :3303-

3306, 2013.

Larsen et al., The effects of auranofin and parenteral gold in the treatment of rheumatoid arthritis: an X-ray analysis. Clinical Rheumatology, 3(Suppl 1):97-104, 1984.

Lee et al., High EGFR gene copy number and skin rash as predictive markers for EGFR tyrosine kinase inhibitors in patients with advanced squamous cell lung carcinoma. Clinical Cancer Research, 18: 1760-1768, 2012.

Lee et al, Impact of EGFR inhibitor in non-small cell lung cancer on progression-free and overall survival: a meta-analysis. J Natl Cancer Inst., 105(9):595-605, 2013a.

Lee et al., Noncovalent wild-type-sparing inhibitors of EGFR T790M. Cancer Discovery, 3: 168-181, 2013b.

Li et al, Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell, 6:459-469, 2004. Li et al., BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene, 27:4702-471 1, 2008.

Li et al, Spectrum of oncogenic driver mutations in lung adenocarcinomas from East Asian never smokers. PLoS ONE, 6:e28204, 201 1.

Lima and Rodriguez, Phosphine-gold(I) compounds as anticancer agents: general description and mechanisms of action. Current Medicinal Chemistry - Anti-Cancer Agents,

11 :921-928, 201 1.

Linardou et al, Assessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systematic review and meta-analysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer. Lancet

Oncol, 9(10):962-972, 2008.

Liu et al, Antitumor activity of a novel STAT3 inhibitor and redox modulator in non-small cell lung cancer cells. Biochem Pharmacol, 83(10): 1456-1464, 2012.

Liu et al, Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974.

Proc. Natl Acad. Sci. USA, 1 10:20224-20229, 2013.

Lo et al, Constitutively activated STAT3 frequently coexpresses with epidermal growth factor receptor in high-grade gliomas and targeting STAT3 sensitizes them to Iressa and alkylators. Clinical Cancer Research, 14:6042-6054, 2008.

Lu et al, IGFBP2/FAK pathway is causally associated with dasatinib resistance in non-small cell lung cancer cells. Molecular Cancer Therapeutics, 12:2864-2873, 2013.

Lynch et al., Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med.,

350(21):2129-2139, 2004.

Madeira et al, The biological activity of auranofin: implications for novel treatment of diseases. Inflammopharmacology, 20:297-306, 2012.

Maemondo et al, Gefitinib or chemotherapy for non-small-cell lung cancer with mutated

EGFR. New England Journal of Medicine, 362:2380-2388, 2010.

Marchetti et al, EGFR mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment. Journal of Clinical Oncology,

23 :857-865, 2005.

Mazieres et al, Lung cancer that harbors an HER2 mutation: epidemiologic characteristics and therapeutic perspectives. Journal of Clinical Oncology, 31 : 1997-2003, 2013. Meng et al., Combination treatment with MEK and AKT inhibitors is more effective than each drug alone in human non-small cell lung cancer in vitro and in vivo. PLoS ONE, 5:el4124, 2010.

Meyer et al, The receptor AXL diversifies EGFR signaling and limits the response to EGFR- targeted inhibitors in triple-negative breast cancer cells. Science Signaling, 6:ra66,

2013.

Miller et al., Afatinib versus placebo for patients with advanced, metastatic non-small-cell lung cancer after failure of erlotinib, gefitinib, or both, and one or two lines of chemotherapy (LUX-Lung 1): a phase 2b/3 randomised trial. [Erratum appears in Lancet Oncol. 2012 May;13(5):el86]. Lancet Oncology, 13:528-538, 2012.

Morelli et al, Prioritizing phase I treatment options through preclinical testing on personalized tumorgraft. Journal of Clinical Oncology, 30:e45-e48, 2012.

Mukohara et al, Differential effects of gefitinib and cetuximab on non-small-cell lung cancers bearing epidermal growth factor receptor mutations. J Natl Cancer Inst., 97(16): 1185-1194, 2005.

Nagaraj et al, Combined blockade of Src kinase and epidermal growth factor receptor with gemcitabine overcomes STAT3 -mediated resistance of inhibition of pancreatic tumor growth. Clinical Cancer Research, 17:483-493, 2011.

Nakamura et al., Association of HER-2 overexpression with prognosis in nonsmall cell lung carcinoma: a metaanalysis. Cancer, 103: 1865-1873, 2005.

Nakaya et al, The gold compound auranofin induces apoptosis of human multiple myeloma cells through both down-regulation of STAT3 and inhibition of NF-kB activity.

Leukemia Research, 35:243-249, 2011.

Nanjundan et al, Proteomic profiling identifies pathways dysregulated in non-small cell lung cancer and an inverse association of AMPK and adhesion pathways with recurrence.

Journal of Thoracic Oncology, 5: 1894-1904, 2010.

Naumov et al, Combined vascular endothelial growth factor receptor and epidermal growth factor receptor (EGFR) blockade inhibits tumor growth in xenograft models of EGFR inhibitor resistance. Clinical Cancer Research, 15:3484-3494, 2009.

O'Brien et al, Ibrutinib as initial therapy for elderly patients with chronic lymphocytic leukaemia or small lymphocytic lymphoma: an open-label, multicentre, phase lb/2 trial. Lancet Oncology 2014, 15, 48-58, 2014.

Paez et al, EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science, 304: 1497-1500, 2004. Pan et al, Discovery of selective irreversible inhibitors for Bruton's tyrosine kinase. Chem

Med Chem., 2(1):58-61, 2007.

Pao et al., EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad

Sci USA, 101(36): 13306-1331 1, 2004.

Pao et al, Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med., 2(3):e73, 2005. Petrelli et al, Relationship between skin rash and outcome in non-small-cell lung cancer patients treated with anti-EGFR tyrosine kinase inhibitors: a literature-based metaanalysis of 24 trials. Lung Cancer, 78:8-15, 2012.

Piao et al, Glioblastoma resistance to anti-VEGF therapy is associated with myeloid cell infiltration, stem cell accumulation, and a mesenchymal phenotype. Neuro-Oncology ,

14: 1379-1392, 2012.

Plowman et al, Human tumor xenograft models in NCI drug development. In: Anticancer Drug Development Guide; Teicher, B. A. Ed.; Humana Press: New Jersey, pp. 101- 125, 1997.

Politi et al, Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the receptors. Genes & Development, 20: 1496-1510, 2006.

Politi et al, Erlotinib resistance in mouse models of epidermal growth factor receptor- induced lung adenocarcinoma. Disease Models & Mechanisms, 3 : 1 11-1 19, 2010.

Ponader et al, The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo. Blood, 1 19(5): 1182- 1189, 2012.

Pounds and Morris, Estimating the occurrence of false positives and false negatives in microarray studies by approximating and partitioning the empirical distribution of p- values. Bioinformatics, 19: 1236-1242, 2003.

Ramalingam et al, Randomized phase II study of dacomitinib (PF-00299804), an irreversible pan-human epidermal growth factor receptor inhibitor, versus erlotinib in patients with advanced non-small-cell lung cancer. Journal of Clinical Oncology, 30:3337- 3344, 2012.

Regales et al, Dual targeting of EGFR can overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer. Journal of Clinical Investigation, 119:3000-3010, 2009. Reva et al., Predicting the functional impact of protein mutations: application to cancer genomics. Nucleic Acids Research, 39:el 18, 2011.

Rosell et al, Screening for epidermal growth factor receptor mutations in lung cancer. New

England Journal of Medicine, 361:958-967, 2009.

Rosell et al, Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer

(EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncology,

13:239-246, 2012.

Rubio-Viqueira et al, An in vivo platform for translational drug development in pancreatic cancer. Clinical Cancer Research, 12:4652-4661, 2006.

Sakuma et al, NF-B signaling is activated and confers resistance to apoptosis in three- dimensionally cultured EGFR-mutant lung adenocarcinoma cells. Biochemical &

Biophysical Research Communications, 423:667-671, 2012.

Sen et al, Targeting Stat3 abrogates EGFR inhibitor resistance in cancer. Clinical Cancer

Research, 18:4986-4996, 2012.

Schuh et al, Gold(I) carbene complexes causing thioredoxin 1 and thioredoxin 2 oxidation as potential anticancer agents. Journal of Medicinal Chemistry, 55:5518-5528, 2012. Sequist et al, Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations. J Clin Oncol, 31(27):3327-

3334, 2013a.

Sequist et al, First-in-human evaluation of CO-1686, an irreversible, selective, and potent tyrosine kinase inhibitor of EGFR T790M. J Clin Oncol, 3 l(Suppl): Abstract 2524,

2013b.

Shalem et al, Genome-scale CRISPR-Cas9 knockout screening in human cells. Science,

343:84-87, 2014.

She et al, The BAD protein integrates survival signaling by EGFR/MAPK and PI3K/Akt kinase pathways in PTEN-deficient tumor cells. Cancer Cell, 8:287-297, 2005.

Shepherd et al, Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med., 353(2): 123-132, 2005.

Shien et al., Acquired resistance to EGFR inhibitors is associated with a manifestation of stem cell-like properties in cancer cells. Cancer Res., 73:3051-3061, 2013.

Shimamura et al, Non-small-cell lung cancer and Ba/F3 transformed cells harboring the ERBB2 G776insV_G/C mutation are sensitive to the dual-specific epidermal growth factor receptor and ERBB2 inhibitor HKI-272. Cancer Res., 66:6487-6491, 2006. Sivanand et al, A validated tumorgraft model reveals activity of dovitinib against renal cell carcinoma. Science Translational Medicine, 4: 137ra75, 2012.

Smith et al, Cabozantinib in patients with advanced prostate cancer: results of a phase II randomized discontinuation trial. Journal of Clinical Oncology, 31 :412-419, 2013. Sos et al, PTEN loss contributes to erlotinib resistance in EGFR-mutant lung cancer by activation of Akt and EGFR. Cancer Res., 69(8):3256-3261, 2009.

Stabile et al, c-Src activation mediates erlotinib resistance in head and neck cancer by stimulating c-Met. Clin Cancer Res., 19(2):380-392, 2013.

Stephens et al, Lung cancer: intragenic ERBB2 kinase mutations in tumours. Nature, 431:525-526, 2004.

Tai et al, Bruton tyrosine kinase inhibition is a novel therapeutic strategy targeting tumor in the bone marrow microenvironment in multiple myeloma. Blood, 120(9): 1877-87,

2012.

Takezawa et al, HER2 amplification: a potential mechanism of acquired resistance to EGFR inhibition in EGFR-mutant lung cancers that lack the second-site EGFRT790M mutation. Cancer Discovery , 2:922-933, 2012.

Tanaka et al, Frequency of and variables associated with the EGFR mutation and its subtypes. International Journal of Cancer, 126:651-655, 2010.

Tanaka et al, Oncogenic EGFR signaling activates an mTORC2-NF-B pathway that promotes chemotherapy resistance. Cancer Discovery, 1:524-538, 2011.

Thatcher et al., Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo- controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet, 366: 1527-1537, 2005.

Tjin et al, In vitro and in vivo characterization of irreversible mutant-selective EGFR inhibitors that are wild-type sparing. Molecular Cancer Therapeutics, 13: 1468-1479, 2014.

van Riel et al, Comparison of auranofin and aurothioglucose in the treatment of rheumatoid arthritis: a single blind study. Clinical Rheumatology, 3(Suppl l):51-56, 1984.

Vermorken et al, A phase lb, open-label study to assess the safety of continuous oral treatment with afatinib in combination with two chemotherapy regimens: cisplatin plus paclitaxel and cisplatin plus 5-fluorouracil, in patients with advanced solid tumors. Annals of Oncology, 24: 1392-1400, 2013. Viola et al, Cabozantinib (XL 184) for the treatment of locally advanced or metastatic progressive medullary thyroid cancer. Future Oncology, 9: 1083-1092, 2013.

Vogelstein et al, Cancer genome landscapes. Science, 339: 1546-1558, 2013.

Wang et al, Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2. Cancer Cell, 6:251-261, 2004.

Wang et al, Targeting BTK with ibrutinib in relapsed or refractory mantle-cell lymphoma. N

Engl J Med., 369(6):507-516, 2013a.

Wang et al, One-step generation of mice carrying mutations in multiple genes by

CRISPR/Cas-mediated genome engineering. Cell, 153:910-918, 2013b.

Ward et al, Structure- and reactivity -based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor

(EGFR). Journal of Medicinal Chemistry, 56:7025-7048, 2013.

Wei et al, Inhibiting ΤΝΚ dephosphorylation and induction of apoptosis by novel anticancer agent NSC-741909 in cancer cells. Journal of Biological Chemistry, 284: 16948- 16955, 2009.

Wei et al, Oxidative stress in NSC-741909-induced apoptosis of cancer cells. Journal of

Translational Medicine, 8:37, 2010.

Woyach et al, Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib.

New England Journal of Medicine, 370:2286-2294, 2014.

Wu et al, Lung cancer with epidermal growth factor receptor exon 20 mutations is associated with poor gefitinib treatment response. Clinical Cancer Research, 14:4877-4882,

2008.

Wu et al, Prodrug oncrasin-266 improves the stability, pharmacokinetics, and safety of NSC- 743380. Bioorganic & Medicinal Chemistry, 22:5234-5240, 2014.

Xu et al., Epidermal growth factor receptor regulates MET levels and invasiveness through hypoxia-inducible factor- 1 alpha in non-small cell lung cancer cells. Oncogene, 29:2616-2627, 2010.

Yoshizawa et al, HER2 status in lung adenocarcinoma: A comparison of immunohistochemistry, fluorescence in situ hybridization (FISH), dual-ISH, and gene mutations. Lung Cancer, 85:373-378, 2014.

Younes et al, Combination of ibrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for treatment-naive patients with CD20- positive B-cell non-Hodgkin lymphoma: a non-randomised, phase lb study. Lancet Oncology, 15: 1019-1026, 2014. Yu and Riely, Second-generation epidermal growth factor receptor tyrosine kinase inhibitors in lung cancers. Journal of the National Comprehensive Cancer Network, 11 : 161-169, 2013.

Yun et al., The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc. Natl. Acad. Sci. USA, 105:2070-2075, 2008.

Yuza et al, Allele-dependent variation in the relative cellular potency of distinct EGFR inhibitors. Cancer Biology & Therapy, 6:661-667, 2007.

Zhang et al, Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer. Nature Genet, 44(8):852-860, 2012.

Zhou et al, Novel mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature, 462(7276): 1070-1074, 2009.

Zhou et al, Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. Lancet Oncol, 12(8):735-742, 2011.

Zhou et al, High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature, 509:487-491, 2014.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of treating a patient having cancer that exhibits (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression, the method comprising administering a therapeutically effective amount of ibrutinib to the patient.

2. The method of claim 1, wherein the cancer exhibits an EGFR mutation.

3. The method of claim 2, wherein the EGFR mutation comprises a T790M substitution.

4. The method of claim 2, wherein the EGFR mutation comprises a L858R substitution.

5. The method of claim 2, wherein the EGFR mutation comprises a deletion in exon 19 of EGFR.

6. The method of claim 2, wherein the EGFR mutation comprises a G719x substitution.

7. The method of claim 2, wherein the EGFR mutation comprises a L861Q substitution.

8. The method of claim 1, wherein the cancer exhibits HER2 gene amplification.

9. The method of claim 1, wherein the cancer exhibits HER2 protein overexpression.

10. The method of claim 1, wherein the cancer is metastatic, recurrent, or multi-drug resistant.

11. The method of claim 1, wherein the cancer is colorectal, breast, prostate, lung, or pancreatic cancer.

12. The method of claim 1 1, wherein the cancer is non-small cell lung cancer.

13. The method of claim 1, further comprising administering at least a second anticancer therapy to the subject.

14. The method of claim 13, wherein the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, or cytokine therapy.

15. The method of claim 14, wherein the chemotherapy is an NF-KB/STAT-3 inhibitor, an AXL inhibitor, an ALK/MET inhibitor, a RAF/VEGFR inhibitor, an AKT inhibitor, a Src inhibitor, a JAK/STAT-3 inhibitor, or a W T/ -catenin inhibitor.

16. The method of claim 15, wherein the NF-KB/STAT-3 inhibitor is auranofin.

17. The method of claim 15, wherein the AXL inhibitor is SGI-7079 or cabozantinib.

18. The method of claim 15, wherein the ALK/MET inhibitor is crizotinib.

19. The method of claim 15, wherein the RAF/VEGFR inhibitor is sorafenib.

20. The method of claim 15, wherein the AKT inhibitor is MK2206.

21. The method of claim 15, wherein the Src inhibitor is dasatinib.

22. The method of claim 15, wherein the JAK/STAT-3 inhibitor is ruxolitinib.

23. The method of claim 15, wherein the WNT/ -catenin inhibitor is LGK974135.

24. The method of claim 1, wherein the patient is a human.

25. The method of claim 1, wherein the patient is a non-human mammal.

26. The method of claim 1, wherein the patient is treated at least a second time.

27. The method of claim 1, wherein the patient is treated over a period of 1 week to 6 months.

28. The method of claim 1, wherein the patient has previously undergone at least one round of anti-cancer therapy.

29. A method of treating a patient having cancer comprising: (a) selecting a patient determined to comprise a cancer comprising (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression; and

(b) administering a therapeutically effective amount of ibrutinib to the patient.

30. The method of claim 29, wherein selecting a patient comprises obtaining a sample of the cancer and determining whether the cancer comprises (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression.

31. The method of claim 30, further comprising providing a report of the determining.

32. The method of claim 31 , wherein the report is a written or electronic report.

33. The method of claim 31, wherein the report is provided to the patient, a health care payer, a physician, an insurance agent, or an electronic system.

34. The method of claim 29, wherein selecting a patient comprises obtaining results for a test that determines whether the cancer comprises (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression.

35. The method of claim 29, comprising selecting a patient determined to comprise a cancer comprising an EGFR mutation.

36. The method of claim 35, wherein the EGFR mutation comprises a T790M substitution.

37. The method of claim 29, comprising selecting a patient determined to comprise a cancer comprising HER2 gene amplification.

38. The method of claim 29, comprising selecting a patient determined to comprise a cancer comprising HER2 protein overexpression.

39. The method of claim 31, wherein the amino acid present at position 790 of the EGFR protein is determined by mass spectrometry, western blot, ELISA, or sequencing a nucleic acid comprising at least a portion of the protein coding sequence of the EGFR protein.

40. The method of claim 29, wherein the cancer further comprises a L858R EGFR mutation, a deletion in exon 19, a G719x substitution, and/or a L861Q substitution.

41. The method of claim 29, wherein the cancer is metastatic, recurrent, or multi-drug resistant.

42. The method of claim 29, wherein the cancer is colorectal, breast, prostate, lung, or pancreatic cancer.

43. The method of claim 29, further comprising administering at least a second anticancer therapy to the subject.

44. The method of claim 43, wherein the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, or cytokine therapy.

45. The method of claim 29, wherein the patient is a human.

46. The method of claim 29, wherein the patient is a non-human mammal.

47. The method of claim 29, wherein the patient is treated at least a second time.

48. The method of claim 29, wherein the patient is treated over a period of 1 week to 6 months.

49. A method of selecting a drug therapy for a cancer patient comprising:

(a) obtaining a sample of the cancer; (b) determining the presence of (i) a mutation in the EGFR protein expressed in the cancer; (ii) amplification of the HER2 gene in the cancer; or (iii) overexpression of the HER2 protein in the cancer; and

(c) selecting ibrutinib if (i) a mutation is determined to be present in the EGFR protein expressed in the cancer; (ii) the HER2 gene is determined to be amplified in the cancer; or (iii) the HER2 protein is determined to be overexpressed in the cancer.

50. The method of claim 49, further comprising administering a therapeutically effective amount of ibrutinib to the patient.

51. The method of claim 49, comprising selecting ibrutinib if a mutation is determined to be present in the EGFR protein expressed in the cancer.

52. The method of claim 51, wherein the mutation in the EGFR protein is a T790M and/or L858R substitution.

53. The method of claim 49, comprising selecting ibrutinib if the HER2 gene is determined to be amplified in the cancer.

54. The method of claim 49, comprising selecting ibrutinib if the HER2 protein is determined to be overexpressed in the cancer.

55. A composition comprising ibrutinib for use in the treatment of a cancer in a subject, wherein the cancer has been determined to comprise (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression.

56. The composition of claim 55, wherein the EGFR mutation comprises a T790M substitution, a L858R substitution, a deletion in exon 19 of EGFR, a G719x substitution, or a L861Q substitution.

57. The composition of claim 55, wherein the cancer is colorectal, breast, prostate, lung, or pancreatic cancer.

58. The composition of claim 55, further comprising at least a second anticancer therapy.

59. The composition of claim 58, wherein the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, or cytokine therapy.

60. The method of claim 59, wherein the chemotherapy is an NF-KB/STAT-3 inhibitor, an AXL inhibitor, an ALK/MET inhibitor, a RAF/VEGFR inhibitor, an AKT inhibitor, a Src inhibitor, a JAK/STAT-3 inhibitor, or a W T/ -catenin inhibitor.

61. Use of ibrutinib in the manufacture of a medicament for the treatment of a cancer, wherein the cancer has been determined to comprise (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression.