WO2012065071A2 - Procédés de prédiction d'une réponse à une thérapie par un anticorps anti-egfr - Google Patents

Procédés de prédiction d'une réponse à une thérapie par un anticorps anti-egfr Download PDF

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WO2012065071A2
WO2012065071A2 PCT/US2011/060385 US2011060385W WO2012065071A2 WO 2012065071 A2 WO2012065071 A2 WO 2012065071A2 US 2011060385 W US2011060385 W US 2011060385W WO 2012065071 A2 WO2012065071 A2 WO 2012065071A2
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egfr
mutant
cancer
peptide
polypeptide
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PCT/US2011/060385
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WO2012065071A3 (fr
Inventor
Matthew Meyerson
Adam Bass
Michael Lawrence
Josep Tabernero
Jose Baslga
Jeonghee Cho
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The Broad Institute Of Mit And Harvard
Dana-Farber Cancer Institute, Inc.
Vall D'hebron University Hospital
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Publication of WO2012065071A2 publication Critical patent/WO2012065071A2/fr
Publication of WO2012065071A3 publication Critical patent/WO2012065071A3/fr

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    • 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
    • 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
    • G01N33/57419—Specifically defined cancers of colon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general

Definitions

  • the invention relates to generally to the predicting a subjects response to EGFR antibody therapy.
  • Colorectal adenocarcinoma is it the second leading cause of cancer death in the United States, leading to approximately 55,000 deaths per year. Although there have been market improvements in our ability to screen for this disease enabling diagnosis when the cancer is curable by surgery, there are still many people who eventually succumb to metastatic disease every fear. Fortunately, improvements in cytotoxic chemotherapy have led to survival improvements for these patients.
  • a useful adjunct to cytotoxic therapy has been the introduction of targeted biologic agents for the treatment of metastatic colorectal cancer.
  • One such agent is Cetuximab, a monoclonal antibody targeting the Epidermal Growth Factor Receptor (EGFR).
  • Cetuximab was shown to increase the likelihood of clinical response and lead to a significant survival benefit when compared to chemotherapy alone in patients who had already failed initial cytotoxic chemotherapy.
  • treatment with Cetuximab alone led to a 10% response rate whereas combination of the antibody with chemotherapy improved response rate to approximately 20%.
  • use of Cetuximab is now FDA approved in combination with chemotherapy in the second line setting after failure of initial therapy.
  • Cetuximab is now widely used in patients with metastatic colorectal cancer, there is a great need to develop a predictive test to enable the drug to be given to those patients with the greatest likelihood of response as early as possible.
  • the invention features method of accessing the effectiveness of an EGFR antibody therapy treatment regimen of a subject having cancer by obtaining a sample from the subject and detecting the presence or absence of a G724S mutation, a G719S mutation, or a L858R mutation in the EGFR polypeptide.
  • the presence of the mutation indicates the subject is responsive to EGFR antibody therapy treatment.
  • EGFR antibodies suitable for therapy include for example Cetuximab, Panitumumab, Zalutumumab, Nimotuzumab, Necitumumab and Matuzumab.
  • the cancer is any cnacer capable of being treated with an EGFR antibody.
  • the cancer is colorectal adenocarcinoma or head and neck cancer.
  • the subject has not received treatment for the cancer.
  • the subject has received treatment for the cancer.
  • FC flow-cytometry
  • IHC immuno-histochemistry
  • IF immuno-fluorescence
  • PCR polymerase chain reaction
  • kits for the detection of mutant (G724S) EGFR polypeptide in a biological sample the kit containing a reagent specifically binds to or detects a mutant EGFR polypeptide but does not bind to or detect wild type EGFR and one or more secondary reagents.
  • Figure 1 depicts graphs establishing that colon cancer derived G719S and G724S EGFR mutants are oncogenic and dependent on dimerization for transforming activity.
  • Panels A- D shows growth of colonies of wild type (A), L858R mutant (B), G724S mutant (C) and G719S mutant (D) in soft agar.
  • Panel E shows the relative colony numbers of G724S dimmers, G724S/L704N dimmers and G724S/I941R dimmers.
  • Panel F shows the relative colony numbers of G719S dimmers, G719S/L704N dimmers and G719S/I941R dimmers.
  • Figure 2 depicts a graph demonstrating that G719S and G724S EGFR mutants are not as sensitive as L858R mutant to Erlotinib.
  • Figure 3 depicts a graph demonstrating that G719S and G724S EGFR mutants are as sensitive as L858R mutant to Cetuximab.
  • the present invention relates to the identification of biomarkers associated with the responsiveness to EGFR antibody therapy.
  • the biomarkers are somatic mutations in the EGFR gene leading to an amino acid substitution at position 724, 719 or 858.
  • the amino acid at position 724 is in the nucleotide binding loop of the EGFR polypeptide.
  • the mutations result in a glycine to a serine at position 724 (G724S) and at position 719 (G719S) and a leucine to an arginine at position 858 (L858R).
  • Anti-EGFR antibodies suitable for human administration are know in the art and include for example, Cetuximab, Panitumumab, Zalutumumab, Nimotuzumab,
  • Necitumumab and Matuzumab are Necitumumab and Matuzumab.
  • Cetuximab also known as Erbitux ® is a chimeric (mouse/human) monoclonal antibody that is an epidermal growth factor receptor (EGFR) inhibitor useful for treatment of colorectal cancer and head and neck cancer.
  • EGFR epidermal growth factor receptor
  • Cetuximab is indicated for the treatment of patients with EGFR expressing, KRAS wild-type metastatic colorectal cancer in combination with chemotherapy or as a single agent in patients who have failed in oxaliplatin- or irinotecan- base therapy and who are intolerant to irinotecan. Cetuximab is also indicated for use in combination with radiation therapy for treating squamous cell carcinoma of the head and neck (SCCHN) or as a single agent in patients who have had prior platinum-based therapy. Panitumumab (Vectibix) is indicated for tthe treatment of EGFR-expressing metastatic colorectal cancer with disease progression despite prior treatment.
  • Zalutumumab (HuMax-EGFr )is indicated for the treatment of patients suffering from SCCHN who have failed standard therapies and have no other options.
  • the drug has undergone pre-clinical and Phase I and II studies and is also in Phases I and II for SCCHN front-line with chemo-radiation and SCCHN with radiation. Additionally, a Phase II is under way for SCCHN and Phase III studies are also being performed for SCCHN and SCCHN front-line with radio therapy.
  • a phase III study reported a non significant improvement in overall survival, and a significant improvement in Progression-free survival.
  • Nimotuzumab (BIOMAb EGFR) is a chimeric monoclonal antibody used to treat squamous cell carcinomas of the head and neck, recurrent/refractory high grade malignant astrocytoma and glioma.
  • the invention provides methods of determining the responsiveness, e.g., sensitivity or resistance, of a cancer cell to EGFR antibody therapy. These methods are also useful for monitoring subjects undergoing treatments and therapies for cancer such as for example colorectal adenocarcinoma or head and neck cancer, and for selecting therapies and treatments that would be efficacious in subjects having cancer, wherein selection and use of such treatments and therapies slow the progression of cancer. More specifically, the invention provides methods of determining the whether a patient with colorectal
  • adenocarcinoma will be responsive to EGFR antibody therapy.
  • genomic discovery was a patient sample with the highest possible likelihood of identification of any genomic projector response.
  • the patients whose tumor we chose to profile for this study was one whose tumor was obtained prior to enrollment in a specific clinical trial whereby patients with metastatic colorectal adenocarcinoma were treated with the use of single agent Cetuximab prior to the receipt of other cytotoxic chemotherapy.
  • “Accuracy” refers to the degree of conformity of a measured or calculated quantity (a test reported value) to its actual (or true) value. Clinical accuracy relates to the proportion of true outcomes (true positives (TP) or true negatives (TN) versus misclassified outcomes
  • FP false positives
  • FN false negatives
  • FP false positives
  • PN positive predictive values
  • NPV negative predictive values
  • Biomarker in the context of the present invention encompasses, without limitation, proteins, nucleic acids, and metabolites, together with their polymorphisms, mutations, variants, modifications, subunits, fragments, protein-ligand complexes, and degradation products, protein-ligand complexes, elements, related metabolites, and other analytes or sample-derived measures. Biomarkers can also include mutated proteins or mutated nucleic acids. Biomarkers also encompass non-blood borne factors or non-analyte physiological markers of health status, such as "clinical parameters” defined herein, as well as “traditional laboratory risk factors”, also defined herein.
  • Biomarkers also include any calculated indices created mathematically or combinations of any one or more of the foregoing measurements, including temporal trends and differences. Where available, and unless otherwise described herein, biomarkers which are gene products are identified based on the official letter abbreviation or gene symbol assigned by the international Human Genome Organization
  • a “Clinical indicator” is any physiological datum used alone or in conjunction with other data in evaluating the physiological condition of a collection of cells or of an organism. This term includes pre-clinical indicators.
  • “Clinical parameters” encompasses all non-sample or non-analyte biomarkers of subject health status or other characteristics, such as, without limitation, age (Age), ethnicity (RACE), gender (Sex), or family history (FamHX).
  • FN is false negative, which for a disease state test means classifying a disease subject incorrectly as non-disease or normal.
  • FP is false positive, which for a disease state test means classifying a normal subject incorrectly as having disease.
  • a “formula,” “algorithm,” or “model” is any mathematical equation, algorithmic, analytical or programmed process, or statistical technique that takes one or more continuous or categorical inputs (herein called “parameters”) and calculates an output value, sometimes referred to as an "index” or “index value.”
  • Parameters continuous or categorical inputs
  • index value sometimes referred to as an "index” or “index value.”
  • Non-limiting examples of “formulas” include sums, ratios, and regression operators, such as coefficients or exponents, biomarker value transformations and normalizations (including, without limitation, those normalization schemes based on clinical parameters, such as gender, age, or ethnicity), rules and guidelines, statistical classification models, and neural networks trained on historical populations.
  • biomarkers Of particular use in combining biomarkers are linear and non-linear equations and statistical classification analyses to determine the relationship between biomarkers detected in a subject sample and the subject's responsiveness to chemotherapy.
  • panel and combination construction of particular interest are structural and synactic statistical classification algorithms, and methods of risk index construction, utilizing pattern recognition features, including established techniques such as cross-correlation, Principal Components Analysis (PCA), factor rotation, Logistic Regression (LogReg), Linear Discriminant Analysis (LDA), Eigengene Linear Discriminant Analysis (ELD A), Support Vector Machines (SVM), Random Forest (RF), Recursive Partitioning Tree (RPART), as well as other related decision tree classification techniques, Shrunken Centroids (SC), StepAIC, Kth-Nearest Neighbor, Boosting, Decision Trees, Neural Networks, Bayesian Networks, Support Vector Machines, and Hidden Markov Models, among others.
  • PCA Principal Components Analysis
  • LogReg Logistic Regression
  • LDA Linear Discriminant Analysis
  • AIC Akaike's Information Criterion
  • BIC Bayes Information Criterion
  • the resulting predictive models may be validated in other studies, or cross-validated in the study they were originally trained in, using such techniques as Bootstrap, Leave-One-Out (LOO) and 10-Fold cross-validation (10-Fold CV).
  • LEO Leave-One-Out
  • 10-Fold cross-validation 10-Fold CV.
  • false discovery rates may be estimated by value permutation according to techniques known in the art.
  • a "health economic utility function" is a formula that is derived from a combination of the expected probability of a range of clinical outcomes in an idealized applicable patient population, both before and after the introduction of a diagnostic or therapeutic intervention into the standard of care.
  • a cost and/or value measurement associated with each outcome, which may be derived from actual health system costs of care (services, supplies, devices and drugs, etc.) and/or as an estimated acceptable value per quality adjusted life year (QALY) resulting in each outcome.
  • the sum, across all predicted outcomes, of the product of the predicted population size for an outcome multiplied by the respective outcome's expected utility is the total health economic utility of a given standard of care.
  • the difference between (i) the total health economic utility calculated for the standard of care with the intervention versus (ii) the total health economic utility for the standard of care without the intervention results in an overall measure of the health economic cost or value of the intervention.
  • This may itself be divided amongst the entire patient group being analyzed (or solely amongst the intervention group) to arrive at a cost per unit intervention, and to guide such decisions as market positioning, pricing, and assumptions of health system acceptance.
  • Such health economic utility functions are commonly used to compare the cost-effectiveness of the intervention, but may also be transformed to estimate the acceptable value per QALY the health care system is willing to pay, or the acceptable cost-effective clinical performance characteristics required of a new intervention.
  • a health economic utility function may preferentially favor sensitivity over specificity, or PPV over NPV based on the clinical situation and individual outcome costs and value, and thus provides another measure of health economic performance and value which may be different from more direct clinical or analytical performance measures.
  • Measurement or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of a subject's non-analyte clinical parameters.
  • NDV Neuronal predictive value
  • hazard ratios and absolute and relative risk ratios within subject cohorts defined by a test are a further measurement of clinical accuracy and utility. Multiple methods are frequently used to defining abnormal or disease values, including reference limits, discrimination limits, and risk thresholds.
  • Performance is a term that relates to the overall usefulness and quality of a diagnostic or prognostic test, including, among others, clinical and analytical accuracy, other analytical and process characteristics, such as use characteristics (e.g. , stability, ease of use), health economic value, and relative costs of components of the test. Any of these factors may be the source of superior performance and thus usefulness of the test, and may be measured by appropriate "performance metrics," such as AUC, time to result, shelf life, etc. as relevant.
  • PSV Positive predictive value
  • “Risk” in the context of the present invention relates to the probability that an event will occur over a specific time period, as in the responsiveness to treatment, cancer recurrence or survival and can mean a subject' s "absolute” risk or “relative” risk.
  • Absolute risk can be measured with reference to either actual observation post- measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no-conversion.
  • Risk evaluation or “evaluation of risk” in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state.
  • Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of cancer, either in absolute or relative terms in reference to a previously measured population.
  • the methods of the present invention may be used to make continuous or categorical measurements of the responsiveness to treatment thus diagnosing and defining the risk spectrum of a category of subjects defined as being responders or non-responders. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for responding. Such differing use may require different HNCMARKER combinations and individualized panels, mathematical algorithms, and/or cut-off points, but be subject to the same aforementioned measurements of accuracy and performance for the respective intended use.
  • sample in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, tissue biopies, whole blood, serum, plasma, blood cells, endothelial cells, lymphatic fluid, ascites fluid, interstitital fluid (also known as "extracellular fluid” and encompasses the fluid found in spaces between cells, including, inter alia, gingival crevicular fluid), bone marrow, cerebrospinal fluid (CSF), saliva, mucous, sputum, sweat, urine, or any other secretion, excretion, or other bodily fluids.
  • a “sample” may include a single cell or multiple cells or fragments of cells.
  • the sample is also a tissue sample.
  • the sample is or contains a circulating endothelial cell or a circulating tumor cell.
  • the sample includes a primary tumor cell, primary tumor, a recurrent tumor cell, or a metastatic tumor cell.
  • Specificity is calculated by TN/(TN+FP) or the true negative fraction of non- disease or normal subjects.
  • Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which presents the probability of obtaining a result at least as extreme as a given data point, assuming the data point was the result of chance alone. A result is considered highly significant at a p-value of 0.05 or less. Preferably, the p-value is 0.04, 0.03, 0.02, 0.01, 0.005, 0.001 or less.
  • a "subject" in the context of the present invention is preferably a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cancer.
  • a subject can be male or female.
  • TN is true negative, which for a disease state test means classifying a non-disease or normal subject correctly.
  • TP is true positive, which for a disease state test means correctly classifying a disease subject.
  • Traditional laboratory risk factors correspond to biomarkers isolated or derived from subject samples and which are currently evaluated in the clinical laboratory and used in traditional global risk assessment algorithms.
  • Traditional laboratory risk factors for tumor recurrence include for example Proliferative index, tumor infiltrating lymphocytes. Other traditional laboratory risk factors for tumor recurrence known to those skilled in the art.
  • the methods disclosed herein are used with subjects undergoing treatment and/or therapies for cancer, subjects who are at risk for developing a reoccurrence of cancer (e.g. colorectal adenocarcinoma or head and neck cancer), and subjects who have been diagnosed with cancer.
  • the methods of the present invention are to be used to monitor or select a treatment regimen for a subject who has cancer, and to evaluate the predicted survivability and/or survival time of a cancer-diagnosed subject.
  • Treatment regimens include EGFR antibody therapys such as Cetuximab, Panitumumab, Zalutumumab, Nimotuzumab, Necitumumab and Matuzumab.
  • Responsiveness e.g., resistance or sensitivity
  • Responsiveness of a cell to EGFR antibody therapy is determined by detecting a mutation associated with responsiveness to EGFR antibody therapy therapy in a test sample (e.g., a subject derived sample).
  • the mutation associated with responsiveness to EGFR antibody therapy includes a somatic mutation in the EGFR gene leading to an amino acid substitution at position 724, at position 719 or at position 858 of the EGFR polypeptide. Specifically the mutation results in a glycine to serine at positions 724 (G724S) and 719 (G719S) and a leucine to an arginine at position 858 (L858R).
  • the mutation associated with responsiveness to EGFR antibody therapy is referred herein as the EGFR G724S mutation, the EGFR G719S mutation or the EGFR L858R mutation.
  • the presence of the EGFR G724S mutation, the EGFR G719S mutation, the EGFR L858R mutation, or any combination thereof indicates the cell will be responsive (i.e. , sensitive) to EGFR antibody therapy.
  • the absence of these mutations indicates the cell will be non- responsive (i.e. , resistant) to EGFR antibody therapy.
  • cells expressing G724S mutant or G719S mutant are as sensitive as L858R mutant to Cetuximab ( Figure 3), but less sensitiveto Erlotinib ( Figure 2).
  • the cell is for example a cancer cell.
  • the cancer is a colorectal
  • adenocarcinoma cancer or a head or neck cancer such as cancer of the nasal cavity, sinuses, lips, mouth, salivary glands, throat, or larynx
  • resistance it is meant that a cell fails to respond to an agent.
  • resistance to EGFR antibody therapy means the cell is not damaged or killed by the drug.
  • sensitivity it is meant that that the cell responds to an agent.
  • sensitivity to EGFR antibody therapy means the cell is damaged or killed by the drug.
  • the methods of the present invention are useful to treat, alleviate the symptoms of, monitor the progression of or delay the onset of cancer.
  • the methods of the present invention are used to identify and/or diagnose subjects who are asymptomatic for a cancer recurrence.
  • “Asymptomatic” means not exhibiting the traditional symptoms.
  • the methods of the present invention are also useful to identify and/or diagnose subjects already at higher risk of developing a head and neck cancer or based on solely on the traditional risk factors.
  • Identification of the EGFR G724S G719S or L858R mutation allows for the determination of whether a subject will derive a benefit from a particular course of treatment.
  • a biological sample is provided from a subject before undergoing treatment, e.g., EGFR antibody therapy or combinations thereof.
  • recipient a benefit it is meant that the subject will respond to the course of treatment.
  • responding it is meant that the treatment decreases in size, prevalence, or metastatic potential of a cancer (e.g. colorectal adenocarcinoma or head and neck cancer) in a subject.
  • a cancer e.g. colorectal adenocarcinoma or head and neck cancer
  • “responding” means that the treatment retards or prevents a cancer (e.g. colorectal adenocarcinoma or head and neck cancer) recurrence from forming or retards, prevents, or alleviates a symptom.
  • a cancer e.g. colorectal adenocarcinoma or head and neck cancer
  • Assessment of cancers are made using standard clinical protocols.
  • the present invention can also be used to screen patient or subject populations in any number of settings.
  • a health maintenance organization, public health entity or school health program can screen a group of subjects to identify those requiring interventions, as described above, or for the collection of epidemiological data.
  • Insurance companies e.g. , health, life or disability
  • Data collected in such population screens, particularly when tied to any clinical progression to conditions like cancer, will be of value in the operations of, for example, health maintenance organizations, public health programs and insurance companies.
  • Such data arrays or collections can be stored in machine- readable media and used in any number of health-related data management systems to provide improved healthcare services, cost effective healthcare, improved insurance operation, etc. See, for example, U.S.
  • Such systems can access the data directly from internal data storage or remotely from one or more data storage sites as further detailed herein.
  • Each program can be implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. The language can be a compiled or interpreted language. Each such computer program can be stored on a storage media or device (e.g., ROM or magnetic diskette or others as defined elsewhere in this disclosure) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein.
  • the health-related data management system of the invention may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform various functions described herein.
  • Differences in the genetic makeup of subjects can result in differences in their relative abilities to metabolize various drugs, which may modulate the symptoms or risk factors of cancer or metastatic events.
  • Subjects that have cancer, or at risk for developing cancer or a metastatic event can vary in age, ethnicity, and other parameters. Accordingly, detection of the EGFR G724S, G719S or L858R mutation disclosed herein, both alone and together in combination with known genetic factors for drug metabolism, allow for a pre-determined level of predictability that a putative therapeutic or prophylactic to be tested in a selected subject will be suitable for treating cancer in the subject. Performance And Accuracy Measures Of The Invention
  • the performance and thus absolute and relative clinical usefulness of the invention may be assessed in multiple ways as noted above.
  • the invention is intended to provide accuracy in clinical diagnosis and prognosis.
  • the accuracy of a diagnostic, predictive, or prognostic test, assay, or method concerns the ability of the test, assay, or method to distinguish between subjects responsive to chemotherapeutic treatment and those that are not, is based on whether the subjects have the EGFR G724S, G719S, L858R mutation, or any combination thereof.
  • an "acceptable degree of diagnostic accuracy” is herein defined as a test or assay in which the AUC (area under the ROC curve for the test or assay) is at least 0.60, desirably at least 0.65, more desirably at least 0.70, preferably at least 0.75, more preferably at least 0.80, and most preferably at least 0.85.
  • a “very high degree of diagnostic accuracy” it is meant a test or assay in which the AUC (area under the ROC curve for the test or assay) is at least 0.80, desirably at least 0.85, more desirably at least 0.875, preferably at least 0.90, more preferably at least 0.925, and most preferably at least 0.95.
  • the predictive value of any test depends on the sensitivity and specificity of the test, and on the prevalence of the condition in the population being tested. This notion, based on Bayes' theorem, provides that the greater the likelihood that the condition being screened for is present in an individual or in the population (pre-test probability), the greater the validity of a positive test and the greater the likelihood that the result is a true positive.
  • pre-test probability the greater the likelihood that the condition being screened for is present in an individual or in the population
  • a positive result has limited value (i.e., more likely to be a false positive).
  • a negative test result is more likely to be a false negative.
  • ROC and AUC can be misleading as to the clinical utility of a test in low disease prevalence tested populations (defined as those with less than 1% rate of occurrences (incidence) per annum, or less than 10% cumulative prevalence over a specified time horizon).
  • absolute risk and relative risk ratios as defined elsewhere in this disclosure can be employed to determine the degree of clinical utility.
  • Populations of subjects to be tested can also be categorized into quartiles by the test's measurement values, where the top quartile (25% of the population) comprises the group of subjects with the highest relative risk for therapeutic unresponsiveness, and the bottom quartile comprising the group of subjects having the lowest relative risk for therapeutic unresponsiveness.
  • values derived from tests or assays having over 2.5 times the relative risk from top to bottom quartile in a low prevalence population are considered to have a "high degree of diagnostic accuracy," and those with five to seven times the relative risk for each quartile are considered to have a "very high degree of diagnostic accuracy.” Nonetheless, values derived from tests or assays having only 1.2 to 2.5 times the relative risk for each quartile remain clinically useful are widely used as risk factors for a disease; such is the case with total cholesterol and for many inflammatory biomarkers with respect to their prediction of future events. Often such lower diagnostic accuracy tests must be combined with additional parameters in order to derive meaningful clinical thresholds for therapeutic intervention, as is done with the aforementioned global risk assessment indices.
  • a health economic utility function is yet another means of measuring the performance and clinical value of a given test, consisting of weighting the potential categorical test outcomes based on actual measures of clinical and economic value for each.
  • Health economic performance is closely related to accuracy, as a health economic utility function specifically assigns an economic value for the benefits of correct classification and the costs of misclassification of tested subjects.
  • As a performance measure it is not unusual to require a test to achieve a level of performance which results in an increase in health economic value per test (prior to testing costs) in excess of the target price of the test.
  • diagnostic accuracy In general, alternative methods of determining diagnostic accuracy are commonly used for continuous measures, when a disease category or risk category has not yet been clearly defined by the relevant medical societies and practice of medicine, where thresholds for therapeutic use are not yet established, or where there is no existing gold standard for diagnosis of the pre-disease.
  • measures of diagnostic accuracy for a calculated index are typically based on curve fit and calibration between the predicted continuous value and the actual observed values (or a historical index calculated value) and utilize measures such as R squared, Hosmer- Lemeshow P-value statistics and confidence intervals.
  • the actual detection of the EGFR G724S, G719S or L858R mutation can be determined at the protein or nucleic acid level using any method known in the art.
  • GenBank accession No: CAA25240.1 GenBank accession No: X00588.1, each of which is incorporated herein by reference in its entirety.
  • a skilled artisan in the field would easily identify the wild type human EGFR nucleic acids and polypeptides sequences.
  • G724S, G719S or L858R mutation -specific reagents useful in the practice of the disclosed methods include, among others, mutant polypeptide specific antibodies and AQUA peptides (heavy-isotope labeled peptides) corresponding to, and suitable for detection and quantification of, mutant (G724S, G719S, L858R, or any combination thereof) polypeptide expression in a biological sample.
  • a mutant polypeptide- specific reagent is any reagent, biological or chemical, capable of specifically binding to, detecting and/or quantifying the presence/level of expressed mutant polypeptide in a biological sample, while not binding to or detecting wild type.
  • the term includes, but is not limited to, the preferred antibody and AQUA peptide reagents discussed below, and equivalent reagents are within the scope of the present invention.
  • Reagents suitable for use in practice of the methods of the invention include a mutant polypeptide- specific antibody.
  • a mutant- specific antibody of the invention is an isolated antibody or antibodies that specifically bind(s) a mutant (G724S, G719S, L858R, or any combination thereof) polypeptide of the invention, but does not substantially bind either wild type or when mutated at other positions.
  • Mutant polypeptide- specific antibodies generated against human mutant may also bind to highly homologous and equivalent epitopic peptide sequences in other mammalian species, for example murine, rat, feline, pig, or rabbit, and vice versa.
  • Antibodies useful in practicing the methods of the invention include (a) monoclonal antibodies, (b) purified polyclonal antibodies that specifically bind to the target polypeptide (e.g. an epitope comprising the G724S, G719S, L858R mutation point, or any combination thereof , (c) antibodies as described in (a)-(b) above that bind equivalent and highly homologous epitopes or phosphorylation sites in other non-human species (e.g. mouse, rat), and (d) fragments of (a)-(c) above that bind to the antigen (or more preferably the epitope) bound by the exemplary antibodies disclosed herein.
  • antibody or “antibodies” as used herein refers to all types of
  • the antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26: 403-11 (1989); Morrision et al., Proc. Natl. Acad. Sci. 81:6851 (1984); Neuberger et al., Nature 312:604 (1984)).
  • the antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No.
  • the antibodies may also be chemically constructed specific antibodies made according to the method disclosed in U.S. Pat. No. 4,676,980 (Segel et al.)
  • the invention is not limited to use of antibodies, but includes equivalent molecules, such as protein binding domains or nucleic acid aptamers, which bind, in a mutant-protein or truncated-protein specific manner, to essentially the same epitope to which a mutant polypeptide- specific antibody useful in the methods of the invention binds. See, e.g.,
  • Polyclonal antibodies useful in practicing the methods of the invention may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen encompassing a desired mutant-protein specific epitope (e.g. the sequence comprising the G724S, G719S, L858R mutation site, or any combination thereof ) collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, and purifying polyclonal antibodies having the desired specificity, in accordance with known procedures.
  • the antigen may be a synthetic peptide antigen comprising the desired epitopic sequence, selected and constructed in accordance with well- known techniques.
  • Polyclonal antibodies produced as described herein may be screened and isolated as further described below.
  • Monoclonal antibodies may also be beneficially employed in the methods of the invention, and may be produced in hybridoma cell lines according to the well-known technique of Kohler and Milstein. Nature 265:495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6: 511 (1976); see also, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al., Eds. (1989). Monoclonal antibodies so produced are highly specific, and improve the selectivity and specificity of assay methods provided by the invention. For example, a solution containing the appropriate antigen (e.g.
  • a synthetic peptide comprising the mutant junction of mutant EGFR polypeptide may be injected into a mouse and, after a sufficient time (in keeping with conventional techniques), the mouse sacrificed and spleen cells obtained.
  • the spleen cells are then immortalized by fusing them with myeloma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells.
  • Rabbit mutant hybridomas for example, may be produced as described in U.S. Pat. No. 5,675,063, K.
  • the hybridoma cells are then grown in a suitable selection media, such as hypoxanthine-aminopterin-thymidine (HAT), and the supernatant screened for monoclonal antibodies having the desired specificity, as described below.
  • a suitable selection media such as hypoxanthine-aminopterin-thymidine (HAT)
  • HAT hypoxanthine-aminopterin-thymidine
  • the secreted antibody may be recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography, or the like.
  • Monoclonal Fab fragments may also be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246:1275-81 (1989); Mullinax et al., Proc. Nat'l Acad. Sci. 87: 8095 (1990). If monoclonal antibodies of one isotype are preferred for a particular application, particular isotypes can be prepared directly, by selecting from the initial mutant, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proc. Nat'l. Acad.
  • the antigen combining site of the monoclonal antibody can be cloned by PCR and single-chain antibodies produced as phage-displayed recombinant antibodies or soluble antibodies in E. coli (see, e.g., ANTIBODY
  • Antibodies useful in the methods of the invention may be screened for epitope and mutant protein specificity according to standard techniques. See, e.g. Czernik et al., Methods in Enzymology, 201: 264-283 (1991).
  • the antibodies may be screened against a peptide library by ELISA to ensure specificity for both the desired antigen and, if desired, for reactivity only with a mutant polypeptide of the invention and not with wild type .
  • the antibodies may also be tested by Western blotting against cell preparations containing target protein to confirm reactivity with the only the desired target and to ensure no appreciable binding to other mutants not containing the G724S point mutation.
  • the production, screening, and use of mutant protein- specific antibodies is known to those of skill in the art, and has been described.
  • Mutant polypeptide- specific antibodies useful in the methods of the invention may exhibit some limited cross-reactivity with similar epitopes in other highly homologous proteins. This is not unexpected as most antibodies exhibit some degree of cross -reactivity, and anti-peptide antibodies will often cross-react with epitopes having high homology or identity to the immunizing peptide. See, e.g., Czernik, supra. Gross -reactivity with other mutant proteins is readily characterized by Western blotting alongside markers of known molecular weight. Amino acid sequences of cross -reacting proteins may be examined to identify sites highly homologous or identical to the mutant polypeptide sequence to which the antibody binds.
  • Undesirable cross-reactivity can be removed by negative selection using antibody purification on peptide columns (e.g. selecting out antibodies that bind either wild type EGFR polypeptide or which bind highly homologous sequences on different proteins).
  • Mutant EGFR polypeptide- specific antibodies of the invention that are useful in practicing the methods disclosed herein are ideally specific for human mutant polypeptide, but are not limited only to binding the human species, per se.
  • the invention includes the production and use of antibodies that also bind conserved and highly homologous or identical epitopes in other mammalian species (e.g. mouse, rat, monkey). Highly homologous or identical sequences in other species can readily be identified by standard sequence
  • Antibodies employed in the methods of the invention may be further characterized by, and validated for, use in a particular assay format, for example FC, IHC, and/or ICC.
  • a particular assay format for example FC, IHC, and/or ICC.
  • Antibodies may also be advantageously conjugated to fluorescent dyes (e.g. Alexa488, PE), or labels such as quantum dots, for use in multi-parametric analyses along with other signal transduction (phospho-AKT, phospho-Erk 1/2) and/or cell marker (cytokeratin) antibodies, as further described below.
  • the expression and/or activity of wild type EGFR in a given biological sample may also be advantageously examined using antibodies (either phospho-specific or total) for these wild type proteins.
  • antibodies may also be produced according to standard methods, as described above.
  • the amino acid sequences of human EGFR are published, as are the sequences of these proteins from other species, including mammalian, as noted above.
  • Detection of wild type EGFR expression and/or activation, along with mutant EGFR polypeptide expression, in a biological sample can provide information on whether the mutant protein alone is driving the tumor, or whether wild type EGFR is also activated and driving the tumor. Such information is clinically useful in assessing whether targeting the mutant protein or the wild type protein(s), or both, or is likely to be most beneficial in inhibiting progression of the tumor, and in selecting an appropriate therapeutic or combination thereof.
  • mutant EGFR polypeptide-specific antibodies together with one or more antibodies specific for another protein, receptor, that is suspected of being, or potentially is, activated in a cancer in which mutant EGFR
  • polypeptide is expressed may be simultaneously employed to detect the activity of such other signaling molecules in a biological sample comprising cells from such cancer.
  • mutant EGFR polypeptides of the present invention and the mutant junction epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • mutant proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature 331: 84-86 (1988)).
  • Mutant proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric mutant EGFR polypeptide alone (Fountoulakis et al., J Biochem 270: 3958-3964 (1995)).
  • Mutant EGFR polypeptide-specific reagents useful in the practice of the disclosed methods may also comprise heavy-isotope labeled peptides suitable for the absolute quantification of expressed mutant EGFR polypeptide in a biological sample.
  • AQUA peptides for the absolute quantification of proteins (AQUA) in complex mixtures has been described. See WO/03016861, "Absolute Quantification of Proteins and Modified Forms Thereof by Multistage Mass Spectrometry," Gygi et al. and also Gerber et al., Proc. Natl. Aced. Sci. U.S.A. 100: 6940-5 (2003) (the teachings of which are hereby incorporated herein by reference, in their entirety).
  • the AQUA methodology employs the introduction of a known quantity of at least one heavy-isotope labeled peptide standard (which has a unique signature detectable by LC-SRM chromatography) into a digested biological sample in order to determine, by comparison to the peptide standard, the absolute quantity of a peptide with the same sequence and protein modification in the biological sample.
  • the AQUA methodology has two stages:
  • the method is a powerful technique for detecting and quantifying a given peptide/protein within a complex biological mixture, such as a cell lysate, and may be employed, e.g., to quantify change in protein phosphorylation as a result of drug treatment, or to quantify differences in the level of a protein in different biological states.
  • a particular peptide (or modified peptide) within a target protein sequence is chosen based on its amino acid sequence and the particular protease to be used to digest.
  • the peptide is then generated by solid-phase peptide synthesis such that one residue is replaced with that same residue containing stable isotopes
  • the result is a peptide that is chemically identical to its native counterpart formed by proteolysis, but is easily distinguishable by MS via a 7-Da mass shift.
  • the newly synthesized AQUA internal standard peptide is then evaluated by LC-MS/MS. This process provides qualitative information about peptide retention by reverse-phase chromatography, ionization efficiency, and fragmentation via collision-induced dissociation. Informative and abundant fragment ions for sets of native and internal standard peptides are chosen and then specifically monitored in rapid succession as a function of chromatographic retention to form a selected reaction monitoring (LC-SRM) method based on the unique profile of the peptide standard.
  • LC-SRM reaction monitoring
  • the second stage of the AQUA strategy is its implementation to measure the amount of a protein or modified protein from complex mixtures.
  • Whole cell lysates are typically fractionated by SDS-PAGE gel electrophoresis, and regions of the gel consistent with protein migration are excised. This process is followed by in-gel proteolysis in the presence of the AQUA peptides and LC-SRM analysis.
  • AQUA peptides are spiked in to the complex peptide mixture obtained by digestion of the whole cell lysate with a proteolytic enzyme and subjected to immunoaffinity purification as described above.
  • the retention time and fragmentation pattern of the native peptide formed by digestion e.g.
  • trypsinization is identical to that of the AQUA internal standard peptide determined previously; thus, LC-MS/MS analysis using an SRM experiment results in the highly specific and sensitive measurement of both internal standard and analyte directly from extremely complex peptide mixtures.
  • the ratio of the areas under the curve can be used to determine the precise expression levels of a protein or phosphorylated form of a protein in the original cell lysate.
  • the internal standard is present during in-gel digestion as native peptides are formed, such that peptide extraction efficiency from gel pieces, absolute losses during sample handling (including vacuum centrifugation), and variability during introduction into the LC-MS system do not affect the determined ratio of native and AQUA peptide abundances.
  • An AQUA peptide standard is developed for a known sequence previously identified by the IAP-LC-MS/MS method within in a target protein.
  • one AQUA peptide incorporating the modified form of the particular residue within the site may be developed, and a second AQUA peptide incorporating the unmodified form of the residue developed.
  • the two standards may be used to detect and quantify both the modified an unmodified forms of the site in a biological sample.
  • Peptide internal standards may also be generated by examining the primary amino acid sequence of a protein and determining the boundaries of peptides produced by protease cleavage. Alternatively, a protein may actually be digested with a protease and a particular peptide fragment produced can then sequenced. Suitable proteases include, but are not limited to, serine proteases (e.g. trypsin, hepsin), metallo proteases (e.g. PUMP1), chymotrypsin, cathepsin, pepsin, thermolysin, carboxypeptidases, etc.
  • a peptide sequence within a target protein is selected according to one or more criteria to optimize the use of the peptide as an internal standard.
  • the size of the peptide is selected to minimize the chances that the peptide sequence will be repeated elsewhere in other non-target proteins.
  • a peptide is preferably at least about 6 amino acids.
  • the size of the peptide is also optimized to maximize ionization frequency.
  • peptides longer than about 20 amino acids are not preferred.
  • the preferred ranged is about 7 to 15 amino acids.
  • a peptide sequence is also selected that is not likely to be chemically reactive during mass spectrometry, thus sequences comprising cysteine, tryptophan, or methionine are avoided.
  • a peptide sequence that does not include a modified region of the target region may be selected so that the peptide internal standard can be used to determine the quantity of all forms of the protein.
  • a peptide internal standard encompassing a modified amino acid may be desirable to detect and quantify only the modified form of the target protein.
  • Peptide standards for both modified and unmodified regions can be used together, to determine the extent of a modification in a particular sample (i.e. to determine what fraction of the total amount of protein is represented by the modified form).
  • peptide standards for both the phosphorylated and unphosphorylated form of a protein known to be phosphorylated at a particular site can be used to quantify the amount of phosphorylated form in a sample.
  • the peptide is labeled using one or more labeled amino acids (i.e. the label is an actual part of the peptide) or less preferably, labels may be attached after synthesis according to standard methods.
  • the label is a mass-altering label selected based on the following considerations: The mass should be unique to shift fragments masses produced by MS analysis to regions of the spectrum with low background; the ion mass signature component is the portion of the labeling moiety that preferably exhibits a unique ion mass signature in MS analysis; the sum of the masses of the constituent atoms of the label is preferably uniquely different than the fragments of all the possible amino acids.
  • the labeled amino acids and peptides are readily distinguished from unlabeled ones by the ion/mass pattern in the resulting mass spectrum.
  • the ion mass signature component imparts a mass to a protein fragment that does not match the residue mass for any of the 20 natural amino acids.
  • the label should be robust under the fragmentation conditions of MS and not undergo unfavorable fragmentation. Labeling chemistry should be efficient under a range of conditions, particularly denaturing conditions, and the labeled tag preferably remains soluble in the MS buffer system of choice. The label preferably does not suppress the ionization efficiency of the protein and is not chemically reactive.
  • the label may contain a mixture of two or more isotopically distinct species to generate a unique mass spectro metric pattern at
  • Stable isotopes such as H, C, N, O, O, or S, are among preferred labels. Pairs of peptide internal standards that incorporate a different isotope label may also be prepared. Preferred amino acid residues into which a heavy isotope label may be incorporated include leucine, proline, valine, and phenylalanine.
  • Peptide internal standards are characterized according to their mass-to-charge (m/z) ratio, and preferably, also according to their retention time on a chromatographic column (e.g., an HPLC column). Internal standards that co-elute with unlabeled peptides of identical sequence are selected as optimal internal standards.
  • the internal standard is then analyzed by fragmenting the peptide by any suitable means, for example by collision-induced dissociation (CID) using, e.g., argon or helium as a collision gas.
  • CID collision-induced dissociation
  • the fragments are then analyzed, for example by multi-stage mass spectrometry (MS) to obtain a fragment ion spectrum, to obtain a peptide fragmentation signature.
  • MS mass spectrometry
  • peptide fragments have significant differences in m/z ratios to enable peaks corresponding to each fragment to be well separated, and a signature is that is unique for the target peptide is obtained. If a suitable fragment signature is not obtained at the first stage, additional stages of MS are performed until a unique signature is obtained.
  • Fragment ions in the MS/MS and MS spectra are typically highly specific for the peptide of interest, and, in conjunction with LC methods, allow a highly selective means of detecting and quantifying a target peptide/protein in a complex protein mixture, such as a cell lysate, containing many thousands or tens of thousands of proteins.
  • a complex protein mixture such as a cell lysate, containing many thousands or tens of thousands of proteins.
  • Any biological sample potentially containing a target protein/peptide of interest may be assayed. Crude or partially purified cell extracts are preferably employed.
  • the sample has at least 0.01 mg of protein, typically a concentration of 0.1-10 mg/mL, and may be adjusted to a desired buffer concentration and pH.
  • a known amount of a labeled peptide internal standard, preferably about 10 femtomoles, corresponding to a target protein to be detected/quantified is then added to a biological sample, such as a cell lysate.
  • the spiked sample is then digested with one or more protease(s) for a suitable time period to allow digestion.
  • a separation is then performed (e.g. by HPLC, reverse-phase HPLC, capillary electrophoresis, ion exchange chromatography, etc.) to isolate the labeled internal standard and its corresponding target peptide from other peptides in the sample.
  • Microcapillary LC is a preferred method.
  • Each isolated peptide is then examined by monitoring of a selected reaction in the MS. This involves using the prior knowledge gained by the characterization of the peptide internal standard and then requiring the MS to continuously monitor a specific ion in the MS/MS or MS n spectrum for both the peptide of interest and the internal standard. After elution, the area under the curve (AUC) for both peptide standard and target peptide peaks are calculated. The ratio of the two areas provides the absolute quantification that can be normalized for the number of cells used in the analysis and the protein's molecular weight, to provide the precise number of copies of the protein per cell. Further details of the AQUA methodology are described in Gygi et al, and Gerber et al. supra.
  • AQUA internal peptide standards may desirably be produced, as described above, to detect and quantify any unique site (e.g. the G724S mutation point) within a mutant EGFR polypeptide of the invention.
  • an AQUA phosphopeptide may be prepared that corresponds to the EGFR peptide sequence immediately encompassing the G724S mutation point.
  • Peptide standards may be produced and such standards employed in the AQUA methodology to detect and quantify the presence of mutant (G724S, G719S, L858R, or any combination thereof) EGFR (i.e. the presence of the peptide sequence encompassing the G724S , G719S, L858R point mutation, or any combination thereof) in a biological sample.
  • an exemplary AQUA peptide of the invention comprises the amino acid sequence EVA, which corresponds to the three amino acids immediately flanking each side of the G724S, G719S or L858R mutation point in the mutant EGFR polypeptide.
  • EVA amino acid sequence
  • larger AQUA peptides comprising the mutant junction sequence (and additional residues downstream or upstream of it) may also be constructed.
  • a smaller AQUA peptide comprising less than all of the residues of such sequence (but still comprising the point of mutant junction itself) may alternatively be constructed.
  • Such larger or shorter AQUA peptides are within the scope of the present invention, and the selection and production of preferred AQUA peptides may be carried out as described above (see Gygi et al., Gerber et al., supra.).
  • Mutant- specific reagents provided by the invention also include nucleic acid probes and primers suitable for detection of a mutant EGFR polynucleotide.
  • the specific use of such probes in assays such as fluorescence in-situ hybridization (FISH) or polymerase chain reaction (PCR) amplification.
  • FISH fluorescence in-situ hybridization
  • PCR polymerase chain reaction
  • kits for the detection of mutant (G724S, G719S, L858R, or any combination thereof) EGFR in a biological sample comprising an isolated mutant EGFR-specific reagent of the invention and one or more secondary reagents.
  • Suitable secondary reagents for employment in a kit are familiar to those of skill in the art, and include, by way of example, buffers, detectable secondary antibodies or probes, activating agents, and the like.
  • the methods of the invention may be carried out in a variety of different assay formats known to those of skill in the art.
  • Immunoassays useful in the practice of the methods of the invention may be homogenous immunoassays or heterogeneous immunoassays.
  • the immunological reaction usually involves a mutant EGFR polypeptide-specific reagent (e.g. a mutant EGFR polypeptide-specific antibody), a labeled analyte, and the biological sample of interest.
  • the signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution.
  • Immunochemical labels that may be employed include free radicals, radio-isotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
  • Semi-conductor nanocrystal labels, or “quantum dots”, may also be advantageously employed, and their preparation and use has been well described. See generally, K. Barovsky, Nanotech. Law & Bus. 1(2): Article 14 (2004) and patents cited therein.
  • the reagents are usually the biological sample, a mutant EGFR polypeptide-specific reagent (e.g., a mutant EGFR-specific antibody), and suitable means for producing a detectable signal.
  • Biological samples as further described below may be used.
  • the antibody is generally immobilized on a support, such as a bead, plate or slide, and contacted with the sample suspected of containing the antigen in a liquid phase.
  • the support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal.
  • the signal is related to the presence of the analyte in the biological sample.
  • Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, enzyme labels, quantum dots, and so forth.
  • an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step.
  • the presence of the detectable group on the solid support indicates the presence of the antigen in the test sample.
  • suitable immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like.
  • Mutant EGFR polypeptide-specific monoclonal antibodies may be used in a "two-site” or “sandwich” assay, with a single hybridoma cell line serving as a source for both the labeled monoclonal antibody and the bound monoclonal antibody. Such assays are described in U.S. Pat. No. 4,376,110.
  • concentration of detectable reagent should be sufficient such that the binding of mutant EGFR polypeptide is detectable compared to background.
  • Antibodies useful in the practice of the methods disclosed herein may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation.
  • Antibodies or other mutant EGFR polypeptide-binding reagents may likewise be conjugated to detectable groups such as radiolabels (e.g., .sup.35S, .sup.1251, .sup.1311), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
  • Cell-based assays such flow cytometry (FC), immuno-histochemistry (IHC), or immunofluorescence (IF) are particularly desirable in practicing the methods of the invention, since such assay formats are clinically-suitable, allow the detection of mutant EGFR polypeptide expression in vivo, and avoid the risk of artifact changes in activity resulting from manipulating cells obtained from, e.g. a tumor sample in order to obtain extracts.
  • FC flow cytometry
  • IHC immuno-histochemistry
  • IF immunofluorescence
  • the methods of the invention are implemented in a flow-cytometry (FC), immuno-histochemistry (IHC), or immunofluorescence (IF) assay format.
  • FC flow-cytometry
  • IHC immuno-histochemistry
  • IF immunofluorescence
  • Flow cytometry may be employed to determine the expression of mutant EGFR polypeptide in a mammalian leukemia sample before, during, and after treatment with a drug targeted at inhibiting EGFR activity.
  • a drug targeted at inhibiting EGFR activity For example, tumor cells from a bone marrow sample may be analyzed by flow cytometry for mutant EGFR polypeptide expression and/or activation, as well as for markers identifying cancer cell types, etc., if so desired.
  • Flow cytometry may be carried out according to standard methods. See, e.g. Chow et al.,
  • Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001). Briefly and by way of example, the following protocol for cytometric analysis may be employed: fixation of the cells with 2% paraformaldehyde for 10 minutes at 37. degree. C. followed by
  • permeabilization in 90% methanol for 30 minutes on ice Cells may then be stained with the primary mutant EGFR polypeptide-specific antibody, washed and labeled with a fluorescent- labeled secondary antibody. The cells would then be analyzed on a flow cytometer (e.g. a Beckman Coulter FC500) according to the specific protocols of the instrument used. Such an analysis would identify the level of expressed mutant EGFR polypeptide in the tumor.
  • a flow cytometer e.g. a Beckman Coulter FC500
  • Immunohistochemical (IHC) staining may be also employed to determine the expression and/or activation status of mutant EGFR polypeptide in a mammalian cancer (e.g. AML) before, during, and after treatment with a drug targeted at inhibiting EGFR activity.
  • IHC may be carried out according to well-known techniques. See, e.g., ANTIBODIES; A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor
  • paraffin-embedded tissue e.g. tumor tissue from a biopsy
  • paraffin-embedded tissue is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slide in primary anti-mutant EGFR polypeptide antibody and secondary antibody; and finally detecting using ABC avidin/biotin method according to manufacturer's instructions.
  • Immunofluorescence assays may be also employed to determine the expression and/or activation status of mutant EGFR polypeptide in a mammalian cancer before, during, and after treatment with a drug targeted at inhibiting EGFR activity.
  • IF may be carried out according to well-known techniques. See, e.g. , J. M. Polak and S. Van Noorden (1997) INTRODUCTION TO IMMUNOCYTOCHEMISTRY, 2nd Ed.; ROYAL MICROSCOPY SOCIETY MICROSCOPY HANDBOOK 37, BioScientific/Springer-Verlag.
  • patient samples may be fixed in paraformaldehyde followed by methanol, blocked with a blocking solution such as horse serum, incubated with the primary antibody against mutant EGFR polypeptide followed by a secondary antibody labeled with a fluorescent dye such as Alexa 488 and analyzed with an epifluorescent microscope.
  • a blocking solution such as horse serum
  • Antibodies employed in the above-described assays may be advantageously conjugated to fluorescent dyes (e.g. Alexa488, PE), or other labels, such as quantum dots, for use in multi-parametric analyses along with other signal transduction (phospho-AKT, phospho-Erk 1/2) and/or cell marker (cytokeratin) antibodies.
  • fluorescent dyes e.g. Alexa488, PE
  • other labels such as quantum dots
  • mutant EGFR polypeptide A variety of other protocols, including enzyme-linked immunosorbent assay (ELISA), radio-Immunoassay (RIA), and fluorescent-activated cell sorting (FACS), for measuring mutant EGFR polypeptide are known in the art and provide a basis for diagnosing altered or abnormal levels of mutant EGFR polypeptide expression.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radio-Immunoassay
  • FACS fluorescent-activated cell sorting
  • mutant EGFR polypeptide-specific reagent comprises a heavy isotope labeled phosphopeptide (AQUA peptide) corresponding to a peptide sequence comprising the mutant junction of mutant EGFR polypeptide, as described above.
  • Mutant EGFR polypeptide-specific reagents useful in practicing the methods of the invention may also be mRNA, oligonucleotide or DNA probes that can directly hybridize to, and detect, mutant or truncated polypeptide expression transcripts in a biological sample.
  • formalin-fixed, paraffin-embedded patient samples may be probed with a fluorescein-labeled RNA probe followed by washes with formamide, SSC and PBS and analysis with a fluorescent microscope.
  • Polynucleotides encoding mutant EGFR polypeptide may also be used for diagnostic purposes.
  • the polynucleotides that may be used include oligonucleotide sequences, antisense RNA and DNA molecules.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of mutant EGFR polypeptide or truncated active EGFR polypeptide may be correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess expression of mutant EGFR polypeptide, and to monitor regulation of mutant EGFR polypeptide levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding mutant EGFR polypeptide or truncated active EGFR polypeptide, or closely related molecules, may be used to identify nucleic acid sequences which encode mutant EGFR polypeptide.
  • genomic sequences including genomic sequences, encoding mutant EGFR polypeptide or truncated active EGFR polypeptide, or closely related molecules
  • the specificity of the probe whether it is made from a highly specific region, e.g., 10 unique nucleotides in the mutant junction, or a less specific region, e.g., the 3' coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding mutant EGFR polypeptide, alleles, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the mutant EGFR polypeptide encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequence and encompassing the G724S, G719S, L858R mutation point, or any combination thereof , or from genomic sequence including promoter, enhancer elements, and introns of the naturally occurring EGFR polypeptides but comprising the G724S, G719S, L858R, or any combination thereof mutation points sequence.
  • a mutant EGFR polynucleotide or truncated EGFR polynucleotide of the invention may be used in Southern or northern analysis, dot blot, or other membrane-based
  • Mutant EGFR polynucleotides may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value.
  • nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding mutant EGFR polypeptide in the sample indicates the presence of the associated disease.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, which encodes mutant EGFR polypeptide, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease.
  • hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • PCR polymerase chain reaction
  • PCR oligomers may be chemically synthesized, generated enzymatically, or produced from a recombinant source. Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5' to 3') and another with antisense (3' to 5'), employed under optimized conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.
  • Methods which may also be used to quantitate the expression of mutant EGFR polypeptide include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby et al, J. Immunol. Methods, 159:235-244 (1993); Duplaa et al. Anal. Biochem. 229-236 (1993)).
  • the speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or calorimetric response gives rapid quantitation.
  • Example 1 Identification of a somatic mutation in the P-loop of the tyrosine kinase of EGFR that is predictive of response to EGFR antibody therapy therapy
  • Fresh frozen adenocarcinoma sampled were obtained from resection of a primary colorectal and a carcinoma as well as tissue from the non-affected Vail d'Hebron Hospital in Barcelona Spain. Patients were consented for the trial and tissue collection in a manner consistent with the local ethics board and following all institutional procedures.
  • the tumor sample was subsequently processed at the biological samples platform of the Broad Institute where DNA and RNA are both obtained from the tissue of tumor as well as the noncancerous normal colon. DNA was subjected to standard quality control measures as well as quantification of DNA contents using picogreen.
  • the DNA from the tumor sample as well as the matched germline DNA were used for the construction of Illumina sequencing libraries using standard library construction approaches. Following library construction, the tumor and normal each subject to whole genome shotgun
  • This sequencing performed on the Illumina genome analyzer was performed with paired 101-base pair reads with the goal of achieving approximately 30 fold coverage of each the tumor and germline genomes.
  • the genomic data were processed by the cancer genome analysis group of the Broad Institute. Briefly, the reads from the tumor and normal sequencing were mapped back to the referenced human genome to create BAM files of each genome. These files were
  • Firehose pipeline which contains distinct algorithms for detection of somatic mutations, somatic insertions and deletions, somatic copy number alterations as well as somatic genomic rearrangements in the tumor sample.
  • G724S three mutations in the tyrosine kinase domain were identified- G724S, G719S and L858R.
  • the mutation, G724S was notably located in a specific subdomain of the tyrosine kinase, the P-loop, which has been noted to be mutated recurrently in non-small cell lung carcinoma.
  • the G724S mutation was present in 10/42 reads from the tumor DNA that covered this specific base. In contrast, in the normal genome a mutation was noted in only one of the genomic reads from sequencing.

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Abstract

Cette présente invention concerne des méthodes de traitement, et d'estimation/surveillance de la réponse d'une cellule cancéreuse vis-à-vis d'une thérapie par un anticorps anti-EGFR.
PCT/US2011/060385 2010-11-12 2011-11-11 Procédés de prédiction d'une réponse à une thérapie par un anticorps anti-egfr WO2012065071A2 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005094357A2 (fr) * 2004-03-31 2005-10-13 The General Hospital Corporation Procede permettant de determiner la reponse d'un cancer a des traitements cibles par le recepteur du facteur de croissance epidermique
US20080286785A1 (en) * 2005-10-05 2008-11-20 Astrazeneca Uk Limited Method to predict or monitor the response of a patient to an erbb receptor drug
US20090181378A1 (en) * 2007-04-27 2009-07-16 Sanders Heather R Nucleic acid detection combining amplification with fragmentation
US20090318480A1 (en) * 2006-09-18 2009-12-24 Boehringer Ingelheim International Gmbh Method for treating cancer harboring egfr mutations
WO2010020618A1 (fr) * 2008-08-18 2010-02-25 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Susceptibilité aux inhibiteurs hsp90

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005094357A2 (fr) * 2004-03-31 2005-10-13 The General Hospital Corporation Procede permettant de determiner la reponse d'un cancer a des traitements cibles par le recepteur du facteur de croissance epidermique
US20080286785A1 (en) * 2005-10-05 2008-11-20 Astrazeneca Uk Limited Method to predict or monitor the response of a patient to an erbb receptor drug
US20090318480A1 (en) * 2006-09-18 2009-12-24 Boehringer Ingelheim International Gmbh Method for treating cancer harboring egfr mutations
US20090181378A1 (en) * 2007-04-27 2009-07-16 Sanders Heather R Nucleic acid detection combining amplification with fragmentation
WO2010020618A1 (fr) * 2008-08-18 2010-02-25 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Susceptibilité aux inhibiteurs hsp90

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