Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57415—Specifically defined cancers of breast
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
  • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
  • G16C20/70—Machine learning, data mining or chemometrics
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/26—Mass spectrometers or separator tubes
  • H01J49/34—Dynamic spectrometers
  • H01J49/40—Time-of-flight spectrometers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
  • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
  • G16C20/20—Identification of molecular entities, parts thereof or of chemical compositions
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
  • G16H10/00—ICT specially adapted for the handling or processing of patient-related medical or healthcare data
  • G16H10/40—ICT specially adapted for the handling or processing of patient-related medical or healthcare data for data related to laboratory analysis, e.g. patient specimen analysis
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
  • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

• This invention relates to methods and systems for predicting whether a cancer patient is likely or not likely to benefit from administration of certain types and classes of drugs, and/or combinations thereof.
• the methods and systems involve using mass spectral data obtained from a blood-based sample of the patient and a computer configured as a classifier operating on the mass spectral data.
• Biodesix, Inc has developed a test known as VeriStrat which predicts whether Non-Small Cell Lung Cancer (NSCLC) patients are likely or not likely to benefit from treatment of Epidermal Growth Factor Receptor (EGFR) pathway targeting drugs.
• NSCLC Non-Small Cell Lung Cancer
• EGFR Epidermal Growth Factor Receptor pathway targeting drugs.
• the test is described in U.S. Patent 7,736,905, the content of which is incorporated by reference herein.
• the test is also described in Taguchi F. et al. 1 , the content of which is also incorporated by reference herein. Additional applications of the test are also described in U.S. Patents 7,858,390; 7,858,389 and 7,867,775, the contents of which are incorporated by reference herein.
• the VeriStrat test is based on serum and/or plasma samples of cancer patients. Through a combination of MALDI-TOF mass spectrometry and data analysis algorithms implemented in a computer, it compares a set of eight integrated peak intensities at predefined m/z ranges with those from a training cohort, and generates a class label for the patient sample: either VeriStrat "good”, VeriStrat "poor”, or VeriStrat "indeterminate.” In multiple clinical validation studies it was shown that patients, whose pre-treatment serum/plasma was VeriStrat "good”, have significantly better outcome when treated with epidermal growth factor receptor inhibitor drugs than those patients whose sample results in a VeriStrat "poor” signature.
• VeriStrat is commercially available from Biodesix, Inc., the assignee of the present invention, and is used in treatment selection for non-small cell lung cancer patients.
• biomarker-based tests are very specific with respect to tumor type and histology, specific interventions, and clinico-pathological factors.
• genetic tests based on tumor tissue such as tests for mutations in the EGFR domain, KRAS mutations, and gene copy number analysis via Fluorescence In-Situ Hybridization (FISH) appear to work only in very specific indications.
• FISH Fluorescence In-Situ Hybridization
• EGFR mutations may give indications for gefitinib response in first line NSCLC cancer with adenocarcinoma, they do not exhibit similar utility for squamous cell carcinoma due to the extreme rarity of these mutations in this type of NSCLC.
• KRAS mutations can be associated with response to cetuximab in colorectal cancer, but attempts to transfer this to NSCLC have been unsuccessful.
• EGFRI EGFR- Inhibitor
• SCCHN head and neck
• VeriStrat test measures the activation of one or more pathways downstream from the growth and survival factors receptors such as EGFR, likely candidate pathways include canonical and non-canonical MAPK (mitogen-activated protein kinase), Akt as well as reactions regulated by PKC (protein kinase C) (see Figure 2).
• MAPK mitogen-activated protein kinase
• Akt as well as reactions regulated by PKC (protein kinase C)
• NF- ⁇ nuclear factor kappa-light-chain-enhancer of activated B-cells
• NF- ⁇ nuclear factor kappa-light-chain-enhancer of activated B-cells
• the VeriStrat test identifies a subset of population with worse prognosis and will predict differential benefit of solid epithelial tumor cancer patient from therapy with therapeutic agents or a combination of therapeutic agents targeting agonists of the receptors, receptors or proteins involved in MAPK pathways or the PKC upstream from or at Akt or ERK/J K/p38 or PKC.
• EGFR inhibitors are the examples of such agents.
• Patients predicted to be likely to benefit from anti- EGFR agents are identified as VeriStrat "good” label; conversely patients predicted as not likely to benefit from anti-EGFR agents are identified with VeriStrat "poor” label.
• the term MAPK (mitogen-activated protein kinase) here is used as a name of at least three related cascades, not of a single enzyme (see Fig. 2).
• VeriStrat "poor” label VeriStrat test is diagnostic for "poor” patients as a subgroup of cancer patients with a poor prognosis. Indeed, the VeriStrat "poor” patients can be considered as having a different disease state from VeriStrat "good” patients.
• VeriStrat "good” label cancer patients having a VeriStrat "good” label are more likely to obtain more benefit from a therapy with therapeutic agent or a combination of therapeutic agents targeting agonists of the receptors, receptors or proteins involved in MAPK pathways; while patients having a VeriStrat "poor” label are not likely to obtain clinical benefit from therapy with such a therapeutic agent; on the other hand, VeriStrat "poor” patients are likely to exhibit benefit from a therapy or combination of therapies that prevents downstream, independent of the receptors, activation of these pathways.
• a method is disclosed of identifying a solid epithelial tumor cancer patient as being likely to benefit from treatment with a therapeutic agent or a combination of therapeutic agents targeting agonists of the receptors, receptors or proteins involved in MAPK pathways or the PKC (protein kinase C) pathway upstream from or at Akt or ERK/JNK/p38 or PKC or not likely to benefit from treatment with the therapeutic agent or the combination of therapeutic agents, comprising the steps of: a) obtaining a mass spectrum from a blood-based sample from the solid epithelial tumor cancer patient; b) performing one or more predefined pre-processing steps on the mass spectrum obtained in step a); c) obtaining integrated intensity values of selected features in said spectrum at one or more predefined m/z ranges after the pre-
• a method for predicting whether a cancer patient is likely to benefit from administration of the combination of a COX2 inhibitor and a EGFR inhibitor comprising the steps of:
• step b) performing one or more predefined pre-processing steps on the mass spectrum obtained in step a);
• step c) obtaining integrated intensity values of selected features in said spectrum at one or more predefined m/z ranges after the pre-processing steps on the mass spectrum in step b) have been performed; and d) using the values obtained in step c) in classification algorithm using a training set comprising class-labeled spectra produced from blood-based samples from other solid epithelial tumor patients to identify the patient as being either likely or not likely to benefit from treatment by administration of a combination of a COX2 inhibitor and a EGFR inhibitor.
• Figure 1 is a flow chart showing the steps for performing the VeriStrat test on a blood-based sample of a patient.
• Figure 2 is a chart showing selected signal transduction pathways in human cells.
• FIG. 3 is a representation of selected biological activity of serum amyloid A (SAA) isoforms and its possible role in cancer progression and therapy resistance.
• SAA serum amyloid A
• Figure 4 is a representation of EGFR signal transduction pathways, their interactions, and possible points of activation by SAA
• Figure 5 is a representation of ErbB family growth factor receptors, including EGFR, and their inhibitors, from Yarden Y, Shilo BZ. SnapShot: EGFR signaling pathway. Cell 2007; 131 : 1018
• Figure 6 is a forest plot showing the hazard ratios between VeriStrat Good and VeriStrat Poor patients by treatment arm for all published VeriStrat analyses.
• Figure 7 is a representation of Kaplan-Meier plots of overall survival (OS) of patents receiving different chemotherapy treatments and the VeriStrat labels ("good” and “poor") for such patients.
• Figure 8 are plots of growth of gefitinib sensitive cell line HCC4006 and gefitinib resistant cell line A549 in VeriStrat “poor” and VeriStrat “good” serum in presence of different concentrations of gefitinib.
• solid epithelial tumor includes but is not necessarily limited to NSCLC, SCCHN, breast cancer, renal cancer, pancreatic cancer, melanoma and colorectal cancer (CRC).
• the term "therapeutic agent or a combination of therapeutic agents targeting agonists of the receptors, receptors or proteins involved in MAPK pathways or the PKC upstream from or at Akt or ERK/JNK/p38 or PKC” includes but is not limited to therapeutic agent or agents targeting erbB receptors family, including EGFR (HER1), HER2, HER3, and HER4, VEGF Receptor (VEGFR2), Hepatocyte growth factor receptor (HGFR or MET), G-protein coupled receptors, Insulin-like Growth Factor (IGF) receptors, VEGF, Growth Factors such as TGFa and EGF, and any other protein upstream from or at Akt, or ERK/JNK7 p38 MAPK or the PKC pathways.
• EGFR HER1
• HER2, HER3, and HER4 VEGF Receptor
• HGFR or MET Hepatocyte growth factor receptor
• IGF Insulin-like Growth Factor
• VEGF Growth Factors
• therapeutic agent or a combination of therapeutic agents targeting agonists of the receptors, receptors or proteins involved in MAPK pathways or the PKC pathway upstream from or at Akt or ERK JNK/p38 or PKC includes known therapeutic agents, as well as therapeutic agents targeting these proteins that are yet to be discovered or disclosed.
• combination of therapeutic agents includes any combination of therapeutic agents, whether they have already been used in combination for treatment of solid epithelial tumors or not. It should be noted that even where an agent is identified as an inhibitor of a particular protein or pathway, such a classification is not meant to represent a description of its mechanism of action because the mechanism of action of many of these agents is not completely understood. As an example, but not as meant as an exhaustive list, these therapeutic agents include:
• TKIs Tyrosine Kinase Inhibitors
• TKIs may target specific molecular receptors, such as the Epidermal Growth Factor receptor (EGFR), and may also target multiple receptors (called “multiple kinase inhibitors"). These include but are not limited to erlotinib, gefitinib, sorafenib, sunitinib, pazopanib, imatinib, nilotinib, lapatinib.
• EGFR Epidermal Growth Factor receptor
• multiple kinase inhibitors include but are not limited to erlotinib, gefitinib, sorafenib, sunitinib, pazopanib, imatinib, nilotinib, lapatinib.
• Antibody-based inhibitors include Cetuximab (anti-EGFR), Panitumumab
• HGFR or MET inhibitors are a potent inhibitor of MET and VEGFR2.
• MET inhibitor includes, but is not limited to: AMG 208, AMG 102, ARQ 197, AV-299, MetMab, GSK 1363089 (XL880), EMD 1214063, EMD 1204831, MGCD265, Crizotanib (PF-02341066), PF-04217903, MP470.
• COX2 inhibitors include, but is not limited to: selective COX2 inhibitors: celecoxib, rofecoxib, valdecoxib, lumiracoxib.
• NSAIDs non-steroidal anti-inflammatory drugs
• COX1 and COX2 include ibuprofen, aspirin, indomethacin, and sulindac.
• ibuprofen such as ibuprofen, aspirin, indomethacin, and sulindac.
• Such drugs have also been shown to suppress NF- ⁇ activation.
• NF- ⁇ inhibitors include, but is not limited to Arsenic trioxide (ATO), thalidomide and its analogues, resveratrol.
• ATO Arsenic trioxide
• COX2 inhibitors also have an inhibitory effect on the NF-kB pathway. Therefore, NSAIDs, such as ibuprofen, aspirin, indomethacin, and sulindac were also shown to suppress NF-kB activation and as such are considered NF-kB inhibitors.
• VEGF inhibitor includes, but is not limited to: Bevacizumab, Cedaranib, Axitinib, Motesanib, BIBF 1 120, Ramucirumab, VEGF Trap, Linifanib (ABT869), Tivozanib, BMS-690514, XL880, Sunitinib, Sorafenib, Brivanib, XL- 184, Pazopanib.
• targeted therapy refers to a type of treatment that uses drugs or other substances, such as monoclonal antibodies or small-molecule inhibitors of specific enzymes, to identify and attack specific molecules, such as receptors.
• drugs or other substances such as monoclonal antibodies or small-molecule inhibitors of specific enzymes, to identify and attack specific molecules, such as receptors.
• EGFR-TKIs erlotinib, gefitinib
• cetuximab cetuximab
• bevacizumab etc.
• non-targeted chemotherapy refers to a therapy interfering with rapidly dividing cells either by interfering with DNA (such as alkylating agents, e.g. cisplatin , carboplatin, oxaliplatin or antimetabolites, e.g. 5-fluoracil or pemetrexed, or topoisomerase inhibitors, such as irinotecan) or interfering with cell division (such as vinorelbine, docetaxel, paclitaxel).
• DNA such as alkylating agents, e.g. cisplatin , carboplatin, oxaliplatin or antimetabolites, e.g. 5-fluoracil or pemetrexed, or topoisomerase inhibitors, such as irinotecan
• cell division such as vinorelbine, docetaxel, paclitaxel.
• prognostic refers to a factor or a measurement that is associated with clinical outcome in the absence of therapy or with the application of standard therapy. It can be thought of as a measurement of a natural history of the disease.
• predictive is a factor or a measurement which is associated with benefit or lack of benefit from a particular therapy.
• a predictive factor implies a differential benefit from the therapy that depends on the status of the predictive marker 2
• disease state means a specific sub-type of the diagnosed condition that can be characterized by differential prognosis and/or differential response to therapy and/or specific molecular and/or metabolic characteristics.
• the VeriStrat test is based on a signature obtained from the mass spectral data of a serum sample, it is able to measure general factors relating to cancer as opposed to most current biomarker-based tests. This fact allows new practical applications for the selection of treatment using the VeriStrat test, which are discussed below.
• the VeriStrat test results in a similar separation of survival curves between patients identified as VeriStrat "good” and patients identified as VeriStrat "poor” regardless of the mechanism of action of EGFR inhibition.
• VeriStrat test used patient sample sets that were treated with the small molecule EGFR-tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva), that inhibit the receptor by blocking the ATP-binding site of the enzyme 1 .
• EGFR-tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva)
• Iressa small EGFR-tyrosine kinase inhibitors gefitinib
• Tarceva erlotinib
• the VeriStrat test shows similar separation between patients identified as VeriStrat "good” and patients identified as VeriStrat "poor” across clinico-pathological characteristics.
• the VeriStrat test can be used in patients whose tumor is an adenocarcinoma, as well as for patients whose tumor is a squamous cell carcinoma..
• the VeriStrat test shows separation between patients identified as VeriStrat "good” and patients identified as VeriStrat “poor” in a variety of solid epithelial tumors. We observed this in NSCLC, squamous cell cancer of the head and neck (SCCHN), and CRC 3 .
• the separation of survival curves by the VeriStrat test classification of in patients treated with non-targeted chemotherapy varies depending on details of the population, intervention type, and tumor type. There is evidence for separation in some non-targeted chemotherapy-treated sets, while the absence of separation in the others. There was also a strong separation seen in placebo arms, i.e., no intervention, indicating that the VeriStrat test has a prognostic component.
• the forest plot of Figure 6 summarizes data from all analysis of the VeriStrat test published or presented to date. It shows the hazard ratio (HR) for overall survival between VeriStrat "good” and VeriStrat “poor” patients for each treatment arm studied. The data can be seen to fall into groupings depending on treatment type. The range of hazard ratios obtained illustrates that VeriStrat is indeed indicative of better or worse outcome as a result of particular types of treatment, and hence has predictive power.
• HR hazard ratio
• VeriStrat test shows a separation with a Hazard ratio between
• EGFRI EGFR inhibitor
• small molecule TKIs erlotinib, gefitinib
• antibody (receptor) inhibitor based EGFRI s e.g. cetuximab.
• histological type e.g. adeno carcinoma, and squamous cell carcinoma
• organ e.g. NSCLC, SCCHN, and CRC.
• genomic marker e.g. EGFR mutation status or KRAS status.
• VeriStrat has a strong prognostic component exhibited by a separation between VeriStrat poor and VeriStrat good subgroups in the absence of treatment.
• VeriStrat defines a novel disease state of clinical significance (worse outcome) in solid epithelial tumors.
• the observed phenomena allow for some tentative conclusions on the molecular state of VeriStrat "poor” tumors: As EGFRIs are not effective in this class of patients, and as the effect is the same for both TKIs and antibody-based therapies, it is likely that in VeriStrat "poor” subjects, pathways below the receptors and the tyrosine-kinase domains are different from VeriStrat "good” subjects, i.e. upregulated. As we observe no correlation with KRAS mutation status, we further conclude that the affected pathway is below RAS.
• VeriStrat test measures the activation of one or more pathways downstream from the receptors of EGF; likely candidate pathways include canonical and non-canonical MAPK, PI3K/Akt as well as reactions regulated by PKC (see Figure 2 at 200A and 200B).
• the VeriStrat test identifies a subset of population with worse prognosis (VeriStrat "poof's) and will predict solid epithelial tumor cancer patient benefit from therapy with therapeutic agents or a combination of therapeutic agents targeting agonists of the receptors, receptors or proteins involved in MAPK pathways or the PKC (protein kinase C) upstream from or at Akt or ERK JNK/p38 or PKC.
• EGFR inhibitors are the examples of such agents.
• Patients predicted to be likely to benefit from anti-EGFR agents are identified as VeriStrat "good” label; conversely patients predicted as not likely to benefit from anti-EGFR agents are identified with VeriStrat "poor” label.
• VeriStrat "poor” patients are not likely to obtain clinical benefit from therapy with such a therapeutic agent targeting at the receptors activating MAPK pathways; on the other hand, VeriStrat "poor” patients are likely to obtain clinical benefit from therapy or combination of therapies that prevents downstream, independent of the receptors, activation of these pathways.
• MAPK mitogen-activate protein kinase
• the VeriStrat test is diagnostic for "poor” patients as a subgroup of cancer patients with a poor prognosis.
• the invention can be considered as a method of identifying a solid epithelial tumor cancer patient as being likely to benefit from treatment with a therapeutic agent or a combination of therapeutic agents targeting agonists of the receptors, receptors or proteins involved in MAPK pathways or the PKC upstream from or at Akt or ERK/JNK/p38 or PKC or not likely to -benefit from treatment with the therapeutic agent or the combination of therapeutic agents, comprising the steps of:
• step b) performing one or more predefined pre-processing steps on the mass spectrum obtained in step a) (e.g., background subtraction, noise estimation, normalization and spectral alignment);
• step b) obtaining integrated intensity values of selected features in said spectrum at one or more predefined m/z ranges (and preferably the m/z ranges described below corresponding to the m/z peaks set forth in Table 1 below) after the pre-processing steps on the mass spectrum in step b) have been performed;
• step d) using the values obtained in step c) in classification algorithm (e.g., K- nearest neighbor) using a training set comprising class-labeled spectra produced from blood-based samples from other solid tumor patients to identify the patient as being either likely or not likely to benefit from treatment with the therapeutic agent or the combination of therapeutic agents.
• classification algorithm e.g., K- nearest neighbor
• COX2 inhibitors e.g. celecoxib or rofecoxib
• EGFR-Is as a treatment regime may overcome the resistance of patients having a VeriStrat "poor” signature to EGFR-Is.
• the VeriStrat test may thus be used as an indicator to prescribe combination therapy including COX2 inhibitors and EGFR-Is.
• VeriStrat "poor" signature is believed to be associated with a specific activation of NF-KB, therefore the test can be used to select patients benefiting most from the NF- ⁇ inhibitors, and, thus, to reduce unnecessary treatment and associated morbidities.
• VeriStrat "poor" signature is believed to be associated with little clinical benefit from specific non-targeted chemotherapies, specifically, the agents interfering with DNA replication and gene expression, such as cisplatin, gemcitabine or pemetrexed, possibly due to the involvement of NF-kB factor in this processes.
• addition of the agents that (1) prevent downstream, independent from the receptors, activation of the MAPK pathways, such as COX2 inhibitors or (2) minimize the inflammatory host-responses, or addition of other targeted agents, that prevent cross-talk pathway activation, can overcome the resistance to the targeted agents.
• the methods for testing a blood-based sample of an solid epithelial tumor cancer patient in order to select such patient for treatment with certain therapeutic agent or a combination of therapeutic agents, such as agents targeting agonists of the receptors, receptors or proteins involved in MAPK pathways or the PKC pathway upstream from or at Akt or ERK/JNK/p38 or PKC in accordance with the present disclosure is illustrated in flow chart form in Figure 1 as a process 100.
• a serum or plasma sample is obtained from the patient.
• the serum samples are separated into three aliquots and the mass spectroscopy and subsequent steps 104, 106 (including sub-steps 108, 1 10 and 1 12), 1 14, 1 16 and 1 18 are performed independently on each of the aliquots.
• the number of aliquots can vary, for example there may be 4, 5 or 10 aliquots, and each aliquot is subject to the subsequent processing steps.
• the sample (aliquot) is subject to mass spectroscopy.
• a preferred method of mass spectroscopy is matrix assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectroscopy, but other methods are possible.
• MALDI matrix assisted laser desorption ionization
• TOF time of flight
• Mass spectroscopy produces data points that represent intensity values at a multitude of mass/charge (m/z) values, as is conventional in the art.
• the samples are thawed and centrifuged at 1500 rpm for five minutes at four degrees Celsius.
• the serum samples may be diluted 1 : 10, or 1 :5, in MilliQ water. Diluted samples may be spotted in randomly allocated positions on a MALDI plate in triplicate (i.e., on three different MALDI targets).
• Mass spectra may be acquired for positive ions in linear mode using a Voyager DE-PRO or DE-STR MALDI TOF mass spectrometer with automated or manual collection of the spectra. Seventy five or one hundred spectra are collected from seven or five positions within each MALDI spot in order to generate an average of 525 or 500 spectra for each serum specimen. Spectra are externally calibrated using a mixture of protein standards (Insulin (bovine), thioredoxin (E. coli), and Apomyglobin (equine)).
• the spectra obtained in step 104 are subject to one or more predefined pre-processing steps.
• the pre-processing steps 106 are implemented in a general purpose computer using software instructions that operate on the mass spectral data obtained in step 104.
• the pre-processing steps 106 include background subtraction (step 108), normalization (step 1 10) and alignment (step 1 12).
• the step of background subtraction preferably involves generating a robust, asymmetrical estimate of background in the spectrum and subtracts the background from the spectrum.
• Step 108 uses the background subtraction techniques described in U.S. patent 7,736,905 B2 and U.S. patent application publication 2005/0267689, which are incorporated by reference herein.
• the normalization step 1 10 involves a normalization of the background subtracted spectrum.
• the normalization can take the form of a partial ion current normalization, or a total ion current normalization, as described in U.S. Patent 7,736,905.
• Step 1 12 aligns the normalized, background subtracted spectrum to a predefined mass scale, as described in U.S. 7,736,905, which can be obtained from investigation of the training set used by the classifier.
• step 1 14 of obtaining values of selected features (peaks) in the spectrum over predefined m/z ranges.
• the normalized and background subtracted amplitudes may be integrated over these m/z ranges and assigned this integrated value (i.e., the area under the curve between the width of the feature) to a feature.
• the integration range may be defined as the interval around the average m/z position of this feature with a width corresponding to the peak width at the current m/z position.
• values are obtained at eight of these m/z ranges shown in Table 1 below, and optionally at all 12 of these ranges. The significance, and methods of discovery of these peaks, is explained in the U.S. patent 7,736,905.
• the values obtained at step 114 are supplied to a classifier, which in the illustrated embodiment is a K-nearest neighbor (KNN) classifier.
• KNN K-nearest neighbor
• the classifier makes use of a training set of class labeled spectra from a multitude of other patients (which may be NSCLC cancer patients, or other solid epithelial cancer patients, e.g., HNSCC, Breast Cancer).
• the application of the KNN classification algorithm to the values at 1 14 and the training set is explained in U.S. patent 7,736,905.
• Other classifiers can be used, including a probabilistic KNN classifier or other classifier.
• the classifier produces a label for the spectrum, either "good",
• steps 104-1 18 are performed in parallel on the three separate aliquots from a given patient sample (or whatever number of aliquots are used).
• a check is made to determine whether all the aliquots produce the same class label. If not, an undefined result is returned as indicated at step 122. If all aliquots produce the same label, the label is reported as indicated at step 124.
• steps 106, 1 14, 1 16 and 1 18 are typically performed in a programmed general purpose computer using software coding the pre-processing step 106, the obtaining of spectral values in step 1 14, the application of the - classification algorithm in step 1 16 and the generation of the class label in step 1 18.
• the training set of class labeled spectra used in step 1 16 is stored in memory in the computer or in a memory accessible to the computer.
• the method and programmed computer may be advantageously implemented at a laboratory test processing center as described in our prior patent application publication U.S. patent 7,736,905.
• VeriStrat measures the intensity of MALDI-TOF MS peaks from serum or plasma.
• the VeriStrat signature consists of 8 mass spectral peaks described in Table 1, below.
• the classification is performed by estimating an intensity, i.e., a feature value, by integrating a sample's mass spectrum over pre- prescribed m/z ranges (see above listing and Table 1), and relating the observed set of 8 feature values to those from the training samples using a 7 nearest neighbor classification algorithm. This procedure uses the feature values in a non-linear combination, and does not allow for a definition of a one-dimensional score. Attempts to generate a score function from linear combinations of feature values have always been unsuccessful, and have always lead to worse performance. It appears that all or most of these eight features are useful in generating clinical utility.
• the peak at 1 1445 is another SAA isoforms related to a sequence of truncations from the C-terminus of the parent SAA protein.
• SAA is a highly conserved sequence through evolution 17 , and the dramatic increase of SAA expression in response to infection, trauma or pathological processes.
• the exact biological functions of the SAA family are still not fully understood.
• SAA is involved in lipid transport and metabolism as a component of HDL, and probably plays a protective role in acute-phase of a disease l8 , while in chronic conditions SAA may become an adverse factor.
• Sustained high expression of SAA leads to amyloid A amyloidosis in some diseases, such as rheumatoid arthritis 19 .
• the range of clinically important function of SAA proteins is much broader, and includes implication in chronic inflammation and carcinogenesis. The latter two are closely related and are discussed in detail in the reviews, of Vlasova and Moshkovskii 20 and Malle et al 21 .
• Involvement of SAA in carcinogenesis can be attributed to its multifaceted biological activity: involvement in inflammation, including supporting chronic processes via pro-inflammatory gene expression activation and cytokine regulation, participation in extracellular matrix degradation, anti-apoptotic properties, and activation of specific pathways, including mitogen-activated protein kinase (MAPK), known to be intricately involved in carcinogenesis.
• MAPK mitogen-activated protein kinase
• SAA is shown to be able to act as extracellular matrix (ECM) adhesion protein 22 and to induce matrix metalloproteiniases (MMPs) l8 , 23 , which play important role in ECM degradation and remodeling, and are associated with the tumorogenesis, metastases and tumor invasion. 24 , 25 .
• ECM extracellular matrix
• MMPs matrix metalloproteiniases
• Immune-related functions of SAA are defined by its cytokine-like activity. It can stimulate production of IL-8, TNF-a and IL- ⁇ ⁇ 26 , 27 (which, probably, induces a positive feedback for the SAA expression), as well as IL-12 and 11-23, which play important role in cell-mediated immune response 28 . It has also been shown that SAA can activate PI3K and p38 MAPK.
• Involvement of SAA in regulation of inflammation can be associated with its ability to induce COX2 expression concurrently with activation of NF- ⁇ and MAPK pathways. 29 , 30 .
• the principal interrelation of cancer and inflammation is a subject of numerous studies and reviews 31"37 .
• the big body of recent data indicates that SAA may play an essential role as one of the mediators between the two processes, because of its ability to activate critical inflammatory and carcinogenic pathways, such as canonical and non-canonical MAPK pathways and of transcriptional factor NF-KB and, probably, participate in their cross-talk.
• the elevated levels of SAA, associated with VeriStrat signature can be a used as a useful method of measuring activation of the pathways.
• the NF-KB transcription factor is known to be constitutively activated in a large number of epithelial and hematologic malignances and is considered to be essential for promoting inflammation-associated cancer 38 ; 3 5 40 5 by regulating anti- and pro-apoptotic target genes, matrix-metal loprotease expression, angiogenesis and cell cycle 41 .
• NF- ⁇ can also exert pro-apoptotic genes activity and can cooperate with tumor suppressor p53 to induce apoptosis. 42 .
• the actual effect is dependent of the stimulus, cell-type, and the subunit involved 43 .
• NF-KB is probably one of the main links between inflammation and cancer because of its association with induction of pro-inflammatory cytokines, such as IL-6 and TNF-a, and chemokines, including MMPs and COX-2 35 , 45 , 46 .
• NF- ⁇ activation can be induced by EGF: EGF stimulation prevents death receptor induced apoptosis trough NF- B activation.
• COX-2 over-expression is observed in broad range of pre-malignant, malignant and metastatic human epithelial cancers 47 , including lung cancer 48 .
• COX2 mediates, via prostaglandin E2 (PGE2), cell proliferation, angiogenesis, apoptosis, and cell migration, and also trans-activates tumorgenic signaling of mitogen- activated protein kinase MAPK cascade 49 , 50 .
• PGE2 prostaglandin E2
• COX2 trans-activates MAPK via Erk activation 49 , 92
• EGF epidermal growth factor
• the mitogen-activated protein kinase (MAPK) cascade plays a crucial role in normal cell biology, as well as in cancer development, because it transduces growth- stimulatory signals from activated growth factors receptors.
• the MAPK signal transduction is often initiated by binding of one of the growth factors to the membrane receptor tyrosine kinase receptor (RTK), leading to the engagement of Raf, MEK and extracellular- signal regulated kinase (ERK) kinases.
• RTK membrane receptor tyrosine kinase receptor
• ERK extracellular- signal regulated kinase
• SAA functionally binds several receptors in various epithelial cells, and this binding can exert downstream activation of both NF- ⁇ and MAPK pathways, that are described above and can lead to the resistance of VeriStrat "poor” patients to the specific treatments (as also discussed above).
• FPRL receptors are expressed in various cells including hepatocytes intestinal epithelium 54 , and lung 55 .
• SAA interacts with FPRL 1- one of the classic G- protein coupled receptor— and triggers signaling networks, essential for regulation of cell function and epithelial proliferation and/ or apoptosis. Binding of SAA to FPRL1 , leads to activation and induction of interleukins. Involvement of FPRL activates protein kinase C (PKC) and the transcriptional factor NF- ⁇ pathway 30 , which is associated with inhibition of apoptosis and progression of cancer. 56 , 57 , 41 .
• PKC protein kinase C
• binding of SAA to FPRL1 leads to apoptosis rescue of neutrophils and rheumatoid syniviocytes, which is mediated by phosphorylation of MAPK ERK 1/2, PI3K/Akt signaling, as well as STAT3 activation and release of intracellular Ca 2+ 58 , 59 ' 60 , thereby promoting cell proliferation and survival.
• SR-BI The scavenger receptor B-I (SR-BI) was identified as a high density lipoprotein receptor, mediating selective cholesterol uptake. 61 SR-BI is expressed most abundantly in steroidogenic tissues and liver, but also was upregulated in macropages and monocytes during inflammation; high SR-BI expression has been demonstrated in lipid-laden macrophages in human atherosclerotic lesion, also characterized by SAA presence. SAA was shown to promote cellular cholesterol efflux mediated by SR-BI 62 .
• SR-BI Human acute monocytic leukemia cell line
• RAGE Receptor for Advanced Glycation Endproducts
• RAGE advanced glycation end product
• TLR4 toll- like receptors
• TLR4 was found to be expressed is some human cancer cells 68 , 69 .
• lung cancer activation of TLR4 was shown to promote production of immunosuppressive cytokines TGF-beta, proangiogenic chemokine IL- 8, and VEGF.
• Increased VEGF and IL-8 secretion is associated with p38MAPK activation.
• Activation of TLR4 by SAA required phosphorylation of p42/44 and p38 MAPK 71 .
• TLR2 was also shown to be a functional receptor for SAA.
• HeLa cells expressing TLR2 responded to SAA with potent activation of NF- ⁇ ; SAA stimulation led to increased phosphorylation of ERK1/2 (P-ERK1/2), p38 MAPK (P- p38), and JNK (P-JNK) MAPKs and accelerated ⁇ (NFKB inhibitor) degradation in TLR2-HeLa cells 72 .
• Stimulation of NF- ⁇ as result of a specific activation by SAA was demonstrated in macropahges.
• Fig. 3 A simplified scheme of possible SAA interactions and its biological effects in cancer development and therapy resistance is presented in Fig. 3.
• the biological functions of SAA can be viewed in light of cross-talk of multiple pathways, triggered by interaction of SAA with various receptors, which eventually converge on activation of at least one of major MAPK pathways: ERK , p38 and JNK, 21 , 41 and/or on NF-KB activation.
• MAPK major MAPK pathways
• EGFR is a tyrosine kinase receptor (TKR) activating several major downstream signaling pathways, including Ras-Raf-Mek and the pathway consisting of phosphoinositide 3-kinase (PI3K), Akt, and PKC.
• PI3K phosphoinositide 3-kinase
• Akt Akt
• PKC phosphoinositide 3-kinase
• SAA may be able to activate these pathways independently of tyrosine-kinase receptor (shown by the wide arrows).
• Overexpression and/or constitutive activation of EGFR is associated with numerous cancers, including brain, breast, intestinal and lung. Alteration of the components of the cascade lead to the activation of the pathways and are considered to be related to cancer induction and progression, e.g. activating mutations of EGFR kinase domain (in non-smokers) or of KRAS (in smokers) are associated with early development of lung cancer 74 , 75 . Ras protein is constitutively activated in about 25%
• NF- ⁇ activation NF- ⁇ activation and subsequent resistance to apoptosis 81 , 19 .
• Inhibition of chemotherapy (gemcitabine) -induced NF- ⁇ activation was shown to restore sensitivity of NSCLC cell line to chemotherapy-induced apoptosis 82 , 81 .
• NF- ⁇ was shown to be associated with sensitivity to chemotherapy, e.g. it has been suggested necessary for paclitaxel-induced cell death 82 .
• NF-KB inhibitors such as arsenic trioxide, curcumin, thalidomide were subject of numerous clinical trials.
• NF- ⁇ inhibitors also enhance the chemotherapy- induced apoptosis of normal hematopoietic progenitors, the use of NF- KB inhibitors as adjuvants in chemotherapy could delay bone marrow recovery. It should be considered that because NF- ⁇ has a critical role in the activation of innate and adaptive immune responses, long-term use inhibitors is likely to be associated with a risk of immunodeficiency 41 .
• VeriStrat "Poor” signature is, in fact, associated with a specific activation of NF-/cB, this signature could be used to select patients benefiting most from the NF- ⁇ inhibitors, and, may reduce unnecessary treatment and associated morbidities.
• EGFR and HER2 belong to the epidermal growth factor receptor (EGFR) family consisting of four members (EGFR (HER1), erbB4 (HER4), erbB3 (HER3), and erbB2 (HER2)). Since the majority of epithelial cancers exhibit abnormal activation of the epidermal growth factor receptor (EGFR) and HER2 receptor, specific inhibition of these receptors became a strategy of the targeted cancer therapy and are the subject of numerous studies.
• EGFR receptors exist in a conformation that suppresses kinase activity. Ligand binding initiates a conformational alteration that unmasks a "dimerization loop", triggering receptor dimerization. These transitions are relayed across the plasma membrane to activate kinase domains. Variations on this activation scheme are found in the ErbB family. ErbB-3 is not a functional kinase, but is able to transactivate dimer partners, whereas HER2/ErbB-2 is a ligand-less oncogenic receptor "locked" in the active conformation.
• Ras protein is constitutively activated in about 25% of tumors, causing mitogeneic signaling independent of upstream .
• tyrosine kinase inhibitors are currently used in clinical practice for a variety of solid tumors, including two small molecule EGFR tyrosine kinase inhibitors - erlotinib and gefitinib, as well as the dual EGFR and HER2 inhibitor lapatinib. Also approved for clinical applications are the humanized monoclonal anti-HER2 antibody trastuzumab and two anti-EGFR antibodies - cetuximab and panitumumab.
• Trans-activation of the pathways was suggested as one of the mechanism of resistance in multiple studies.
• IGF-1R insulin-like growth factor-I receptor
• An alternative downstream signaling, in particular through Akt activation, such as by an oncogenic PIK3CA or by other RTK has been described as one of the mechanisms of resistance to TKIs in NSCLC.
• Indirect action of S AA may be explained by acting via FPRL receptor, leading to the release of interleukins 116, and 118, which in turn, reacting with G-protein coupled receptor, activate PKC.
• Activation of PKC leads to cell proliferation and vasopermeablity, and to activation of MEK in the MAPK pathway ). Besides, it induces VEGF expression.
• SAA is a ligand for TRL4 in lung endothelial cells and macrophages. Ligation of TLRs expressed in tumor cells reportedly also increases VEGF levels .
• COX2 overexpression in lung cancer was first reported by Huang et al 87 , it is observed in approximately 70% of adenocarcinomas 88 , and was confirmed in many other studies.
• EGF epidermal growth factor
• MAPK mesenchymal growth factor
• PGE2 prostaglandin E2
• NSCLC G-protein coupled receptor and protein kinase C
• COX2 inhibitors were shown to inhibit NF- ⁇ pathway: celecoxib conferred its effect through suppression if Akt and IKK.
• celecoxib was shown to suppress NF- ⁇ , as well as TNF- induced JNK, p38 MAPK, and ERK activation through inhibition of IKK and Akt activation, leading to down-regulation of synthesis of COX-2 and other genes needed for inflammation, proliferation, and carcinogenesis 46 , 90 .
• Other NSAIDs including aspirin and ibuprofen, were shown to act by suppressing IKK activation and ⁇ degradation. Combined, these consideration provided strong rationale for addition of COX2 to standard cancer therapy.
• VeriStrat "poor” serum can cause a biological effect in tumor cells, in particular, it can increase resistance of cells to gefitinib in drug-sensitive cell lines.
• the experiments were carried out on the gefitinib sensitive line HCC4006 (it has EGFR exon 19 deletion) and the resistant line A549 (EGFR wild type).
• Human sera were from stage IIIB/IV NSCLC patients and characterized as VS 'good" or "poor”. Pools were created by combining sera within each classification and used in growth inhibition assays. Cells were plated (10 replicates/drug concentration; 2,000 cells/well) using two media compositions; RPMI with 10% Good serum or RPMI with 10% Poor serum. After 24 hours, gefitinib was added and the plates were incubated for 6 days. The MTT assay was used to measure growth inhibition. The results are presented in Table 2 below and in Fig. 8.
• Figure 8 depicts graphs showing the growth of gefitinib sensitive cell line HCC4006, and gefitinib resistant cell line A549 in VeriStrat Poor and VeriStrat Good serum in presence of different concentrations of gefitinib.
• % Control was calculated from the ratio of the absorbance at the given concentration of gefitinib relative to the mean absorbance in the absence of the drug in the corresponding growth medium. Error bars correspond to standard deviation of the normalized measurements.
• VeriStrat "poor” serum but no significant change in resistant tumor cells.
• VeriStrat "poor" signature is associated with poor response to some non-targeted therapies, while not to others.
• VeriStrat classification is likely to be correlated with outcomes in chemotherapies, that interfere with DNA replication or with transcription of genes regulated by NF-kB (such as cisplatin, gemcitabine, etc), however concrete areas of VeriStrat usability in non-targeted therapies need to be determined experimentally.
• VeriStrat test provides a method for predicting whether a cancer patient is not likely to benefit from administration of certain non-targeted chemotherapy regimes, such as one interacting with replication of DNA and/or activation of genes regulated by NF-kB transcription factor comprising: conducting the VeriStrat test on a sample ( Figure 1) and if the result is "poor" class label generating a result that the patient is not likely to benefit.
• VeriStrat signature may correlate with the cancer primary resistance to radiation therapy, and with patient's response to chemotherapy.
• NF-KB inhibitors such as arsenic trioxide, curcumin, thalidomide are being evaluated in clinical trials as anti-cancer agents.
• their usability can be limited by the absence of biomarkers of response to these agents, as well as by their side effects.
• VeriStrat can be useful as biomarker of the elevated activation of NF-kB, hence, for selection of patients (presumably, VeriStrat "poor") potentially benefiting most from NF-kB inhibitors.
• the present invention encompasses additional uses of the VeriStrat test of Figure 1.
• the VeriStrat test will predict cancer patient benefit from therapy with any agent or combination of therapeutic agents, which is targeting agonists of the receptors, receptors or proteins involved in the MAPK pathways or the PKC (protein kinase C) pathway upstream from or at Akt or ERK/JNK/p38 or PKC.
• the magnitude of prediction will depend on a particular drug or drugs combination.
• the VeriStrat test will not predict effects of drugs targeting downstream regulations.
• the invention can be considered as a method of identifying a solid epithelial tumor cancer patient as being likely to benefit from treatment with a therapeutic agent or a combination of therapeutic agents targeting agonists of the receptors, receptors or proteins involved in MAPK pathways or the PKC pathway upstream from or at Akt or ERK/JNK/p38 or PKC or not likely to benefit from treatment with the therapeutic agent or the combination of therapeutic agents, comprising the steps of:
• step b) performing one or more predefined pre-processing steps on the mass spectrum obtained in step a) (e.g., background subtraction, normalization and spectral alignment);
• step b) obtaining integrated intensity values of selected features in said spectrum at one or more predefined m/z ranges (and preferably the m/z ranges described previously corresponding to the m/z peaks set forth in Table 1) after the pre-processing steps on the mass spectrum in step b) have been performed;
• step d) using the values obtained in step c) in classification algorithm (e.g., K- nearest neighbor) using a training set comprising class-labeled spectra produced from blood-based samples from other solid epithelial tumor patients to identify the patient as being either likely or not likely to benefit from treatment with the therapeutic agent or the combination of therapeutic agents.
• classification algorithm e.g., K- nearest neighbor
• the addition of targeted agents blocking the downstream activation of MAPK pathway to EGFR-Is may overcome the resistance of patients having a VeriStrat "poor" signature to EGFR-Is.
• COX2 inhibitors colecoxib or rofecoxib
• EGFR-Is EGFR-Is
• the VeriStrat test may thus be used as an indicator to prescribe combination therapy including COX2 inhibitors and EGFRIs.
• the method for predicting whether a cancer patient is likely to benefit from administration of a COX2 inhibitor and a EGFRI comprises the steps of a) obtaining a mass spectrum from a blood-based sample from the cancer patient; b) performing one or more predefined pre-processing steps on the mass spectrum obtained in step a) (e.g., background subtraction, normalization and spectral alignment); c) obtaining integrated intensity values of selected features in said spectrum at one or more predefined m/z ranges (and preferably the m/z ranges described previously corresponding to the m/z peaks set forth in Table 1) after the pre-processing steps on the mass spectrum in step b) have been performed; and d) using the values obtained in step c) in classification algorithm (e.g., K-nearest neighbor) using a training set comprising class-labeled spectra produced from blood- based samples from other solid epithelial tumor patients to identify the patient as being either likely or not likely to benefit from treatment by administration of
• the VeriStrat "Poor" signature is believed to be associated with a specific activation of NF- ⁇ , therefore the test can be used to select patients benefiting most from the NF-f B inhibitors and the addition of COX2 inhibitors to the standard chemotherapy treatment, and, at the same time, to reduce unnecessary treatment and associated morbidities.
• the methods of this disclosure can be implemented as a laboratory test center that receives blood-based samples from cancer patients (or mass spectral data from such samples), stores such mass spectral data in machine readable memory, and implements the processing and classification steps as shown in Figure 1 in a machine, e.g., using a programmed computer, to generate the class label (VeriStrat "good” or "poor"), thereby providing the prediction of identification of the patient as likely to benefit from treatment from the therapeutic agent or combination of therapeutic agents as described above.
• the invention can be configured as an apparatus configured to identify or predict whether a cancer patient is likely to benefit from administration of the combination of a COX2 inhibitor and an EGFR inhibitor.
• the apparatus consists in combination of a storage device, computer memory or database, storing a mass spectrum of a blood-based sample from the cancer patient, and a processor (e.g., conventional CPU of a programmed general purpose computer) executing software instructions configured to a) perform one or more predefined pre-processing steps on the mass spectrum (See Figure 1); b) obtain integrated intensity values of selected features in said spectrum at one or more predefined m/z ranges after the pre-processing steps on the mass spectrum in step a) have been performed (such as ranges encompassing the list of peaks of Table 1 or the m/z ranges set forth above); and c) use the values obtained in step b) in classification algorithm (e.g.
• KNN classification algorithm using a training set comprising class- labeled spectra produced from blood-based samples from other cancer patients to identify the patient as being either likely or not likely to benefit from treatment by administration of a combination of a COX2 inhibitor and an EGFR inhibitor.
• the invention can be embodied as an apparatus configured to identify a solid epithelial tumor cancer patient as being likely to benefit from treatment with a therapeutic agent or a combination of therapeutic agents targeting agonists of the receptors, receptors or proteins involved in MAPK (mitogen-activated protein kinase) pathways or the PKC (protein kinase C) pathway upstream from or at Akt or ERK/JNK/p38 or PKC or not likely to benefit from treatment with the therapeutic agent or combination of therapeutic agents.
• MAPK mitogen-activated protein kinase
• PKC protein kinase C pathway upstream from or at Akt or ERK/JNK/p38 or PKC or not likely to benefit from treatment with the therapeutic agent or combination of therapeutic agents.
• the apparatus takes the form of a storage device storing a mass spectrum of a blood-based sample from the solid epithelial tumor cancer patient, and a processor executing software instructions configured to a) perform one or more predefined pre-processing steps on the mass spectrum (See Figure 1), b) obtain integrated intensity values of features in said mass spectrum at one or more predefined m/z ranges (such as ranges encompassing the list of peaks of Table 1 or the m/z ranges set forth above); and c) use the values obtained in step b) in a classification algorithm using a training set comprising class-labeled spectra produced from blood-based samples from other solid epithelial tumor cancer patients to identify the patient as being either likely or not likely to benefit from the therapeutic agent or a combination of therapeutic agents.
• Clark GM Zborowski DM, Culbertson JL, et al. Clinical utility of epidermal growth factor receptor expression for selecting patients with advanced non-small cell lung cancer for treatment with erlotinib. J Thorac Oncol 2006; 1 :837-46.
• Serum amyloid A an acute-phase protein involved in tumour pathogenesis. Cell Mol Life Sci 2009;66:9-26.
• Serum amyloid A is an endogenous ligand that differentially induces IL-12 and IL-23. J Immunol 2006;177:4072-9.
• Serum amyloid A (SAA) protein enhances formation of cyclooxygenase metabolites of activated human monocytes. FEBS Lett 1997;419:215-9.
• Greten FR Eckmann L
• Greten TF et al. IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell 2004; 1 18:285-96.
• the c-Rel transcription factor can both induce and inhibit apoptosis in the same cells via the upregulation of
• TLR2 is a functional receptor for acute-phase serum amyloid A. J Immunol 2008;181 :22-6.
• Gazdar AF Personalized Medicine and Inhibition of EGFR Signaling in Lung Cancer. N Engl J Med 2009.
• Gadgeel SM Gadgeel SM, Ruckdeschel JC, Heath EI, Heilbrun LK, Venkatramanamoorthy R, Wozniak A. Phase II study of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), and celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, in patients with platinum refractory non-small cell lung cancer (NSCLC). J Thorac Oncol 2007;2:299-305.
• EGFR-TKI epidermal growth factor receptor tyrosine kinase inhibitor
• COX-2 cyclooxygenase-2

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