# Patent

## Title
Biomarkers for cancer treatment

## Abstract

The present invention provides identification of a thirty-five gene set that predicts the anticancer activity of inhibitors of the RAF/MEK/MAPK pathway, methods of qualifying cancer status in a subject, methods of identifying an anti-tumor response in a subject, methods of monitoring the efficacy of a therapeutic drug in a subject, and methods of identifying an agent useful in the treatment of a cancer based on expression of the thirty-five gene set.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS/PATENTS & INCORPORATION BY REFERENCE
This application claims priority to U.S. Provisional Application Ser. No. 60/788,014, filed Mar. 31, 2006, the contents of which are incorporated herein by reference.
Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; “application cited documents”), and each of the PCT and foreign applications or patents corresponding to and/or paragraphing priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference. More generally, documents or references are cited in this text, either in a Reference List before the paragraphs, or in the text itself; and, each of these documents or references (“herein-cited references”), as well as each document or reference cited in each of the herein-cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.


BACKGROUND OF THE INVENTION
One approach to the study of cancer is genetic profiling, an effort aimed at identifying perturbations in gene expression that lead to the malignant phenotype. However, cancers differ widely in their genetic “signature”, leading to difficulty in diagnosis and treatment, as well as in the development of effective therapeutics.
Genetic profiling of tumors may provide a more effective approach to cancer management and/or treatment. Accordingly, there is a need in the art to better understand the genetic profile of specific classes of tumors, in an effort to provide improved therapeutics, diagnostics and screening methods.
The kinase pathway comprising RAS, RAF, mitogen-activated protein kinase kinase (MEK) and extracellular signal regulated kinase (ERK) is activated in most human tumours, often through gain-of-function mutations of RAS and RAF family members (Davies, H. et al. 2002 Nature 417, 949-954). It has previously been shown that mutation of BRAF is associated with enhanced and selective sensitivity to MEK inhibition. This MEK dependency was observed in BRAF mutant cells regardless of tissue lineage. Since BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma and other cancers.
SUMMARY OF THE INVENTION
Biomarkers whose levels change depending on cancer status following treatment with a therapeutic drug have been identified and characterized herein. Profiles based on the same have been generated, and methods of using such profiles for qualification of cancer status defined.
In one aspect, the invention provides a biomarker indicative of the therapeutic efficacy of a Ras/Raf/MEK/ERK pathway inhibitor comprising one or more of Markers 1-35. In one embodiment of the invention, the biomarker is selected from the group consisting of SPRY2, ETV1, ETV4, ETV5, and DUSP6. In further embodiments of the invention, the Ras/Raf/MEK/ERK pathway inhibition is inhibition of MEK or Raf.
In another aspect, the invention provides a panel of biomarkers indicative of the therapeutic efficacy of a Ras/Raf/MEK/ERK pathway inhibitor comprising two or more of Markers 1-35. In one embodiment of the invention, the two or more biomarkers are selected from the group consisting of SPRY2, ETV1, ETV4, ETV5, and DUSP6. In further embodiments of the invention, the Ras/Raf/MEK/ERK pathway inhibition is inhibition of MEK or Raf.
In another aspect, the invention provides a method for selecting an individual for treatment with a Ras/Raf/MEK/ERK pathway inhibitor, comprising: obtaining a cancerous tumor sample from the individual; measuring the amount of a biomarker in the tumor sample, wherein the biomarker is selected from the group consisting of DUSP6 (Marker 1, Table 1), SPRY2 (Marker 15), ETV4 (Marker 23), ETV5 (Marker 21), PLK3 (Marker 12), and MAP2K3 (Marker 7); and selecting the individual for treatment with a Ras/Raf/MEK/ERK pathway inhibitor based on the measured amount of the biomarker. In one embodiment of the invention, the cancerous tumor is from an organ selected from the group consisting of skin, colon, thyroid, ovarian, lung, and pancreas. In another embodiment of the invention, the skin tumor is a melanoma. In further embodiments of the invention, cells of the tumor comprise an activated Ras/Raf/MEK/ERK pathway or a B-Raf mutation. In still further embodiments of the invention, the step of measuring comprises measuring the amount of nucleic acid encoding the biomarker in the sample or measuring the amount of a biomarker polypeptide.
In another aspect, the invention provides a method of qualifying cancer status in a subject comprising: measuring the amount of at least one biomarker in a tumor sample obtained from the subject, wherein the biomarker is selected from the group consisting of Markers 1-35; and correlating the measurement with cancer status, thereby qualifying cancer status in a subject. In one embodiment of the invention, the step of measuring comprises measuring the amount of at least one biomarker in a tumor sample obtained from the subject after treatment with a therapeutic drug, and comparing the amount to an amount of the biomarker in a tumor sample from the subject prior to treatment with the therapeutic drug. In one embodiment, the invention further comprise managing treatment of the subject based on cancer status. In an additional embodiment, the invention comprises obtaining the tumor sample.
In yet another embodiment of the invention, the subject is human. In further embodiments of the invention, cells of the tumor comprise an activated Ras/Raf/MEK/ERK pathway or have a B-Raf mutation. In another embodiment of the invention, the cancerous tumor is from an organ selected from the group consisting of skin, colon, thyroid, ovarian, lung, and pancreas. In a specific embodiment, the skin tumor is a melanoma. In another embodiment of the invention, the tumor is selected from the group consisting of: prostate carcinoma, lung carcinoma, glioma, acute myelogenous leukemia, pancreatic carcinoma, head and neck carcinomas. In further embodiments of the method of the invention, the step of measuring comprises measuring the amount of nucleic acid encoding the biomarker in the sample or measuring the amount of a biomarker polypeptide.
In another aspect, the invention provides a method for identifying an anti-tumor response in a subject exposed to a therapeutic drug, the method comprising determining the amount of a biomarker selected from the group consisting of Markers 1-35, or combinations thereof, in a tumor, wherein an increase in the amount of Markers 32-35, or a decrease in the amount of Markers 1-31, or combinations thereof, after exposure to the drug identifies an anti-tumor response in the subject.
In yet another aspect, the invention provides a method of monitoring the efficacy of a therapeutic drug in a subject comprising determining the amount of a biomarker selected from the group consisting of Markers 1-35, or combinations thereof, in a tumor, wherein an increase in the amount of Markers 32-35, or a decrease in the amount of Markers 1-31, or combinations thereof, after exposure to the drug indicates that the drug is effective.
In one embodiment of the invention, the subject is human. In further embodiments of the invention, cells of the tumor have a B-Raf mutation or an activated Ras/Raf/MEK/ERK pathway. In another embodiment of the invention, the cancerous tumor is from an organ selected from the group consisting of skin, colon, thyroid, ovarian, lung, and pancreas. In a specific embodiment, the skin tumor is a melanoma.
In one embodiment of the invention, the tumor is selected from the group consisting of: prostate carcinoma, lung carcinoma, glioma, acute myelogenous leukemia, pancreatic carcinoma, head and neck carcinomas. In yet another embodiment of the invention the therapeutic drug is an inhibitor of the Ras/Raf/MEK/ERK pathway. In further embodiments of the invention, the therapeutic drug is selected from the group consisting of: Ras, Raf, MEK or MAPK inhibitors. In another embodiment of the method of the invention, the step of measuring comprises measuring each of Markers 1-35.
In another aspect, the invention provides a method of identifying an agent useful in the treatment of a cancer, comprising: contacting a cancerous cell with a test agent; determining the amount of a biomarker selected from the group consisting of Markers 1-35; and comparing the amount of the biomarker with the amount of the biomarker in the cancerous cell prior to the step of contacting, wherein an increase in the amount of Markers 32-35, or a decrease in the amount of Markers 1-31, or combinations thereof, identifies the test agent as useful in the treatment of cancer. In one embodiment of the invention, the test agent is a Ras/Raf/MEK/ERK inhibitor. In specific embodiments, the test agent is a MEK inhibitor or a Raf inhibitor.
In another aspect, the invention provides a method for identifying a biomarker for determining the efficacy of a cancer treatment, comprising: contacting a cancer cell with a Ras, Raf, MEK or ERK inhibitor; measuring the amount of a candidate biomarker in the cancer cell; and comparing the amount of the biomarker to the amount of the biomarker in the cell prior to contacting the cancer cell with a Ras, Raf, MEK or MAPK inhibitor, wherein a difference identifies the candidate as a biomarker for determining the efficacy of a cancer treatment. In one embodiment of the invention, the cancer cell comprises a B-Raf mutation. In further embodiments of the invention, the inhibitor is a MEK inhibitor or a Raf inhibitor.
In another aspect, the invention provides a kit for determining the efficacy of a cancer therapy, comprising an adsorbent, wherein the adsorbent retains one or more biomarkers selected from one or more of Markers 1-35, and written instructions for use of the kit for determining the efficacy of a cancer therapy. In one embodiment of the invention, the adsorbent is an antibody, single or double stranded oligonucleotide, amino acid, protein, peptide or fragments thereof. In another embodiment of the invention, one or more biomarkers is detected using mass spectrometry, immunoassay, or PCR.
The invention also includes an biomarker profile derived from a biological sample exposed to a Ras/Raf/MEK/ERK pathway inhibitor, wherein the profile indicates therapeutic efficacy of a Ras/Raf/MEK/ERK pathway inhibitor, comprising an increased amount of a biomarker selected from the group consisting of Markers 32-35, and a decreased amount of a biomarker selected from the group consisting of Markers 1-31, or combinations thereof.
Other aspects of the invention are described in or are obvious from the following disclosure and are within the ambit of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
The following Detailed Description, given by way of example, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying drawings, incorporated herein by reference. Various preferred features and embodiments of the present invention will now be described by way of non-limiting example and with reference to the accompanying drawings in which:
FIG. 1 shows the nucleic acid and amino acid sequences corresponding to Markers 1-35.
FIG. 2 shows a graph depicting the relative enrichment significance as a function of time of exposure to MEK inhibitor, with each line corresponding to genes of a functional class based on biological process according to Gene Ontology (GO) classification.
FIG. 3 shows (A) a graph depicting the significance of difference between the two groups (control and MEK-inhibited) of genes, with the identified 45 probe sets (closed red circles) representing 35 genes which change significantly in either direction in response to MEK inhibition. Of these, 31 genes are downregulated (to the right of the 45 degree angle dotted line), and 4 genes are upregulated (to the left of the 45 degree angle dotted line) in response to MEK inhibition; and (B) a heatmap representation of expression of those genes represented by the red filled circles in A in individual cell lines in the two conditions (control, MEK inhibited), where red represents higher expression and blue represents lower gene expression.
FIG. 4A shows bar graphs depicting the relative levels of mRNA in cells as a function of time (2 hrs, 4 hrs, 8 hrs, and 24 hrs) following exposure to the MEK inhibitor (PD0325901). Decrease in mRNA levels are seen for five of the target genes, including DUSP6, SPRY2, ETV1, ETV5, and CCND1, in the cell line SkMel 5 (V600E BRAF). FIG. 4B shows the relative protein expression in cells as a function of time following exposure to MEK inhibitor (PD0325901 50 nM).
FIG. 5 schematically depicts a molecular interaction map of the Ras/Raf/MEK/ERK signaling pathway, which was constructed for the major components of the pathway, including RAS, RAF, MEK and MAPK, and the primary downstream targets of MAPK signaling. Of note, those genes identified in FIG. 3, which have associations with MAPK signaling are also shown, and are highlighted. At least 15 of the genes delineated in Table 1 are directly implicated as effectors of MAPK signaling.
In FIG. 6A, each cell line or xenograft tumor is represented as a single vertical lane (7+2) or (6+2) for each of the four conditions as identified. The last two lanes in each condition are the xenografts (SkMel 28 tumors for the V600E BRAF, and BT474 tumors for the WT BRAF). Plotted above is the shared vector of expression across 18 genes in four independent conditions, between wild-type and mutant BRAF status and control versus MEK inhibition. Data is represented as mean±SEM of the 18 genes per sample. Boxes represent cell lines, while triangles represent xenografts. Below is the heatmap representation of individual gene expression stratified by sample membership in the given conditions (center). FIG. 6B shows the ranked signed differential expression of all genes.
FIG. 7 shows a table providing sequence identifying information for the 35 biomarkers listed in Table 1.



DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Lackie and Dow, The Dictionary of Cell & Molecular Biology (3 ed. 1999); Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
As used herein, the “Ras/Raf/MEK/ERK pathway” refers to the intracellular kinase cascade comprising RAS, RAF, mitogen-activated protein kinase kinase (MEK) and extracellular signal regulated kinase (ERK). The Ras/Raf/MEK/ERK signaling pathway is known in the art and described in McCormick F. 1993 Nature 363(6424):15-6. As used herein, an “activated Ras/Raf/MEK/ERK pathway” refers to a detectable increase in levels of phosphorylated MAPK, and/or increased expression and/or activation of its primary targets (Favres, G. 2006 Bulletin du Cancer 93(4):25-30; Kolch, W. 2000 Biochem J 351(2):289-305; Peysonnaux, C. and Eychece, A. 2001 Biol Cell 93:53-62; Satyamoorthy, K., et al. 2003 Cancer Res 63:756-759; Houben, M., et al. 2004 J Carcinog 3:6). An “activated Ras/Raf/MEK/ERK pathway is also defined as an increase in kinase activity of members of the pathway. Methods for measuring kinase activity of Ras/Raf/MEK/ERK pathway members are taught, for example, in Davies et al., 2002, Nature 417:949.
As used herein, an “inhibitor” of the Ras/Raf/MEK/ERK pathway refers to a compound, such as a small molecule, chemical compound, biologic compound, antibody, antisense, siRNA, or the like that reduces the level of Ras/Raf/MEK/ERK pathway activation by at least 10%. Where Ras/Raf/MEK/ERK pathway activation is determined by measuring the amount of phosphorylated MAPK, an inhibitor of the pathway is a compound that will decrease the amount of phosphorylated MAPK by 10%. Similarly, “inhibition” of the Ras/Raf/MEKIERK pathway refers to a decrease in pathway activity, as measured by phosphorylated MAPK levels, by at least 10%.
A “subject” is a vertebrate, preferably a mammal, more preferably a primate and still more preferably a human. Mammals include, but are not limited to, primates, humans, farm animals, sport animals, and pets.
As used herein, “sample” or “biological sample” refers to a tumor sample.
As used herein, the term “panel” refers to a composition, such as an array, comprising one or more biomarkers. A “panel” can also refer to a profile or index of expression patterns of one or more biomarkers described herein. For example, a panel can refer to a solid surface comprising biomarker nucleic acid or polypeptide molecules. A panel can also be a description of the expression of one or more biomarkers for a given tumor or cancer type, or for a given patient. That is, by way of example, a panel of biomarkers for a given patient or given tumor or cancer may be as follows: Marker 1: downregulated; Marker 2: downregulated; Marker 10: downregulated; Marker 20: downregulated; Marker 32: upregulated.
The term “qualifying cancer status” refers to the association of the expression level of a given biomarker, or the expression pattern of a plurality of biomarkers, with the status of a cancer, wherein “status” refers to stage, degree, severity, operability, or prognosis. Qualifying cancer status can include quantifying the amount of a given biomarker, or can include a qualitative analysis of an expression pattern of multiple biomarkers.
“Managing subject treatment” refers to the behavior of the clinician or physician subsequent to the determination of cancer status. For example, if the result of the methods of the present invention is inconclusive or there is reason that confirmation of status is necessary, the physician may order more tests. Alternatively, if the status indicates that treatment is appropriate, the physician may schedule the patient for treatment, e.g., surgery, administer one or more therapeutic agents or radiation. Likewise, if the status is negative, e.g., late stage cancer or if the status is acute, no further action may be warranted. Furthermore, if the results show that treatment has been successful, a maintenance therapy or no further management may be necessary. For example, detection of the differential presence of one or more biomarkers in a tumor sample from an individual may indicate to a clinician the efficacy of a therapeutic drug (such as a MEK inhibitor) and, based on that, the clinician can manage the subject's treatment by continuing treatment with the therapeutic drug.
“Monitoring” refers to observing, measuring, and/or recording changes in a varying parameter (e.g. monitoring the efficacy of a Ras/Raf/MEK/ERK pathway inhibitor in a subject).
As used herein, the phrase “prior to treatment” can refer to a time prior to the commencement of treatment, but can also refer to a time prior to the current treatment. That is, “prior to treatment” can refer to a prior treatment.
“Marker” or “biomarker” in the context of the present invention refer to a polypeptide (of a particular apparent molecular weight) or nucleic acid, which is differentially present in a sample taken from subjects having cancer as compared to a comparable sample taken from control subjects (e.g., a person with a negative diagnosis or undetectable cancer, normal or healthy subject); or which is differentially present in a sample taken from a subject having cancer, prior to treatment as compared to after exposure to a therapeutic drug. The term “biomarker” is used interchangeably with the term “marker.” Polypeptide biomarkers can be identified by molecular mass in Daltons, and include the masses centered around the identified molecular masses for each marker. Nucleic acid biomarkers can be identified by sequence.
The term “measuring” means methods which include detecting or observing the presence or absence of marker(s) in the sample, quantifying the amount of marker(s) in the sample, and/or qualifying the type of biomarker. Measuring can be accomplished by methods known in the art and those further described herein, including but not limited to microarray analysis (with Significance Analysis of Microarrays (SAM) software), SELDI, PCR, and immunoassay. Any suitable methods can be used to detect and measure one or more of the markers described herein. These methods include, without limitation, mass spectrometry (e.g., laser desorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasmon resonance, ellipsometry, atomic force microscopy, and PCR (including quantitative PCR).
“Detect” refers to identifying the presence, absence or amount of the object to be detected.
The phrase “differentially present” refers to differences in the quantity and/or the frequency of a marker present in a sample taken from subjects having cancer as compared to a control subject, or in a sample taken from cancer subjects prior to treatment as compared to after exposure to a therapeutic drug. For example, some markers described herein are present at an elevated level in samples of subjects compared to samples from control subjects, or in samples of cancer subjects before vs. after exposure to a therapeutic drug. In contrast, other markers described herein are present at a decreased level in samples of cancer subjects compared to samples from control subjects, or in samples of cancer subjects before vs. after exposure to a therapeutic drug.
A marker can be differentially present in terms of quantity, frequency or both.
A polypeptide is differentially present between two samples if the amount of the polypeptide in one sample is statistically significantly different from the amount of the polypeptide in the other sample. For example, a polypeptide is differentially present between the two samples if it is present at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% greater than it is present in the other sample, or if it is detectable in one sample and not detectable in the other.
Alternatively or additionally, a polypeptide is differentially present between two sets of samples if the frequency of detecting the polypeptide in the cancer subjects' samples is statistically significantly higher or lower than in the control samples. For example, a polypeptide is differentially present between the two sets of samples if it is detected at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% more frequently or less frequently observed in one set of samples than the other set of samples.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms “polypeptide,” “peptide” and “protein” include glycoproteins, as well as non-glycoproteins.
“Gas phase ion spectrometer” refers to an apparatus that detects gas phase ions. Gas phase ion spectrometers include an ion source that supplies gas phase ions. Gas phase ion spectrometers include, for example, mass spectrometers, ion mobility spectrometers, and total ion current measuring devices. “Gas phase ion spectrometry” refers to the use of a gas phase ion spectrometer to detect gas phase ions.
“Mass spectrometer” refers to a gas phase ion spectrometer that measures a parameter that can be translated into mass-to-charge ratios of gas phase ions. Mass spectrometers generally include an ion source and a mass analyzer. Examples of mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these. “Mass spectrometry” refers to the use of a mass spectrometer to detect gas phase ions.
“Laser desorption mass spectrometer” refers to a mass spectrometer that uses laser energy as a means to desorb, volatilize, and ionize an analyte.
“Tandem mass spectrometer” refers to any mass spectrometer that is capable of performing two successive stages of m/z-based discrimination or measurement of ions, including ions in an ion mixture. The phrase includes mass spectrometers having two mass analyzers that are capable of performing two successive stages of m/z-based discrimination or measurement of ions tandem-in-space. The phrase further includes mass spectrometers having a single mass analyzer that is capable of performing two successive stages of m/z-based discrimination or measurement of ions tandem-in-time. The phrase thus explicitly includes Qq-TOF mass spectrometers, ion trap mass spectrometers, ion trap-TOF mass spectrometers, TOF-TOF mass spectrometers, Fourier transform ion cyclotron resonance mass spectrometers, electrostatic sector—magnetic sector mass spectrometers, and combinations thereof.
“Mass analyzer” refers to a sub-assembly of a mass spectrometer that comprises means for measuring a parameter that can be translated into mass-to-charge ratios of gas phase ions. In a time-of-flight mass spectrometer the mass analyzer comprises an ion optic assembly, a flight tube and an ion detector.
“Ion source” refers to a sub-assembly of a gas phase ion spectrometer that provides gas phase ions. In one embodiment, the ion source provides ions through a desorption/ionization process. Such embodiments generally comprise a probe interface that positionally engages a probe in an interrogatable relationship to a source of ionizing energy (e.g., a laser desorption/ionization source) and in concurrent communication at atmospheric or subatmospheric pressure with a detector of a gas phase ion spectrometer.
Forms of ionizing energy for desorbing/ionizing an analyte from a solid phase include, for example: (1) laser energy; (2) fast atoms (used in fast atom bombardment); (3) high energy particles generated via beta decay of radionucleides (used in plasma desorption); and (4) primary ions generating secondary ions (used in secondary ion mass spectrometry). The preferred form of ionizing energy for solid phase analytes is a laser (used in laser desorption/ionization), in particular, nitrogen lasers, Nd-Yag lasers and other pulsed laser sources. “Fluence” refers to the energy delivered per unit area of interrogated image. A high fluence source, such as a laser, will deliver about 1 mJ/mm2 to 50 mJ/mm2. Typically, a sample is placed on the surface of a probe, the probe is engaged with the probe interface and the probe surface is struck with the ionizing energy. The energy desorbs analyte molecules from the surface into the gas phase and ionizes them.
Other forms of ionizing energy for analytes include, for example: (1) electrons that ionize gas phase neutrals; (2) strong electric field to induce ionization from gas phase, solid phase, or liquid phase neutrals; and (3) a source that applies a combination of ionization particles or electric fields with neutral chemicals to induce chemical ionization of solid phase, gas phase, and liquid phase neutrals.
“Solid support” refers to a solid material that can be derivatized with, or otherwise attached to, a capture reagent. Exemplary solid supports include probes, microtiter plates and chromatographic resins.
“Probe” in the context of this invention refers to a device adapted to engage a probe interface of a gas phase ion spectrometer (e.g., a mass spectrometer) and to present an analyte to ionizing energy for ionization and introduction into a gas phase ion spectrometer, such as a mass spectrometer. A “probe” will generally comprise a solid substrate (either flexible or rigid) comprising a sample presenting surface on which an analyte is presented to the source of ionizing energy.
“Surface-enhanced laser desorption/ionization” or “SELDI” refers to a method of desorption/ionization gas phase ion spectrometry (e.g., mass spectrometry) in which the analyte is captured on the surface of a SELDI probe that engages the probe interface of the gas phase ion spectrometer. In “SELDI MS,” the gas phase ion spectrometer is a mass spectrometer. SELDI technology is described in, e.g., U.S. Pat. No. 5,719,060 (Hutchens and Yip) and U.S. Pat. No. 6,225,047 (Hutchens and Yip).
“Surface-Enhanced Affinity Capture” or “SEAC” is a version of SELDI that involves the use of probes comprising an absorbent surface (a “SEAC probe”). “Adsorbent surface” refers to a surface to which is bound an adsorbent (also called a “capture reagent” or an “affinity reagent”). An adsorbent is any material capable of binding an analyte (e.g., a target polypeptide or nucleic acid). “Chromatographic adsorbent” refers to a material typically used in chromatography. Chromatographic adsorbents include, for example, ion exchange materials, metal chelators (e.g., nitriloacetic acid or iminodiacetic acid), immobilized metal chelates, hydrophobic interaction adsorbents, hydrophilic interaction adsorbents, dyes, simple biomolecules (e.g., nucleotides, amino acids, simple sugars and fatty acids) and mixed mode adsorbents (e.g., hydrophobic attraction/electrostatic repulsion adsorbents). “Biospecific adsorbent” refers an adsorbent comprising a biomolecule, e.g., a nucleic acid molecule (e.g., an aptamer), a polypeptide, a polysaccharide, a lipid, a steroid or a conjugate of these (e.g., a glycoprotein, a lipoprotein, a glycolipid, a nucleic acid (e.g., DNA)-protein conjugate). In certain instances the biospecific adsorbent can be a macromolecular structure such as a multiprotein complex, a biological membrane or a virus. Examples of biospecific adsorbents are antibodies, receptor proteins and nucleic acids. Biospecific adsorbents typically have higher specificity for a target analyte than chromatographic adsorbents. Further examples of adsorbents for use in SELDI can be found in U.S. Pat. No. 6,225,047 (Hutchens and Yip, “Use of retentate chromatography to generate difference maps,” May 1, 2001).
In some embodiments, a SEAC probe is provided as a pre-activated surface that can be modified to provide an adsorbent of choice. For example, certain probes are provided with a reactive moiety that is capable of binding a biological molecule through a covalent bond. Epoxide and carbodiimidizole are useful reactive moieties to covalently bind biospecific adsorbents such as antibodies or cellular receptors.
“Adsorption” refers to detectable non-covalent binding of an analyte to an adsorbent or capture reagent.
“Eluant” or “wash solution” refers to an agent, typically a solution, which is used to affect or modify adsorption of an analyte to an adsorbent surface and/or remove unbound materials from the surface. The elution characteristics of an eluant can depend on, for example, pH, ionic strength, hydrophobicity, degree of chaotropism, detergent strength and temperature.
“Analyte” refers to any component of a sample that one wishes to detect. The term can refer to a single component or a plurality of components in the sample.
The “complexity” of a sample adsorbed to an adsorption surface of an affinity capture probe means the number of different protein species that are adsorbed.
“Array,” “microarray,” or “Biochip” refers to a solid substrate having a generally planar surface to which an adsorbent is attached. Frequently, the surface of the biochip comprises a plurality of addressable locations, each of which location has the adsorbent bound there. Biochips can be adapted to engage a probe interface, and therefore, function as probes. For a DNA microarray or polynucleotide array, one or more DNA molecules, preferably a collection of DNA spots, is attached to a solid surface. A DNA microarray can be used for expression profiling, monitoring expression levels for thousands of genes simultaneously, or for comparative genomic hybridization. A “protein biochip” refers to a biochip adapted for the capture of polypeptides.
“Protein biochip” refers to a biochip adapted for the capture of polypeptides. Many protein biochips are described in the art. These include, for example, protein biochips produced by Ciphergen Biosystems (Fremont, Calif.), Packard BioScience Company (Meriden Conn.), Zyomyx (Hayward, Calif.) and Phylos (Lexington, Mass.). Examples of such protein biochips are described in the following patents or patent applications: U.S. Pat. No. 6,225,047 (Hutchens and Yip, “Use of retentate chromatography to generate difference maps,” May 1, 2001); International publication WO 99/51773 (Kuimelis and Wagner, “Addressable protein arrays,” Oct. 14, 1999); U.S. Pat. No. 6,329,209 (Wagner et al., “Arrays of protein-capture agents and methods of use thereof,” Dec. 11, 2001) and International publication WO 00/56934 (Englert et al., “Continuous porous matrix arrays,” Sep. 28, 2000). Protein biochips produced by Ciphergen Biosystems comprise surfaces having chromatographic or biospecific adsorbents attached thereto at addressable locations. Biochips are further described in: WO 00/66265 (Rich et al., “Probes for a Gas Phase Ion Spectrometer,” Nov. 9, 2000); WO 00/67293 (Beecher et al., “Sample Holder with Hydrophobic Coating for Gas Phase Mass Spectrometer,” Nov. 9, 2000); U.S. patent application US20030032043A1 (Pohl and Papanu, “Latex Based Adsorbent Chip,” Jul. 16, 2002) and U.S. patent application 60/350,110 (Um et al., “Hydrophobic Surface Chip,” Nov. 8, 2001).
Upon capture on a biochip, analytes can be detected by a variety of detection methods selected from, for example, a gas phase ion spectrometry method, an optical method, an electrochemical method, atomic force microscopy and a radio frequency method. Gas phase ion spectrometry methods are described herein. Of particular interest is the use of mass spectrometry, and in particular, SELDI. Optical methods include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry). Optical methods include microscopy (both confocal and non-confocal), imaging methods and non-imaging methods. Immunoassays in various formats (e.g., ELISA) are popular methods for detection of analytes captured on a solid phase. Electrochemical methods include voltametry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy.
“Immunoassay” is an assay that uses an antibody to specifically bind an antigen (e.g., a marker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
“Antibody” refers to a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen). The recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad immunoglobulin variable region genes. Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab-′′ and F(ab)-′′ 2 fragments. The term “antibody,” as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. “Fc” portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CH1, CH2 and CH3, but does not include the heavy chain variable region.
The phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to marker “X” from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with marker “X” and not with other proteins, except for polymorphic variants and alleles of marker “X”. This selection may be achieved by subtracting out antibodies that cross-react with marker “X” molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
The term “obtaining” as in “obtaining the tumor sample” is intended to include any method of acquiring the tumor sample.
The terms “comprises”, “comprising”, and the like are intended to have the broad meaning ascribed to them in U.S. Patent Law and can mean “includes”, “including” and the like.
It is to be understood that this invention is not limited to the particular component parts of a device described or process steps of the methods described, as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly indicates otherwise.
Other definitions appear in context throughout the specification.
II. Biomarkers of the Invention
The present invention is based in part on the discovery that in cancers characterized by activation of the Ras/Raf/MEK/ERK pathway, inhibition of the pathway results in the differential presence of specific biomarkers. These biomarkers can then be correlated with disease status, be used to identify subjects as candidates for certain treatments, or be used to monitor treatment efficacy.
In one aspect, the present invention identifies biomarkers that are indicative of the therapeutic efficacy of a drug in a cancer subject. Indeed, it has been discovered that a number of genes are expressed at an increased or decreased level in tumor samples of cancer subjects after exposure to a therapeutic drug. These genes are involved in DNA replication, ribosome biogenesis, regulation of signal transduction, regulation of progression through the cell cycle, MAP kinase phosphatase activity, transcription factor activity, cell proliferation, cell-cell signaling, and regulation of transcription from PolII promoters (FIG. 2). Included are the biomarkers listed, without limitation, in Table 1, below.








TABLE 1





Marker
















1
DUSP6


2
ARID5A


3
BRS1


4
MYC


5
FOSL1


6
SPRED2


7
MAP2K3


8
EGR1


9
CD3EAP


10
PHLDA2


11
POLR1C


12
PLK3


13
HES1


14
PYCRL


15
SPRY2


16
SH3BP1


17
IER2


18
CCND1


19
IER3


20
SPRY4


21
ETV5


22
ETV1


23
ETV4


24
SLC4A7


25
RKHD2


26
GJB1


27
DUSP4


28
LNK


29
HEY1


30
TNC


31
ST8SIAI


32
HYDIN


33
NFKB2


34
GRINI


35
SORBS2









While initially identified using genetic analysis, the biomarkers described herein include both nucleic acid and amino acid biomarker sequences. For example, the nucleic acid sequence (e.g., mRNA) a differentially present gene can be used as a biomarker, but the polypeptide sequence encoded thereby can also be used as a biomarker. Accordingly, reference to detection or measurement of a biomarker can refer to detection or measurement of either or both of a polynucleotide or polypeptide sequence (e.g., by using PCR or SELDI, respectively, to detect the biomarker).
While the present disclosure is focused on the biomarkers noted in Table 1, these markers can be classified based on function. For example, the biomarkers of Table 1 can be classified into functional groups including biomarkers involved in DNA replication, ribosome biogenesis, regulation of signal transduction, regulation of progression through the cell cycle, MAP kinase phosphatase activity, transcription factor activity, cell proliferation, cell-cell signaling, and regulation from a PolII promoter. Accordingly, it is contemplated, that additional genes and encoded proteins that fall within these functional classifications could be useful biomarkers according to the invention.
The biomarkers of the invention represent known genes, the sequences of which are available through public databases known to those of skill in the art. FIG. 7 provides exemplary accession numbers for the biomarkers of Table 1. Also contemplated within the scope of the instant invention are variants of the biomarkers of Table 1. Proteins frequently exist in a sample in a plurality of different forms. These forms can result from either or both of pre- and post-translational modification. Pre-translational modified forms include allelic variants, splice variants and RNA editing forms. Post-translationally modified forms include forms resulting from proteolytic cleavage (e.g., fragments of a parent protein), glycosylation, phosphorylation, lipidation, oxidation, methylation, cysteinylation, sulphonation and acetylation. When detecting or measuring a protein in a sample, the ability to differentiate between different forms of a protein depends upon the nature of the difference and the method used to detect or measure. For example, an immunoassay using a monoclonal antibody will detect all forms of a protein containing the epitope and will not distinguish between them. However, a sandwich immunoassay that uses two antibodies directed against different epitopes on a protein will detect all forms of the protein that contain both epitopes and will not detect those forms that contain only one of the epitopes. In diagnostic assays, the inability to distinguish different forms of a protein has little impact when the forms detected by the particular method used are equally good biomarkers as any particular form. However, when a particular form (or a subset of particular forms) of a protein is a better biomarker than the collection of different forms detected together by a particular method, the power of the assay may suffer. In this case, it is useful to employ an assay method that distinguishes between forms of a protein and that specifically detects and measures a desired form or forms of the protein. Distinguishing different forms of an analyte or specifically detecting a particular form of an analyte is referred to as “resolving” the analyte.
Mass spectrometry is a particularly powerful methodology to resolve different forms of a protein because the different forms typically have different masses that can be resolved by mass spectrometry. Accordingly, if one form of a protein is a superior biomarker for a disease than another form of the biomarker, mass spectrometry may be able to specifically detect and measure the useful form where traditional immunoassay fails to distinguish the forms and fails to specifically detect to useful biomarker.
One useful methodology combines mass spectrometry with immunoassay. First, a biospecific capture reagent (e.g., an antibody, aptamer or Affibody that recognizes the biomarker and other forms of it) is used to capture the biomarker of interest. Preferably, the biospecific capture reagent (e.g., an adsorbent) is bound to a solid phase, such as a bead, a plate, a membrane or a chip. After unbound materials are washed away, the captured analytes are detected and/or measured by mass spectrometry. (This method also will also result in the capture of protein interactors that are bound to the proteins or that are otherwise recognized by antibodies and that, themselves, can be biomarkers.) Various forms of mass spectrometry are useful for detecting the protein forms, including laser desorption approaches, such as traditional MALDI or SELDI, and electrospray ionization (discussed further below).
Also included with the scope of the invention are variants of the nucleic acid or amino acid biomarker sequences, including, but not limited to amino acid or nucleic acid sequences having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to the sequences shown in FIG. 1.
To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions.times. 100%). In one embodiment, the two sequences are the same length.
The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilised for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, word length=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, word length=3 to obtain amino acid sequences homologous to a protein molecule of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilised as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search, which detects distant relationships between molecules (Id.). When utilising BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm utilised for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilising the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
III. Methods of the Invention
The invention further provides that the biomarkers described herein, used individually or in combination with other biomarkers, can be employed in various methods. In the first, biomarkers of the invention are employed in methods for selecting subjects for treatment with a Ras/Raf/MEK/ERK pathway inhibitor. As such, the amount of at least one biomarker delineated herein may be predictive of a subject's response to treatment. Biomarkers particularly useful in such an aspect of the invention include, without limitation, DUSP6 (Marker 1, Table 1), SPRY2 (Marker 15), ETV4 (Marker 23), ETV5 (Marker 21), PLK3 (Marker 12), and MAP2K3 (Marker 7). A subject is identified as a candidate for treatment with a Ras/Raf/MEK/ERK pathway inhibitor if the one or more of the biomarkers are detected in an amount greater than twice the average value (of an unselected population of tumors or patient samples, or cell lines).
In another aspect, the amount of at least one biomarker delineated herein provides information for qualifying cancer status in a subject. The determination of status may be the result of an isolated measurement, or it may precede and follow exposure of the subject to a therapeutic drug, thus providing indication of a potentially changing cancer status in the subject. For example, isolated measurement of one or more biomarkers may be used to determine whether a patient's cancer is of a type that is amenable to treatment with a Ras/Raf/MEK/ERK pathway inhibitor, whereas a repeated measurement may be used to determine a patient's responsiveness to a treatment regime using a Ras/Raf/MEK/ERK pathway inhibitor.
The biomarkers of the invention find further use in methods for identifying anti-tumor response and for monitoring the efficacy of a therapeutic drug in a cancer subject. For example, the biomarkers described herein have been shown to be differentially present in cancer cells following treatment with inhibitors of the Ras/Raf/MEK/ERK pathway. Thus, the level of one or more biomarkers in a patient that has been treated with a Ras/Raf/MEK/ERK pathway inhibitor can be determined, wherein the differential presence of the biomarker can be indicative of the efficacy of the treatment. Based on this information, a physician or other medical professional, can manage treatment of the patient. Managing patient treatment refers to the behavior of the clinician or physician subsequent to the determination of cancer status. For example, if the result of the methods of the present invention is inconclusive or there is reason that confirmation of status is necessary, the physician may order more tests. Alternatively, if the status indicates that treatment is appropriate, the physician may schedule the patient for treatment, e.g., surgery, administer one or more therapeutic agents or radiation. Likewise, if the status is negative, e.g., late stage cancer or if the status is acute, no further action may be warranted. Furthermore, if the results show that treatment has been successful, a maintenance therapy or no further management may be necessary. For example, detection of the differentially presence of one or more biomarkers in a tumor sample from an individual may indicate to a clinician the efficacy of a therapeutic drug (such as a MEK inhibitor) and, based on that, the clinician may manage the subjects treatment by continuing treatment with the therapeutic drug.
Conversely, therapeutic agents useful in cancer treatment may be identified using methods employing the biomarkers delineated herein. For example, a patient or other subject with cancer may be treated with a Ras/Raf/MEK/ERK pathway inhibitor (such as a chemical or small molecule), for a given period of time. The amount of one or more biomarkers present in cancer cells can then be determined in the subject wherein the differential presence of the biomarker in the subject relative to some earlier time (e.g., prior to administration of the inhibitor, or an earlier time of inhibitor administration) indicates that the pathway inhibitor may be a useful therapeutic. For example, the presence (or differential presence) of a biomarker may be determined following treatment of a subject with a Ras/Raf/MEK/ERK using a biochip. Although the variability of genes across biochips can be large, an about 2-fold or greater change in the context of the entirety of 22,000 genes (on the chip) is significant, when the gene is scored in at least one of the two conditions as “present” (with a raw signal intensity about 100 or greater), and the difference between the two conditions (within the context of the whole microarray) is associated with a p-value of about ≦0.001. Similarly, the presence (or differential presence) of a biomarker may be determined using PCR, wherein a candidate agent is identified where a 50% reduction or increase in mRNA levels for a given biomarker is observed.
Therapeutic Drugs
A “therapeutic drug,” as used herein, refers to a compound (such as a small molecule, biologic compound, or chemical compound) that exerts an inhibitory effect on the Ras/Raf/MEK/ERK signaling pathway in a cell. Preferred therapeutic drugs contemplated herein include Ras inhibitors, Raf inhibitors, MEK inhibitors, and ERK inhibitor. Exemplary MEK inhibitors include, without limitation, AZD6244 (Astra Zeneca), PD0325901 (Pfizer), XL518 (Exelixis), hypothemycin, and anthrax lethal factor. Exemplary RAF inhibitors include, without limitation, RAF265 (Novartis), PLX4032 (Plexxikon), XL281 (Exelixis), and Bay 43-9006 (Bayer). Exemplary Ras inhibitors include farnesyl transferase inhibitors such as Zarnestra (Johnson & Johnson)
Therapeutic drugs useful in the invention, in addition to the foregoing, include compounds that may act on other components of the Ras/Raf/MEK/ERK pathway other than Ras, Raf, MEK, or ERK, and that ultimately result in a decrease in pathway activation, for example, by inhibiting an activator of Ras, Raf, MEK, or ERK, or by inhibiting an effector molecule downstream from Ras, Raf, MEK, or ERK, or by activating an inhibitory effector molecule downstream or upstream from Ras, Raf, MEK, or ERK.
Cancers
Cancers contemplated for the methods described herein are cancers characterized by BRAF mutations and/or an activated Ras/Raf/MEK/ERK pathway. In particular, such cancers include, without limitation, melanoma, colon cancer, thyroid cancer, ovarian cancer, breast cancer, lung cancer, and pancreatic cancer.
Tumors/cancers sampled for the methods described herein constitute any tumor (cancer) with an activating mutation or lesion in the pathway that results in MAPK activation. This includes, without limitation, sarcomas, carcinomas and other solid tumor cancers, including, but not limited to germ line tumors, tumors of the central nervous system, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, glioma, pancreatic cancer, stomach cancer, liver cancer, colon cancer, melanoma, renal cancer, bladder cancer, esophageal cancer, cancer of the larynx, cancer of the parotid, cancer of the biliary tract, rectal cancer, endometrial cancer, squamous cell carcinomas, adenocarcinomas, small cell carcinomas, neuroblastomas, mesotheliomas, adrenocortical carcinomas, epithelial carcinomas, desmoid tumors, desmoplastic small round cell tumors, endocrine tumors, Ewing sarcoma family tumors, germ cell tumors, hepatoblastomas, hepatocellular carcinomas, lymphomas, melanomas, non-rhabdomyosarcome soft tissue sarcomas, osteosarcomas, peripheral primative neuroectodermal tumors, retinoblastomas, rhabdomyosarcomas, Wilms tumors, and the like.
A. Sample Collection and Preparation
The biomarkers can be measured in tumor samples from cancer subjects before and after exposure of the subject to a therapeutic drug. Accordingly, tumor samples may be collected from a subject over a period of time. Furthermore, obtaining numerous samples from a subject over a period of time can be used to verify results from earlier detections and/or identify a differential expression as a result of exposure to a therapeutic drug. Generally, tumor samples are typically collected from a subject via biopsy, but may be collected using other known clinical methods specific for particular types of cancers, such as the collection of peripheral blood or bone marrow for hematological malignancies, or the collection of peripheral blood mononuclear cells for surrogate assays of target inhibition/biomarker measurement.
In one embodiment of the invention, the tumor samples are analyzed without additional preparation and/or separation procedures. In another embodiment of the invention, sample preparation and/or separation can involve, without limitation, any of the following procedures, depending on the type of sample collected and/or types of biomarkers searched: removal of high abundance polypeptides; addition of preservatives and calibrants, desalting of samples; concentration of sample substances; protein digestions; and fraction collection. In yet another embodiment of the invention, sample preparation techniques concentrate information-rich biomarkers and deplete polypeptides or other substances that would carry little or no information such as those that are highly abundant in or native to the tumor.
In another embodiment of the invention, sample preparation takes place in a manifold or preparation/separation device. Such a preparation/separation device may, for example, be a microfluidics device. In yet another embodiment of the invention, the preparation/separation device interfaces directly or indirectly with a detection device. Such a preparation/separation device may, for example, be a fluidics device.
In another embodiment of the invention, the removal of undesired polypeptides (e.g., high abundance, uninformative, or undetectable polypeptides) can be achieved using high affinity reagents, high molecular weight filters, column purification, ultracentrifugation and/or electrodialysis. High affinity reagents include antibodies that selectively bind to high abundance polypeptides or reagents that have a specific pH, ionic value, or detergent strength. High molecular weight filters include membranes that separate molecules on the basis of size and molecular weight. Such filters may further employ reverse osmosis, nanofiltration, ultrafiltration and microfiltration.
Ultracentrifugation constitutes another method for removing undesired polypeptides. Ultracentrifugation is the centrifugation of a sample at about 60,000 rpm while monitoring with an optical system the sedimentation (or lack thereof) of particles. Finally, electrodialysis is an electromembrane process in which ions are transported through ion permeable membranes from one solution to another under the influence of a potential gradient. Since the membranes used in electrodialysis have the ability to selectively transportions having positive or negative charge and reject ions of the opposite charge, electrodialysis is useful for concentration, removal, or separation of electrolytes.
In another embodiment of the invention, the manifold or microfluidics device performs electrodialysis to remove high molecular weight polypeptides or undesired polypeptides. Electrodialysis can be used first to allow only molecules under approximately 30 kD to pass through into a second chamber. A second membrane with a very small molecular weight (roughly 500 D) allows smaller molecules to egress the second chamber.
Upon preparation of the samples, biomarkers of interest may be separated in another embodiment of the invention. Separation can take place in the same location as the preparation or in another location. In one embodiment of the invention, separation occurs in the same microfluidics device where preparation occurs, but in a different location on the device. Samples can be removed from an initial manifold location to a microfluidics device using various means, including an electric field. In another embodiment of the invention, the samples are concentrated during their migration to the microfluidics device using reverse phase beads and an organic solvent elution such as 50% methanol. This elutes the molecules into a channel or a well on a separation device of a microfluidics device.
Chromatography constitutes another method for separating subsets of substances. Chromatography is based on the differential absorption and elution of different substances. Liquid chromatography (LC), for example, involves the use of fluid carrier over a non-mobile phase. Conventional LC columns have an in inner diameter of roughly 4.6 mm and a flow rate of roughly 1 ml/min. Micro-LC has an inner diameter of roughly 1.0 mm and a flow rate of roughly 40 ul/min. Capillary LC utilizes a capillary with an inner diameter of roughly 300 im and a flow rate of approximately 5 ul/min. Nano-LC is available with an inner diameter of 50 um-1 mm and flow rates of 200 nl/min. The sensitivity of nano-LC as compared to HPLC is approximately 3700 fold. Other types of chromatography contemplated for additional embodiments of the invention include, without limitation, thin-layer chromatography (TLC), reverse-phase chromatography, high-performance liquid chromatography (HPLC), and gas chromatography (GC).
In another embodiment of the invention, the samples are separated using capillary electrophoresis separation. This will separate the molecules based on their eletrophoretic mobility at a given pH (or hydrophobicity).
In another embodiment of the invention, sample preparation and separation are combined using microfluidics technology. A microfluidic device is a device that can transport liquids including various reagents such as analytes and elutions between different locations using microchannel structures.
A biomarker can be modified before analysis to improve its resolution or to determine its identity. For example, the biomarker may be subject to proteolytic digestion before analysis. Any protease can be used. Proteases, such as trypsin, that are likely to cleave the biomarkers into a discrete number of fragments are particularly useful. The fragments that result from digestion function as a fingerprint for the products, thereby enabling their detection indirectly. This is particularly useful where there are biomarkers with similar molecular masses that might be confused for the product in question. Also, proteolytic fragmentation is useful for high molecular weight products, because smaller products are more easily resolved by mass spectrometry. In specific embodiments, the proteases occur or naturally exist in the tumor sample.
To improve detection resolution of the biomarkers, neuraminidase can, for instance, be used to remove terminal sialic acid residues from glycoproteins to improve binding to an anionic adsorbent (e.g., cationic exchange ProteinChip® arrays) and to improve detection resolution. In another example, the biomarkers can be modified by the attachment of a tag of particular molecular weight that specifically bind to molecular markers, further distinguishing them. Optionally, after detecting such modified products, the identity of the products can be further determined by matching the physical and chemical characteristics of the modified products in a protein database (e.g., SwissProt).
It has been found that proteins frequently exist in a sample in a plurality of different forms characterized by a detectably different mass. These forms can result from either, or both, of pre- and post-translational modification. Pre-translational modified forms include allelic variants, slice variants and RNA editing forms. Post-translationally modified forms include forms resulting from proteolytic cleavage (e.g., fragments of a parent protein), glycosylation, phosphorylation, lipidation, oxidation, methylation, cystinylation, sulphonation and acetylation. Modified forms of any biomarker of this invention also may be used, themselves, as biomarkers in the profiles. In certain cases, the modified forms may exhibit better discriminatory power in diagnosis than the specific forms set forth herein.
For some of the method embodiments of the invention, it may be helpful to purify the biomarker whose differential presence has been detected by the methods disclosed herein prior to subsequent analysis. Nearly any means known to the art for the purification and separation of small molecular weight substances, e.g., anion or cation exchange chromatography, gas chromatography, liquid chromatography or high pressure liquid chromatography may be used. Methods of selecting suitable separation and purification techniques and means of carrying them out are known in the art (see, e.g., Labadarious et. al., J Chromatography (1984) 310:223-231, and references cited therein; and Shahrokhin and Gehrke, J. Chromatography (1968) 36:31-41, and Niessen J. Chromatography (1998) 794:407-435). To the extent that it is desired to determine the differential presence of a nucleic acid biomarker, the biomarker may be purified using known methods including, slab or capillary gel electrophoresis.
In another embodiment of the method of the invention, purification of the biomarker comprises fractioning a sample comprising one or more protein products by size-exclusion chromatography and collecting a fraction that includes the one or more products; and/or fractioning a sample comprising the one or more products by anion exchange chromatography and collecting a fraction that includes the one or more products. Fractionation is monitored for purity on normal phase and immobilized nickel arrays. Generating data on immobilized biomarker fractions on an array is accomplished by subjecting the array to laser ionization and detecting intensity of signal for mass/charge ratio; and transforming the data into computer readable form. Preferably, fractions are subjected to gel electrophoresis and correlated with data generated by mass spectrometry. In one aspect, gel bands representative of potential biomarkers are excised and subjected to enzymatic treatment and are applied to biochip arrays for peptide mapping.
B. Detection and Quantitation of Biomarkers
Any suitable method can be used to detect (a differential presence of) one or more of the biomarkers described herein. Successful practice of the invention can be achieved with one or a combination of methods that can detect and, preferably, quantify the biomarkers. These methods include, without limitation, hybridization-based methods including those employed in biochip arrays, mass spectrometry (e.g., laser desorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasmon resonance, ellipsometry and atomic force microscopy. For nucleic acid biomarkers, methods for detection and quantitation include PCR, quantitative PCR, northern blot analysis, southern blot analysis, mass spectrometry and the like.
Methods may further include, by one or more of electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)n, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)n, quadrupole mass spectrometry, fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero. Spectroscopic methods for detecting and quantifying protein biomarkers are known in the art and are described, for example in U.S. Pat. Nos. 5,719,060; 6,225,047; 5,719,060; 6,124,137 and PCT International Publication No. WO 03/64594.
In another embodiment of the invention, the biomarkers of the invention are measured by a method other than mass spectrometry or other than methods that rely on a measurement of the mass of the biomarker. In one such embodiment that does not rely on mass, the biomarkers of this invention are measured by immunoassay. Immunoassay requires biospecific capture reagents, such as antibodies, to capture the biomarkers. Antibodies can be produced by methods well known in the art, e.g., by immunizing animals with the biomarkers. Biomarkers can be isolated from samples based on their binding characteristics. Alternatively, if the amino acid sequence of a polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies by methods well known in the art.
This invention contemplates traditional immunoassays including, for example, sandwich immunoassays including ELISA or fluorescence-based immunoassays, as well as other enzyme immunoassays. Nephelometry is an assay done in liquid phase, in which antibodies are in solution. Binding of the antigen to the antibody results in changes in absorbance, which is measured. In the SELDI-based immunoassay, a biospecific capture reagent for the biomarker is attached to the surface of an MS probe, such as a pre-activated ProteinChip array. The biomarker is then specifically captured on the biochip through this reagent, and the captured biomarker is detected by mass spectrometry.
In one embodiment, the biomarker can be a nucleic acid, wherein the nucleic acid can be detected and/or quantified using methods known in the art. For example, nucleic acid biomarkers may be detected using PCR (disclosed in U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,965,188 and others in detail). In one embodiment, a real time PCR method is used to enable a convenient and reliable quantitative measurement of biomarker nucleic acids having a wide dynamic range. The real time PCR technique includes the method by using a TaqMan probe using ABIPRISM7700.TM. (Applied Biosystems) and the method by using LightCycler.TM. (Ropche Diagnostics). Particularly in the latter case, in a high rate reaction cycle in which a temperature cycle of PCR is completed for some 10 minutes, a change of an amplified amount of a DNA synthesized for every cycle can be detected in a real time. DNA detection method of the real time PCR method includes 4 methods using a DNA-binding pigment (intercalator), a hybridization probe (kissing probe), TaqMan probe, or Sunrise Uniprimer (molecular beacon). On the other hand, the expression level of a biomarker gene can be analyzed by using a DNA-binding pigment such as SYBR GreenI. SYBR GreenI is a binding pigment specific to a double strand of the DNA and, when bound to a double strand, an inherent fluorescence intensity is reinforced. By adding SYBR GreenI at the PCR reaction and measuring the fluorescence intensity at the end of each cycle of an elongation reaction, the increase in a PCR product can be detected. For detection of a biomarker gene, similar to normal PCR, a primer is designed by using a commercialized gene analysis software on the basis of a sequence of the biomarker gene. SYBR GreenI detects a nonspecific product and, thus, requires designing an optimal primer. Required designing standards are a length of an oligomer, a base composition of the sequence, a GC content, and a Tm value.
Microarray-/Biochip-Based Methods
Detection methods may include use of a microarray/biochip array. Biochip arrays useful in the invention include protein and nucleic acid arrays. One or more biomarkers are captured on the biochip array and subjected to laser ionization to detect the molecular weight of the products. Analysis of the products is, for example, by molecular weight of the one or more biomarkers against a threshold intensity that is normalized against total ion current.
The biochip surfaces may, for example, be ionic, anionic, hydrophobic; comprised of immobilized nickel or copper ions, comprised of a mixture of positive and negative ions; and/or comprised of one or more antibodies, single or double stranded nucleic acids, proteins, peptides or fragments thereof, amino acid probes, or phage display libraries. Many protein biochips are described in the art. These include, for example, protein biochips produced by Ciphergen Biosystems (Fremont, Calif.), Packard BioScience Company (Meriden Conn.), Zyomyx (Hayward, Calif.) and Phylos (Lexington, Mass.). Examples of such protein biochips are described in the following patents or patent applications: U.S. Pat. No. 6,225,047 (Hutchens and Yip, “Use of retentate chromatography to generate difference maps,” May 1, 2001); International publication WO 99/51773 (Kuimelis and Wagner, “Addressable protein arrays,” Oct. 14, 1999); U.S. Pat. No. 6,329,209 (Wagner et al., “Arrays of protein-capture agents and methods of use thereof,” Dec. 11, 2001) and International publication WO 00/56934 (Englert et al., “Continuous porous matrix arrays,” Sep. 28, 2000).
Biomarkers may be captured with capture reagents immobilized to a solid support, such as a biochip, a multiwell microtiter plate, a resin, or nitrocellulose membranes that are subsequently probed for the presence of proteins. Capture can be on a chromatographic surface or a biospecific surface. For example, a tumor sample containing the biomarkers may be placed on the active surface of a biochip for a sufficient time to allow binding. Then, unbound molecules are washed from the surface using a suitable eluant, such as phosphate buffered saline. In general, the more stringent the eluant, the more tightly the proteins must be bound to be retained after the wash.
Upon capture on a biochip, analytes can be detected by a variety of detection methods selected from, for example, a gas phase ion spectrometry method, an optical method, an electrochemical method, atomic force microscopy and a radio frequency method. Also of interest is the use of mass spectrometry, for example, SELDI. Optical methods include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry). Optical methods include microscopy (both confocal and non-confocal), imaging methods and non-imaging methods. Immunoassays in various formats (e.g., ELISA) are popular methods for detection of analytes captured on a solid phase. Electrochemical methods include voltametry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy.
C. Qualification of Cancer Status
As indicated above, the invention provides methods for qualifying cancer status in a subject using a biomarker profile, as specified herein. The biomarkers can be used alone or in combination with other products. The biomarkers are differentially present in tumor samples of a cancer patient before and after exposure to a therapeutic drug. For example, some of the markers are expressed at an elevated level and/or are present at a higher frequency after treatment, while some of the products are expressed at a decreased level and/or are present at a lower frequency after treatment. Therefore, generating a biomarker profile for a subject would provide useful information regarding cancer status.
In some embodiments, isolated (i.e., not before and after treatment, but, rather, at a single timepoint) quantitation of a biomarker is useful and can be correlated with a qualification of cancer status. Thus, the amount of the biomarker detected in a subject being tested may qualify cancer status.
In certain embodiments of the methods of qualifying cancer status, the methods further comprise managing subject treatment based on the status. The invention also provides for such methods where the biomarkers (or specific combination of biomarkers) are measured again after such subject management. In these cases, the methods are used to monitor the status of the cancer, e.g., candidacy for treatment with a Ras/Raf/MEK/ERK pathway inhibitor, response to cancer treatment, remission of the disease or progression of the disease.
The biomarkers of the present invention have a number of other uses. For example, identification of an anti-tumor response or monitoring the efficacy of a therapeutic drug in a cancer subject takes into account the amount of the biomarker(s) in a tumor sample before and after exposure of the subject to a therapeutic drug (up or down regulation of the biomarker(s)). The amounts are measured under the same or substantially similar experimental conditions but at different time periods preceding and following treatment. The biomarkers of the invention can also be used to identify an agent useful in the treatment of cancer.
The detection of a differential presence of a plurality of biomarkers in a tumor sample may improve the indication of therapeutic efficacy of a therapeutic drug in the treatment of a cancer.
In another embodiment of the invention, the output from a detection device can subsequently be processed, stored, and further analyzed or assayed using a bio-informatics system. A bio-informatics system may include one or more of the following, without limitation: a computer; a plurality of computers connected to a network; a signal processing tool(s); a pattern recognition tool(s); a tool(s) to control flow rate for sample preparation, separation, and detection.
The data processing utilizes mathematical foundations. In another embodiment of the invention, dynamic programming is used to align a separation axis with a standard separation profile. Intensities may be normalized, for example, by fitting roughly 90% of the intensity values into a standard spectrum. The data sets can then be fitted using wavelets designed for separation and mass spectrometer data. In yet another embodiment of the invention, data processing filters out some of the noise and reduces spectrum dimensionality, potentially allowing for pattern recognition.
Following data processing, pattern recognition tools can be utilized to identify subtle differences between phenotypic states. Pattern recognition tools are based on a combination of statistical and computer scientific approaches, which provide dimensionality reduction. Such tools are scalable. Data so obtained may be stored on a computer readable medium.
D. Kits
In one aspect, the invention provides kits for qualifying cancer status in a subject, wherein the kits can be used to detect the differential presence of the biomarkers described herein. For example, the kits can be used to detect a differential presence of any combination of the biomarkers in tumor samples of cancer subjects before and after exposure to a therapeutic drug. The kits of the invention have many applications. For example, the kits can be used to monitor efficacy of a therapeutic drug in a cancer subject. The kits can also be used to identify agents useful in the treatment of cancer.
In specific embodiments, kits of the invention contain a biomarker, which is optionally isotopically or fluorescently labeled.
The kits of the invention may include instructions, reagents, testing equipment (test tubes, reaction vessels, needles, syringes, etc.), standards for calibration, and/or equipment. Reagents may include acids, bases, oxidizing agents, and marker species. The instructions provided in a kit according to the invention may be directed to suitable operational parameters in the form of a label or a separate insert.
The kits may also include an adsorbent, wherein the adsorbent retains one or more biomarkers described herein (polynucleotide or polypeptide), and written instructions for use of the kit for qualification of cancer status in a subject. Such a kit could, for example, comprise: (a) a substrate comprising an adsorbent thereon, wherein the adsorbent is suitable for binding a biomarker, and (b) instructions to detect the biomarker(s) by contacting a tumor sample with the adsorbent and detecting the product(s) retained by the adsorbent. Accordingly, the kit could comprise (a) a DNA probe that specifically binds to a biomarker; and (b) a detection reagent. Such a kit could further comprise an eluant (as an alternative or in combination with instructions) or instructions for making an eluant, wherein the combination of the adsorbent and the eluant allows detection of the biomarker using, for example, gas phase ion spectrometry.
This invention is further illustrated by the following examples, which should not be construed as limiting. A skilled artisan should readily understand that other similar instruments with equivalent function/specification, either commercially available or user modified, are suitable for practicing the instant invention. Rather, the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.
EXAMPLES
Example 1
Response of BRAF Mutant and Wild Type Cell Lines to MEK Inhibitor
Tumors with high MAPK activity due to activating BRAF mutations demonstrate sensitivity to pharmacologic inhibition by MEK (Solit, et al. Nature. January 19; 439(7074):358-62). Eleven cells lines, including five BRAF mutants, are characterized by their tissue of lineage and mechanism of MAPK activation, were used for the experiments described herein, and are listed in Table 2. All the cell lines have high levels of MAPK activity.







TABLE 2







Characteristics of cell lines used on experiments













Alteration





of Receptor


Cell line
Tissue of origin
BRAF status
tyrosine kinase





SkMel 1
Melanoma
V600E



SkMel 5
Melanoma
V600E


SkMel 19
Melanoma
V600E


SkMel 28
Melanoma
V600E


MALME 3M
Melanoma
V600E


Colo205
Colorectal carcinoma
V600E


HT29
Colorectal carcinoma
V600E


SkMel 31
Melanoma
WT
Increase in copy





number of EGFR


BT474
Breast cancer
WT
HER2 amplification


SkBr3
Breast cancer
WT
HER2 amplification


MDA-468
Breast cancer
WT
EGFR amplification


A431
Squamous cell
WT
EGFR amplification



carcinoma


H1650
Non-small cell lung
WT
EGFR mutation



cancer









Tumors in which high MAPK activity is due to activating mutations in B-RAF (V600EBRAF, the most common activating mutation of RAF serine/threonine kinases) are sensitive to pharmacologic inhibition by MEK. Treatment of tumor cell lines and xenograft models with small molecule inhibitors of MEK (CI-1040, PD0325901, Pfizer) caused a decline in cyclin D levels and hypophosphorylation of RB, resulting in G1 arrest and inhibition of proliferation. Tumor growth delay was achieved, compared to vehicle-treated mice, in several V600E B-RAF xenograft models of melanoma and colon carcinoma, and analysis of tumor samples by western blot and immunohistochemistry revealed decline in both cyclin D and Ki-67, a marker of proliferation. In contrast, tumors with high MAPK activity under the control of activated receptors were resistant to MEK inhibition in both in vitro assays and xenograft models. Although effectiveness of the drug at its expected target, phosphorylated MAPK, was not measurably different in this group, an accompanying decline in cyclin D, change in cell cycle distribution, or anti-proliferative response in the HER2/EGFR (WT BRAF) tumors was not observed.
Example 2
Enrichment Significance of Genes Altered in Response to MEK Inhibition as a Function of Time in a MEK Inhibitor Sensitive V600E BRAF Melanoma Cell Line
SkMel 28 cells (a melanoma cell line with the V600E BRAF mutation) were treated with the MEK inhibitor, PD0325901 (50 nM), for 2, 8, or 24 hours, or no treatment at all (=time 0 hours). Total cellular RNA was analyzed using an Affymetrix U133A 2.0 platform. Pairwise comparisons of gene expression at each individual time point to reference sample (time 0 hours) were performed, and genes were selected using a threshold of two-fold or greater change in either direction. The number of genes changing significantly increased as a function of time following exposure to MEK inhibitor (2 hours—91 genes; 8 hours—327 genes; 24 hours—1191 genes).
Thus, genes which changed greater than or equal to two-fold in either direction in response to PD0325901 compared to control were identified at three time points following MEK inhibition. All genes were assigned to a functional class based on biological process, according to Gene Ontology (GO) classification (FIG. 2). The number of genes changing in each functional classification was assigned a p-value based on the significance of the number of genes changing given the total number of genes changed at that time point, compared to the total number of genes in the GO functional class, and the total number of genes on the chip. An enrichment score was assigned to each functional class, and was defined as the −log 10(p-value).
As an example, for MAPK phosphatase genes at 2 hours, 3 were found to be changed, of 63 unique genes changed at this timepoint. There are 9 genes representing this functional class out of a total of 13,116 genes on the chip, so fewer genes would have been expected to have changed due to chance alone. Their deviation from expected is highly significant, with a p-value of 8.69×10−6 (fisher's exact test), and, therefore, is assigned an enrichment score of 5.06).
The genes most significantly enriched at early time points include those whose roles include regulation of signal transduction and MAP kinase phosphatase activity. Also highly enriched are genes whose functions include transcription factor activity, cell proliferation, and regulation of progression through the cell cycle. At the later time point (24 hours), these functional classifications lose their significance, whereas other functional classes of genes not expected to result from direct consequences of MEK inhibition, are increased in their enrichment. These data form the justification for examining the profiles of genes changed in response to MEK inhibition in a large panel of cell lines at 8 hours.
Example 3
Determination of Genes Whose Expression Changes in Response to MEK Inhibition in a Panel of Cell Lines with V600E BRAF
Mean expression of genes was determined for a panel of V600E BRAF cell lines in each of two conditions, MEK-inhibited (PD0325901 50 nM) or DMSO control, both at 8 hours. The panel of V600E BRAF cell lines included 5 melanoma cell lines (SkMel 1, SkMel 5, SkMel 19, SkMel 28, and MALME 3M) and two colorectal carcinoma cell lines (Colo205 and HT29).
Significance analysis of microarray (SAM) methods were used to determine the significance of difference between the two groups of genes (using a false discovery rate of 9%). 45 probe sets (closed red circles) were identified, representing 35 genes which change significantly in either direction in response to MEK inhibition (FIG. 3A). Of these, 31 genes are downregulated, and 4 genes are upregulated, in response to MEK inhibition. Expression of the genes was also presented as a heatmap representation (FIG. 3B).
Of note, all 35 genes are known to be associated with MAPK signaling (i.e., are effectors of MAPK activity) (FIG. 5), indicating a highly non-random signature.
Example 4
RNA and Protein Levels of a Subset of Genes Identified in FIG. 3 as a Function of Time Following MEK Inhibition
Quantitative RT-PCR was used to determine relative levels of mRNA in cells as a function of time following exposure to the MEK inhibitor (PD0325901 50 nM). SkMel 5 cells were treated with MEK inhibitor (PD0325901 50 nM) for the times indicated (2 hrs, 4 hrs, 8 hrs, and 24 hrs). Following total RNA extraction and an in vitro reverse transcription step, synthesized cDNA was used in a PCR reaction containing pre-designed probe-primer sets purchased from Applied Biosystems and run on an ABI 7500 RT-PCR machine, using software supplied by Applied Biosystems. Relative levels of mRNA compared to the reference sample (time 0) were determined using the ΔΔCT calculation, using HPRT as a housekeeping gene for normalization.
In brief, each gene for each sample is assigned a value based on the number of PCR amplification cycles required for the amplified gene product to cross a detection threshold (i.e., a gene detectable at 25 amplification cycles is present in greater starting quantity than a gene product detectable at 30 cycles). The cycle value for each sample for the gene of interest is subtracted from the cycle value for a reference sample (untreated, or time 0), and this value is the delta CT for the gene of interest. The same is performed for a housekeeping gene (such as GAPDH or HPRT), the expression of which is not expected to change significantly by the perturbation (i.e. addition of the MEK inhibitor). This is the delta CT for the housekeeping gene. The delta CT (housekeeping gene) is subtracted from the delta CT (gene of interest), and this is the delta delta CT. Thus, a small difference in the expression of the housekeeping gene from sample to sample is taken into account to minimize differences due to sample preparation.
Decreases in mRNA levels were seen for five of the target genes, including DUSP6, SPRY2, ETV1, ETV5, and CCND1, in the cell line SkMel 5 (V600E BRAF) (FIG. 4A).
Following protein isolation using NP40 1% lysis buffer, Western blot analysis was used to determine relative protein expression in cells as a function of time following exposure to MEK inhibitor (PD0325901 50 nM). A decrease in protein expression was seen for two of the target genes, including DUSP6 and SPRY2, in three cell lines, SkMel 5, SkMel 19, and Colo205, all harboring the V600E BRAF mutation (FIG. 4B). For the Western Blot analysis, DUSP6 antibody (SantaCruz (#8598)), and SPRY2 antibody (Upstate Cell Signaling Solutions).
Example 5
A Multi-Gene Vector of Expression Shared Between Cell Line and Xenograft Models
An 18 gene subset of the 35 genes from FIG. 3 was defined, comprised of those genes present in a mixed lineage (colon plus melanoma) analysis of mean expression across cell lines in MEK inhibited and control conditions. Expression of this gene profile was extracted from Affymetrix U133A gene chips for 7 cell lines with V600E BRAF, 2 xenograft tumors with V600E BRAF, 6 cell lines with WT BRAF, and 2 xenograft tumors with WT BRAF, each in the two conditions—control (untreated) or MEK-inhibited (PD0325901 50 nM for 8 hours for cell lines, or 25 mg/kg for 8 hours for murine xenografts) (FIG. 6A).
Those 35 genes identified as most significantly changed in V600E BRAF cell lines were ranked among the entire profile of 22,000 genes on the microarray with regard to the degree to which they change in response to MEK inhibition, in each of three other conditions: WT BRAF cell lines, V600E BRAF xenografts, and WT BRAF xenografts. The 35 genes, which cluster at the top in the V600E BRAF cell lines (not shown), are widely distributed in the rank of their change in the WT BRAF cell lines and xenografts, whereas they cluster among the most significantly changed genes in the V600E BRAF xenografts, suggesting a high degree of concordance between cell line and xenograft data for the V600E BRAF class (p-value=10−36) (FIG. 6B).
Example 6
Predicting Candidacy for Ras/Raf/MEK/ERK Pathway Inhibitor Treatment
The following experiments were performed to identify biomarkers useful to identify promising candidates for treatment with Ras/Raf/MEK/ERK pathway inhibitor. These studies were conducted to determine whether the greater magnitude in change in the B-RAF mutant cell lines for this select group of genes was due to higher basal expression in B-RAF mutant cells compared to B-RAF WT/RTK activated cells. In a test of significance on each of the 23 probes generated in the B-RAF mutant mixed lineage analysis of cell lines upon MEK inhibition, we evaluated the background difference in expression between wild-type and mutant BRAF independent of MEK inhibition. Six of these, including DUSP6, SPRY2, ETV4 and ETV5, MAP2K3 and PLK3, had higher expression in the B-RAF mutant group, with fold change difference between 2.17 and 6.09 (p≦0.02; one-tailed Student's t-test). Both SPRY4 and SPRED2, as well as MYC, were also significant yet with reduced fold change difference (fold change of 1.77 to 1.92; p=0.018 to 0.057), and the balance did not show differential expression between BRAF status uncoupled from MEK inhibition (0.57<FC<1.23 and 0.057≦p≦0.49; not shown). Relative expression, shown as mean log transformed expression values, for 14 most significant probe sets, representing 10 unique genes, are shown in Table 3, and illustrate the differences in expression in B-RAF mutant and B-RAF wild-type in a MEK-independent fashion.







TABLE 3







log expression












Probe set
Gene






ID
symbol
V600E
WT
fold diff
p-value















203348_s_at
ETV5
10.904
7.491
6.086
0.004


211603_s_at
ETV4
9.229
6.481
5.957
0.001


208893_s_at
DUSP6
10.175
5.404
5.288
0.005


203349_s_at
ETV5
11.119
8.201
4.781
0.004


220098_at
HYDIN
2.919
4.169
3.664
0.047


208892_s_at
DUSP6
10.896
8.162
2.952
0.018


208891_at
DUSP6
10.826
8.188
2.930
0.022


204011_at
SPRY2
10.180
7.573
2.802
0.011


204958_at
PLK3
7.473
6.332
2.260
0.012


215498_s_at
MAP2K3
10.747
9.602
2.173
0.000


212466_at
SPRED2
6.417
5.087
1.919
0.057


221489_s_at
SPRY4
10.044
8.557
1.909
0.042


202431_s_at
MYC
11.443
10.232
1.881
0.040


212458_at
SPRED2
10.288
9.309
1.768
0.018









Each of the 10 genes changes in response to MEK inhibition in each of the B-RAF mutant, MEK sensitive cell lines tested, but that only a subset of the genes are overexpressed in those cell lines compared to the RTK activated cells. When analyzed as a group, statistically significant differences upon MEK inhibition were not noted for the WT B-RAF group, but small changes were detected for individual cell line pairs. Thus we have functionally classified the genes which move in a MEK-ERK dependent fashion into at least three groups: 1) those that are both ERK dependent and B-RAF dependent for expression, 2) those that are ERK dependent only it the B-RAF mutant cells but not B-RAF dependent for expression, and 3) those that are B-RAF independent and ERK dependent in both groups of tumor cells. In other words, some genes tend to have higher expression in B-RAF mutant transformed cells, and are downregulated on exposure to MEK inhibition (DUSP6, ETV5). Others do not have higher expression in B-RAF mutant tumors compared to BRAF wild type, and thus their expression could be a result of transformation via a variety of molecular events (EER3), but they are ERK dependent in only MEK sensitive B-RAF mutant cell lines, whereas their expression does not change as a result of MEK inhibition in the WT B-RAF group of tumors. It is the former subset of genes which may be predictive of response to MEK inhibitors, in those tumors both with and without BRAF mutation. (i.e., the example of a WT BRAF melanoma without an identified lesion in the pathway, but with high expression of the output genes, would be expected to be MEK inhibitor sensitive even in the absence of the V600E BRAF mutation). So while these genes are markers of BRAF mutation, they may also be markers of cells with or without other mutations, that for some other reason (lineage/unidentified mutation/amplification of a WT kinase), have high output and are MEK dependent.

## Claims

1. A panel of isolated biomarkers consisting of DUSP6 (Marker 1), SPRY2 (Marker 15) and one or more biomarkers selected from the group consisting of Markers 2-14 and 16-35.

2. The panel of claim 1, wherein said one or more biomarkers are selected from the group consisting of ETV1, ETV4, and ETV5.

3. The panel of claim 1, wherein the differential expression of a biomarker in response to an agent is indicative of the therapeutic efficacy of the agent as an inhibitor of the Ras/Raf/MEK/ERK pathway.

4. The panel of claim 3, wherein said agent is a Raf inhibitor or MEK inhibitor.

5. The panel of claim 3, wherein said inhibitor is selected from the group consisting of AZD6244, PD0325901, XL518, hypothemycin, anthrax lethal factor, RAF265, PLX4032, XL281, Bay 43-9006, and a farnesyl transferase inhibitor.

6. A panel of isolated biomarkers consisting of DUSP6 (Marker 1), SPRY2 (Marker 15), ETV1 (Marker 22), ETV4 (Marker 23), and ETV5 (Marker 21) and one or more biomarkers selected from the group consisting of Markers 2-14, 16-20, and 24-35.

7. The panel of claim 6, wherein the differential expression of a biomarker in response to an agent is indicative of the therapeutic efficacy of the agent as an inhibitor of the Ras/Raf/MEK/ERK pathway.

8. The panel of claim 6, wherein said Ras/Raf/MEK/ERK pathway inhibitor is a Raf inhibitor or a MEK inhibitor.

9. The panel of claim 6, wherein said inhibitor is selected from the group consisting of AZD6244, PD0325901, XL518, hypothemycin, anthrax lethal factor, RAF265, PLX4032, XL281, Bay 43-9006, and a farnesyl transferase inhibitor.