WO2017120376A1 - Quantification de protéine pour thérapie anticancéreuse optimale - Google Patents

Quantification de protéine pour thérapie anticancéreuse optimale Download PDF

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WO2017120376A1
WO2017120376A1 PCT/US2017/012389 US2017012389W WO2017120376A1 WO 2017120376 A1 WO2017120376 A1 WO 2017120376A1 US 2017012389 W US2017012389 W US 2017012389W WO 2017120376 A1 WO2017120376 A1 WO 2017120376A1
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peptide
level
amol
protein
biological sample
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Fabiola CECCHI
Manish MONGA
Todd Hembrough
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Expression Pathology Inc
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Expression Pathology Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5752—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the lungs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57575—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

  • New and improved methods for treating cancer patients are provided.
  • the methods provide new assays for measuring the levels of specific proteins in tumor tissue from patients and then, based upon those levels, treating the patients with an optimized medication regimen. More specifically, new quantitative assays for the proteins XRCC1, ECAD, CAT and NQOl are provided.
  • Measurements from these assays are used to identify those patients that will respond to, or are most likely to respond to, treatment with regimens that include the anticancer drug pemetrexed, and/or other anti-cancer drugs in the antifolate class of drugs, such as lometrexol, AG2034, LY309887 and pelitrexol, that function to kill tumor cells in a similar manner by attacking the GART protein.
  • regimens that include the anticancer drug pemetrexed, and/or other anti-cancer drugs in the antifolate class of drugs, such as lometrexol, AG2034, LY309887 and pelitrexol, that function to kill tumor cells in a similar manner by attacking the GART protein.
  • Pemetrexed is a member of the antifolate class of drugs that enter the tumor cell via the Folate Receptor- alpha (FR-a), the Receptor-beta (FR- ⁇ ), proton-coupled folate transporter (PCFT), and the Folate transporter 1 (RFC).
  • FR-a Folate Receptor- alpha
  • FR- ⁇ the Receptor-beta
  • PCFT proton-coupled folate transporter
  • RRC Folate transporter 1
  • GART glycinamide ribonucleotide synthetase
  • TS thymidylate synthase
  • DHFR dihydrofolate reductase
  • the presence and/or quantitative levels of XRCC1, ECAD, CAT and NQOl protein expression in the tumor tissue is determined by quantitating a specified fragment peptide derived from subsequences of each of the full-length proteins in digests prepared from formalin-fixed tissue samples.
  • the presence and/or quantitative level of KRAS and GART also can be measured using specific fragment peptides derived from the proteins as described in PCT/US2015/040208 (filed July 13, 2015), PCT/U2016/45072 (filed August 1, 2016) and US application 62/199,202, (filed July 30, 2015), the contents of each of which are hereby incorporated by reference in their entireties.
  • the patient is treated with a regimen that includes the pemetrexed therapeutic agent, and, optionally, other drugs that function similarly to pemetrexed.
  • the KRAS level is below the level of detection and levels of the GART protein are found to be above a specified quantitative level, the patient is treated with a regimen that does not include pemetrexed therapeutic agent, nor other drugs that function similarly such as lometrexol, AG2034, LY309887 and pelitrexol.
  • specified levels of XRCC1, ECAD, CAT and NQOl are identified, such that when expression of the respective proteins in a tumor sample is observed to be below the specified level then the patient is treated with a regimen that includes the pemetrexed therapeutic agent, and, optionally, other drugs that function similarly to pemetrexed. If levels of the protein are found to be above a specified quantitative level, the patient is treated with a regimen that does not include pemetrexed therapeutic agent, nor other drugs that function similarly.
  • the specified XRCC1, ECAD, CAT, NQOl, KRAS and GART fragment peptides are detected using mass spectrometry-based Selected Reaction Monitoring (SRM), also referred to as Multiple Reaction Monitoring (MRM), and referred to herein as an SRM/MRM assay.
  • SRM mass spectrometry-based Selected Reaction Monitoring
  • MRM Multiple Reaction Monitoring
  • An SRM/MRM assay is used to detect the presence and quantitatively measure the amount of the specified fragment peptides, directly in cells procured from cancer patient tissue, such as, for example formalin fixed cancer tissue.
  • the amount of the specific peptides is then used to quantitate the amount of intact XRCC1, ECAD, CAT, NQOl, KRAS and GART proteins in the tumor sample.
  • Specific and optimized therapeutic agents and treatment strategies are then determined and implemented to treat an individual cancer patient's disease based on how much of the XRCC1, ECAD, CAT, NQOl, KRAS and
  • Methods are provided for treating a patient suffering from lung cancer comprising:
  • the reference level of the GART fragment peptide may be, for example, 900 amol ⁇ g., +/- 250 amol ⁇ g, +/- 150 amol ⁇ g, +/- 100 amol ⁇ g, +/- 50 amol ⁇ g, or +/- 25 amol ⁇ g, of biological sample protein analyzed.
  • the KRAS fragment peptide is either detected above the lower limit of detection or is not detected.
  • the protein digest may be a protease digest, such as a trypsin digest prepared, for example, using the liquid tissue protocol.
  • the mass spectrometry may be, for example, tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, hybrid ion trap/quadrupole mass spectrometry and/or time of flight mass spectrometry.
  • the mode of mass spectrometry used may be, for example, Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), Parallel Reaction Monitoring (PRM), intelligent Selected Reaction Monitoring (iSRM), and/or multiple Selected Reaction Monitoring (mSRM).
  • the specified KRAS peptide may have the amino acid sequence as set forth as SEQ ID NO: 6.
  • the specified GART peptide may have the amino acid sequence as set forth as SEQ ID NO:5.
  • the tumor sample may be, for example, a cell, collection of cells, or a solid tissue.
  • the tumor sample may be formalin fixed solid tissue, and may be paraffin embedded tissue.
  • quantifying the specified GART fragment peptide may include determining the amount of the GART peptide in the sample by comparing to a spiked internal standard peptide of known amount, where both the native peptide in the biological sample and the internal standard peptide corresponds to the same amino acid sequence of the GART fragment peptide as shown in SEQ ID NO:5.
  • the internal standard peptide may be, for example, an isotopically labeled peptide.
  • the isotopically labeled internal standard peptide may comprise one or more heavy stable isotopes selected from 18 0, 17 0, 15 N, 13 C, 2 H or combinations thereof.
  • Detecting and quantitating the specified GART fragment peptide and/or detecting the KRAS peptide can be combined with detecting and quantitating other peptides from other proteins in a multiplex format so that the treatment decision about which agent used for treatment is based upon specific levels of the specified GART fragment peptide, or detection of the KRAS peptide, in combination with other peptides/proteins in the biological sample.
  • the therapeutic strategy may include the combination of pemetrexed and cisplatin agents.
  • Methods are also provided for measuring the level of the human XRCC1 protein in a human biological sample of formalin-fixed tissue, comprising detecting and/or quantifying the amount of an XRCC1 fragment peptide in a protein digest prepared from the human biological sample using mass spectrometry; and calculating the level of XRCC1 protein in the sample; where the XRCC1 fragment peptide is SEQ ID NO:l, and where the level is a relative level or an absolute level.
  • Methods are also provided for measuring the level of the human CAT protein in a human biological sample of formalin-fixed tissue, comprising detecting and/or quantifying the amount of an CAT fragment peptide in a protein digest prepared from the human biological sample using mass spectrometry; and calculating the level of CAT protein in the sample; where the CATT fragment peptide is SEQ ID NO:2, and where the level is a relative level or an absolute level.
  • Quantifying the specified CAT fragment peptide may include determining the amount of the CAT peptide in the sample by comparing to a spiked internal standard peptide of known amount, where both the native peptide in the biological sample and the internal standard peptide corresponds to the same amino acid sequence of the CAT fragment peptide as shown in SEQ ID NO:2.
  • Methods also are provided for measuring the level of the human NQOl protein in a human biological sample of formalin-fixed tissue, comprising detecting and/or quantifying the amount of an NQOl fragment peptide in a protein digest prepared from the human biological sample using mass spectrometry; and calculating the level of NQOlprotein in the sample; where the NQOlfragment peptide is SEQ ID NO:3, and where the level is a relative level or an absolute level.
  • Quantifying the specified NQOl fragment peptide may include determining the amount of the NQOl peptide in the sample by comparing to a spiked internal standard peptide of known amount, where both the native peptide in the biological sample and the internal standard peptide corresponds to the same amino acid sequence of the NQOl fragment peptide as shown in SEQ ID NO:3.
  • Methods are also provided for measuring the level of the human ECAD protein in a human biological sample of formalin-fixed tissue, for example, paraffin-embedded tissue, comprising detecting and/or quantifying the amount of an ECAD fragment peptide in a protein digest, such as a protease digest, prepared from the human biological sample using mass spectrometry; and calculating the level of ECAD protein in the sample; where the ECAD fragment peptide is SEQ ID NO:4, and where the level is a relative level or an absolute level.
  • the tissue may be obtained from a tumor.
  • the method may further include the step of fractionating the protein digest prior to detecting and/or quantifying the amount of the fragment peptide.
  • Quantifying the fragment peptide may include comparing the amount of the fragment peptide in one biological sample to the amount of the same fragment peptide in a different and separate biological sample, or may include determining the amount of the fragment peptide in a biological sample by comparison to an added internal standard peptide of known amount, where the fragment peptide in the biological sample is compared to an internal standard peptide having the same amino acid sequence; and where the internal standard peptide is an isotopically labeled peptide.
  • detecting and/or quantifying the amount of the fragment peptide in the protein digest indicates the presence of the corresponding modified or unmodified protein and an association with cancer in the subject, and the results may be correlated to the diagnostic stage/grade/status of the cancer.
  • the correlating step may be combined with detecting and/or quantifying the amount of other proteins or peptides from other proteins in a multiplex format to provide additional information about the diagnostic stage/grade/status of the cancer.
  • the patient from which the biological sample was obtained is administered a therapeutically effective amount of a therapeutic agent, where the therapeutic agent and/or amount of the therapeutic agent administered is based upon the amount of the fragment peptide or the level of the protein.
  • Also provides are methods of treating a patient suffering from lung cancer comprising:
  • the reference level of the CAT fragment peptide may be 960 amol ⁇ g, +/- 250 amol ⁇ g, +/- 150 amol ⁇ g, +/- 190 amol ⁇ g, +/- 50 amol ⁇ g, or +/- 25 amol ⁇ g of biological sample protein analyzed and the reference level of the NQOl fragment peptide may be 24000 amol ⁇ g, +/- 250 amol ⁇ g, +/- 150 amol ⁇ g, +/- 190 amol ⁇ g, +/- 50 amol ⁇ g, or +/- 25 amol ⁇ g of biological sample protein analyzed.
  • the mass spectrometry may be, for example, tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, MALDI-TOF mass spectrometry,
  • MALDI mass spectrometry hybrid ion trap/quadrupole mass spectrometry and/or time of flight mass spectrometry.
  • the mode of mass spectrometry used may be, for example, Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), Parallel
  • PRM PRM
  • intelligent Selected Reaction Monitoring iSRM
  • mSRM multiple Selected Reaction Monitoring
  • the tumor sample may be a cell, collection of cells, or a solid tissue, and may be formalin fixed solid tissue, which may be paraffin embedded tissue.
  • the internal standard peptide may be an isotopically labeled peptide, containing, for example, one or more heavy stable isotopes selected from 18 0, 17 0, 15 N, 13 C,
  • Detecting and quantitating the specified CAT and/or NQOl fragment peptides can be combined with detecting and quantitating other peptides from other proteins in a multiplex format so that the treatment decision about which agent used for treatment is based upon specific levels of the specified CAT and/or NQOl fragment peptide in combination with other peptides/proteins in the biological sample.
  • the therapeutic strategy may include the combination of pemetrexed and cisplatin agents.
  • Also provided are methods of treating a patient suffering from lung cancer comprising:
  • the reference level of the ECAD fragment peptide may be 2450 amol ⁇ g, +/- 250 amol ⁇ g, +/- 150 amol ⁇ g, +/- 190 amol ⁇ g, +/- 50 amol ⁇ g, or +/- 25 amol ⁇ g, of biological sample protein analyzed.
  • the protein digest of the biological sample may be prepared by the liquid tissue protocol and may include a protease digest, such as a trypsin digest.
  • the specified CAT peptide has the amino acid sequence as set forth as SEQ ID NO:4.
  • the mass spectrometry may be tandem mass spectrometry, ion trap mass
  • SRM Selected Reaction Monitoring
  • MRM Multiple Reaction Monitoring
  • PRM Parallel Reaction Monitoring
  • iSRM intelligent Selected Reaction Monitoring
  • mSRM multiple Selected Reaction Monitoring
  • Figure 1 shows an overall survival curve showing that patients whose tumor cells express levels of the GART protein below 900 amol/ug of protein analyzed have much greater probability of longer overall survival than patients whose tumor cells express above 900amol/ug of protein analyzed when treated with a therapeutic combination of pemetrexed and cisplatin.
  • Figure 2 shows an overall survival curve showing that patients whose tumor cells express detectable levels of KRAS and whose tumor cells also express greater than
  • 900amol/ug GART analyzed have a much lower probability of longer overall survival than patients whose tumor cells do not express detectable levels of KRAS and also express less than 900amol/ug GART protein analyzed when treated with a therapeutic combination of pemetrexed and cisplatin.
  • Figure 3 is an overall survival curve that shows that patients whose tumor cells express levels of the ECAD protein over 2450 amol ⁇ g have a lower probability of longer survival than patients whose tumor cells express lower levels of ECAD when treated with the combination of pemetrexed and cisplatin.
  • Figure 4 provides an overall survival curve that shows that patients whose tumor cells express NQOl protein levels below 2400 amol/ug protein analyzed have a slightly greater probability of longer survival than patients whose tumor cells express NQOl protein levels above 2400amol/ug when treated with the combination of pemetrexed and cisplatin
  • Figure 5 provides an overall survival curve that shows that patients whose tumor cells express XRCC1 protein levels below 521amol/ug protein analyzed have a slightly greater probability of longer survival than patients whose tumor cells express XRCC1 protein levels above 521amol/ug when treated with the combination of pemetrexed and cisplatin.
  • Figure 6 provides an overall survival curve that shows that patients whose tumor cells express CAT protein levels above 960 amol/ug protein analyzed have a greater probability of longer survival than patients whose tumor cells express CAT protein levels below 960 amol/ug when treated with the combination of pemetrexed and cisplatin.
  • Figure 7 provides an overall survival curve that shows that patients whose tumor cells exhibit high CAT expression (>960 amol ⁇ g) and low NQOl expression ( ⁇ 24,000 amol ⁇ g) have a significantly higher OS compared to the rest of the patients (20.3 vs 11.2 months).
  • Methods are provided for identifying and optimizing treatment strategies for a cancer patient by determining whether or not a cancer patient will clinically respond in a favorable manner to the therapeutic cancer agent pemetrexed and therapeutic regimens containing pemetrexed.
  • Pemetrexed is also referred to as Alimta.
  • diagnostic methods for measuring expression of the combination of the KRAS and GART proteins in a tumor sample or samples from the patient are provided.
  • Methods of measuring expression of XRCC1, EC AD, CAT and NQOl in a tumor sample also are provided, together with methods of measuring combinations of these proteins, including the combination of CAT and NQOl.
  • the tumor sample is advantageously a formalin-fixed sample.
  • the amount of the KRAS, GART, XRCC1, ECAD, CAT and/or NQOl proteins in cells derived from formalin fixed paraffin embedded (FFPE) tissue is determined.
  • the peptide fragments derive from the full-length proteins; the specific peptide sequence that is used for KRAS is SFEDIHHYR and the peptide sequence used for detecting and quantitating the GART protein is VLAVTAIR.
  • the fragment used to detect and quantify XRCC1 is TPATAPVPAR, for CAT it is LNVITVGPR, for NQOl it is ALIVLAHSER, and for ECAD it is NTGVISVVTTGLDR.
  • this SRM/MRM assay can measure these peptides directly in complex protein lysate samples prepared from cells procured from patient tissue samples, such as formalin fixed cancer patient tissue.
  • patient tissue samples such as formalin fixed cancer patient tissue.
  • Methods of preparing protein samples from formalin-fixed tissue are described in U.S. Pat. No. 7,473,532, the contents of which are hereby incorporated by reference in their entirety.
  • the methods described in U.S. Pat. No. 7,473,532 may conveniently be carried out using Liquid Tissue® reagents and protocol available from Expression Pathology Inc. (Rockville, Md.).
  • formalin fixed, paraffin embedded tissue The most widely and advantageously available form of tissue, and cancer tissue, from cancer patients is formalin fixed, paraffin embedded tissue. Formaldehyde/formalin fixation of surgically removed tissue is by far the most common method of preserving cancer tissue samples worldwide and is the accepted convention in standard pathology practice.
  • Aqueous solutions of formaldehyde are referred to as formalin. "100%" formalin consists of a saturated solution of formaldehyde (about 40% by volume or 37% by mass) in water. A small amount of stabilizer, usually methanol, is added to limit oxidation and degree of polymerization.
  • tissue is preserved in aqueous formaldehyde, commonly termed 10% neutral buffered formalin, followed by embedding the fixed whole tissue in paraffin wax for long term storage at room temperature.
  • aqueous formaldehyde commonly termed 10% neutral buffered formalin
  • Results from the SRM/MRM assay(s) can be used to correlate accurate and precise quantitative levels of the KRAS, GART, XRCC1, CAT, NQOl, and/or EC AD proteins within the specific cancer of the patient from whom the tissue was collected and preserved, including lung cancer tissue. This not only provides diagnostic information about the cancer, but also permits a physician or other medical professional to determine appropriate therapy for the patient.
  • utilizing this assay can provide information about specific levels of KRAS, GART, XRCC1, CAT, NQOl, and/or ECAD protein expression in cancer tissue from a patient and makes it possible to determine whether or not the patient will respond favorably to therapy with the anti-cancer therapeutic agent pemetrexed, and potentially with other similar drugs in the antifolate class, designed to specifically inhibit the function of the GART, TS, and/or DHFR proteins.
  • Treating cancer patients with pemetrexed is one of the most common and effective strategies for preventing cancer from growing and thus prolonging the lives of cancer patients, especially lung cancer patients.
  • the KRAS protein is a GTPase that performs an essential function in normal tissue signaling, and mutation of the KRAS gene is an essential step in the development of many cancers.
  • the GART protein is a multifunctional transferase/ligase protein that is involved in purine metabolism and thus is an integral part of the nucleic acid synthesis function of the cell, and without which the cell cannot synthesize nucleic acids and grow/divide. Accordingly, inhibiting the function of the GART protein with pemetrexed prevents the tumor cell from synthesizing nucleic acids and leads to tumor cell death. It therefore is useful for a clinician to know the level of the GART protein in a patient's tumor cells to determine if pemetrexed will have a toxic effect on the tumor cells.
  • XRCC1 also known as X-ray repair cross-complementing protein 1
  • X-ray repair cross-complementing protein 1 is involved in
  • XRCC1 DNA repair where it complexes with DNA ligase III.
  • XRCC1 is involved in repair of DNA single-strand breaks formed by exposure to ionizing radiation and alkylating agents.
  • XRCC1 is over-expressed in non- small-cell lung carcinoma (NSCLC).
  • CAT catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen, and is involved in protecting cells from oxidative damage by reactive oxygen species (ROS). It has been proposed that catalase may regulate cathepsin activity by controlling the production of ROS, leading to variation in migration and invasion ability of lung cancer cells.
  • ROS reactive oxygen species
  • NQOl (NAD(P)H dehydrogenase [quinone] 1) is an enzyme that is a member of the NAD(P)H dehydrogenase (quinone) family. It is a flavoenzyme that protects against endogenous and exogenous quinones by catalyzing two- or four-electron reductions of these substrates and protecting cells from unwanted oxidative damage. NQOl has been shown to be overexpressed in non-small-cell lung carcinoma (NSCLC).
  • NSCLC non-small-cell lung carcinoma
  • ECAD E-cadherin
  • Mutations of the ECAD gene are associated with gastric, breast, colorectal, thyroid, and ovarian cancers. Loss of function is thought to contribute to progression in cancer by increasing proliferation, invasion, and/or metastasis.
  • IHC immunohistochemistry
  • Inaccurate IHC test results may mean that patients diagnosed with cancer do not receive the best possible care. If all or part of a cancer is positive for a specific target oncoprotein but test results classify it as negative, physicians are unlikely to implement the correct therapeutic treatment, even though the patient could potentially benefit from agents that target the oncoprotein. If a cancer is oncoprotein target negative but test results classify it as positive, physicians may use a specific therapeutic treatment, even though the patient is not only unlikely to receive any benefit but also is exposed to the agent's secondary risks.
  • Quantitative levels of the analyzed proteins are then determined by the SRM/MRM methodology whereby the SRM/MRM signature chromatographic peak area of an individual specified peptide from each of the proteins in one biological sample is compared to the SRM/MRM signature chromatographic peak area of a known amount of a "spiked" internal standard for each of the individual specified fragment peptides.
  • the internal standard is a synthetic version of the same fragment peptides where the synthetic peptides contain one or more amino acid residues labeled with one or more heavy isotopes, such as 2 H, 18 0, 17 0, 15 N, 13 C, or combinations thereof.
  • isotope labeled internal standards are synthesized so that mass spectrometry analysis generates a predictable and consistent SRM/MRM signature chromatographic peak that is different and distinct from the native fragment peptide chromatographic signature peaks and which can be used as comparator peaks.
  • the SRM/MRM signature chromatographic peak area of the native peptide is compared to the SRM/MRM signature chromatographic peak area of the internal standard peptide, and this numerical comparison indicates either the absolute molarity and/or absolute weight of the native peptide present in the original protein preparation from the biological sample.
  • Quantitative data for fragment peptides are displayed according to the amount of protein analyzed per sample.
  • a mass spectrometer that additional information is used to direct and instruct the mass spectrometer, (e.g., a triple quadrupole mass spectrometer) to perform the correct and focused analysis of the specified fragment peptides.
  • An SRM/MRM assay may be effectively performed on a triple quadrupole mass spectrometer.
  • That type of a mass spectrometer may be considered to be one of the most suitable instruments for analyzing a single isolated target peptide within a very complex protein lysate that may consist of hundreds of thousands to millions of individual peptides from all the proteins contained within a cell.
  • the additional information provides the mass spectrometer, such as a triple quadrupole mass spectrometer, with the correct directives to allow analysis of a single isolated target peptide within a very complex protein lysate.
  • SRM/MRM assays also can be developed and performed on other types of mass spectrometer, including MALDI, ion trap, ion trap/quadrupole hybrid, or triple quadrupole instruments, but presently the most advantageous instrument platform for SRM/MRM assay is often considered to be a triple quadrupole instrument platform.
  • the additional information about target peptides in general, and in particular about the specified fragment peptides, may include one or more of the mono isotopic mass of each peptide, its precursor charge state, the precursor m z value, the m z transition ions, and the ion type of each transition ion.
  • the peptide sequence of the specified KRAS, GART, CAT, NQOl, XRCCl and ECAD fragment peptides and the necessary additional information as described for these specified fragment peptides is shown in Table 1 below.
  • tumor samples were obtained from a cohort of patients suffering from cancer, in this case lung cancer.
  • the lung tumor samples were formalin- fixed using standard methods and the level of KRAS, GART, CAT, NQOl, XRCCl and ECAD in the samples was measured using the methods as described above.
  • the tissue samples may also be examined using IHC and FISH using methods that are well known in the art.
  • the patients in the cohort were treated with the pemetrexed therapeutic agent and the response of the patients was measured using methods that are well known in the art, for example by recording the overall survival (OS) of the patients at time intervals after treatment.
  • OS overall survival
  • a suitable reference level was determined using statistical methods that are well known in the art, for example by determining the lowest p value of a log rank test. Once a reference level was determined it was used to identify those patients whose protein expression levels indicate that they may likely benefit from a pemetrexed therapeutic regimen as measured by extending the life of the patient.
  • pemetrexed may also be used as part of a treatment regimen that includes additional drugs or combinations of drugs, such as a combination with the drug cisplatin.
  • Therapeutic regimens for treating lung cancer are known in the art and a very common and widely-used drug combination is cisplatin combined with pemetrexed.
  • Levels of KRAS, GART, CAT, NQOl, XRCCl and ECAD proteins in patient tumor samples are typically expressed in amol ⁇ g, although other units can be used.
  • a reference level can be expressed as a range around a central value, for example, +/- 250, 150, 100, 50 or 25 amol/ ⁇ g.
  • a suitable reference level for the GART protein was found to be 900amol ⁇ g.
  • levels higher or lower than these reference levels can be selected based on clinical results and experience.
  • both nucleic acids and protein can be analyzed from the same Liquid Tissue biomolecular preparation it is possible to generate additional information about disease gnosis and drug treatment decisions from the nucleic acids in same sample upon which proteins were analyzed. For example, if the KRAS, GART, CAT, NQOl, XRCCl and ECAD proteins are expressed by certain cells at increased levels, when assayed by SRM the data can provide information about the state of the cells and their potential for uncontrolled growth, choice of optimal therapy, and potential drug resistance.
  • nucleic acids and proteins they encode can be obtained from nucleic acids present in the same Liquid TissueTM biomolecular preparation.
  • Nucleic acids can be assessed simultaneously to the SRM analysis of proteins, including the KRAS, GART, CAT, NQOl, XRCCl and ECAD proteins.
  • information about one or more of the KRAS, GART, CAT, NQOl, XRCCl and ECAD proteins and/or one, two, three, four or more additional proteins may be assessed by examining the nucleic acids encoding those proteins.
  • nucleic acids can be examined, for example, by one or more, two or more, or three or more of: sequencing methods, polymerase chain reaction methods, restriction fragment polymorphism analysis, identification of deletions, insertions, and/or determinations of the presence of mutations, including but not limited to, single base pair polymorphisms, transitions, transversions, or combinations thereof.
  • Example Determination of a predictive value of protein expression levels for pemetrexed sensitivity in a population of lung cancer patients.
  • NSCLC non-small cell lung cancer
  • FFPE paraffin-embedded
  • Tumor cells from FFPE tumor tissue were procured and isolated from the tumor tissue by tissue microdissection and solubilized for downstream mass spectrometry analysis using the Liquid Tissue® reagents as described above. Protein levels were quantitated using selected reaction monitoring mass spectrometry (SRM-MS). Overall survival curves of the patients in this study as related to levels of various proteins, including KRAS, GART, CAT, NQOl, XRCCl and ECAD proteins, were developed. Results
  • Quantitative SRM/MRM data across a number of proteins including those proteins specifically targeted by the therapeutic drug pemetrexed indicate a strong and significant correlation of the expression levels of KRAS, GART, CAT, NQOl, XRCC1 and ECAD with positive therapeutic outcome by treatment of the cancer patient with pemetrexed, as evidenced by extended survival after initial diagnosis and initiation of treatment regime with pemetrexed.
  • a cutoff level of 900 amol ⁇ g GART was established. Expression of GART above this level was associated with lower progression-free survival (PFS) and lower overall survival (OS).

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Abstract

L'invention concerne des méthodes pour déterminer si une tumeur du poumon réagira au traitement avec les agents thérapeutiques tels que la cisplatine et le pemetrexed. Des peptides de fragments protéiques spécifiques sont détectés et quantifiés avec précision par spectrométrie de masse SRM directement dans des cellules de tumeur du poumon recueillies dans un tissu de tumeur du poumon qui été obtenu auprès d'un patient atteint d'un cancer, puis sont comparés à des niveaux de référence pour déterminer si le patient atteint d'un cancer du poumon va réagir positivement au traitement à l'aide de la combinaison d'agents thérapeutiques de cisplatine et pemetrexed.
PCT/US2017/012389 2016-01-05 2017-01-05 Quantification de protéine pour thérapie anticancéreuse optimale Ceased WO2017120376A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130108711A1 (en) * 2010-05-07 2013-05-02 Medicus Biosciences, Llc Methods for treating diseases of the lung
US20140018254A1 (en) * 2008-09-16 2014-01-16 Caris Mpi, Inc. Theranostic and diagnostic methods using sparc and hsp90
US20150376678A1 (en) * 2013-03-15 2015-12-31 Expression Pathology, Inc. SRM Assay to Indicate Cancer Therapy

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140018254A1 (en) * 2008-09-16 2014-01-16 Caris Mpi, Inc. Theranostic and diagnostic methods using sparc and hsp90
US20130108711A1 (en) * 2010-05-07 2013-05-02 Medicus Biosciences, Llc Methods for treating diseases of the lung
US20150376678A1 (en) * 2013-03-15 2015-12-31 Expression Pathology, Inc. SRM Assay to Indicate Cancer Therapy

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