WO2019108922A1 - Dosage srm/mrm destiné au sous-typage d'une histologie du cancer de la tête et du cou - Google Patents

Dosage srm/mrm destiné au sous-typage d'une histologie du cancer de la tête et du cou Download PDF

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WO2019108922A1
WO2019108922A1 PCT/US2018/063292 US2018063292W WO2019108922A1 WO 2019108922 A1 WO2019108922 A1 WO 2019108922A1 US 2018063292 W US2018063292 W US 2018063292W WO 2019108922 A1 WO2019108922 A1 WO 2019108922A1
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cdk6
protein
amount
mass spectrometry
quantifying
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PCT/US2018/063292
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Sheeno Thyparambil
Dongyao YAN
Shankar SELLAPPAN
Fabiola CECCHI
Todd Hembrough
Yeoun Jin KIM
Andrew Chambers
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Nantomics, Llc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material

Definitions

  • the field of invention is personalized cancer therapy treatment. Improved methods for cancer treatment are provided wherein detection and quantitation of protein expression in patient tumor tissue can be used to guide the cancer treatment strategies.
  • Head and neck cancer is a broad category of diverse tumor types arising from various anatomic structures including the craniofacial bones, soft tissues, salivary glands, skin, and mucosal membranes.
  • SCC squamous cell carcinomas
  • head and neck cancer is often used to describe all carcinomas arising from the epithelium lining the sinonasal tract, oral cavity, pharynx, and larynx and showing microscopic evidence of squamous differentiation.
  • ACC adenoid cystic carcinoma
  • Adenoid cystic carcinoma is a relatively uncommon cancer with an annual incidence of approximately 1200 cases in the United States. Adenoid cystic carcinoma occurs primarily in the major and minor salivary glands, but can also be found at other sites. The disease usually presents in a localized manner, but is known for its propensity for late metastasis (up to 15 years after initial diagnosis). Although ACC can be found at any age, it typically presents in the 5th decade (median age: 57.4 years).
  • Methods are provided to detect and/or quantitate specific fragment peptides from the full length CDK6 protein directly in cancer patient tumor cells in order to distinguish between ACC or SCC head and neck cancer.
  • Improved methods of treatment of head and neck cancer are provided using a cancer treatment regimen comprising agents that specifically target the CDK6 protein.
  • a method for detecting and measuring the level of the CDK6 protein in a human biological sample, such as a sample of formalin-fixed tissue, where the method includes detecting and quantifying by mass spectrometry the amount of one or more CDK6 fragment peptides in a protein digest prepared from the biological sample and calculating the level of CDK6 protein in the sample, where the amount is a relative amount or an absolute amount.
  • the method may be used for diagnosis, evaluation, and/or determining treatment of adenoid cystic carcinoma (ACC) of the head and neck.
  • ACC adenoid cystic carcinoma
  • the protein digest may be fractionated prior to detecting and/or quantifying the amount of the one or more fragment peptides by, for example, liquid chromatography, nano-reverse phase liquid chromatography, high performance liquid chromatography, and/or reverse phase high performance liquid chromatography.
  • the protein digest may contain a protease digest, such as a trypsin digest.
  • the protein digest advantageously may be prepared by the Liquid Tissue protocol.
  • the mass spectrometry methods may include tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, orbitrap mass spectrometry, hybrid ion trap/quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, and/or time of flight mass spectrometry.
  • the mode of mass spectrometry used to determine the absolute amount of CDK6 protein may be, for example, Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), intelligent Selected Reaction Monitoring (iSRM), Parallel Reaction Monitoring (PRM), and/or multiple Selected Reaction Monitoring (mSRM).
  • the method of mass spectrometry may be the Data Independent Acquisition method.
  • the CDK6 fragment peptide or peptides that are measured may be, for example, one or more of the peptides set forth in SEQ ID NO: 1-7.
  • the tissue used to prepare the digest may be paraffin embedded tissue, and may be obtained from a tumor, such as a primary tumor or a secondary tumor.
  • One method of quantifying the at least one CDK6 fragment peptide includes comparing an amount of the fragment peptide in one biological sample to the amount of the same fragment peptide in a different and separate biological sample.
  • a further method of quantifying the at least one CDK6 fragment peptide includes comparing an amount of the fragment peptide in the biological sample to other and different fragment peptides from other and different proteins in the same biological sample.
  • a further method of quantifying at least one CDK6 fragment peptide comprises determining the amount of the fragment peptide in a biological sample by comparison to an added internal standard peptide of known amount having the same amino acid sequence.
  • the internal standard peptide may be an isotopically labeled peptide, which may include one or more heavy stable isotopes selected from 18 0, 17 0, 34 S, 15 N, 13 C, 2 H or combinations thereof.
  • detecting and quantifying at least one CDK6 fragment peptide in the protein digest indicates the presence of the CDK6 protein and an association with cancer in a patient or subject.
  • the method may further include correlating the results of the detecting and quantifying at least one CDK6 fragment peptide, or the amount of the CDK6 protein, to the diagnostic histology/stage/grade/status of the cancer. Correlating the results of the detecting and quantifying at least one CDK6 fragment peptide to the diagnostic histology/stage/grade/status of the cancer may be combined with detecting and/or quantifying the amount of other proteins or fragment peptides from other proteins in a multiplex format to provide additional information about the diagnostic histology/stage/grade/status of the cancer.
  • These methods may further include administering to a patient or subject from which the biological sample was obtained a therapeutically effective amount of a therapeutic agent, where the therapeutic agent and/or amount of the therapeutic agent administered is based upon the detect of and/or amount at least one CDK6 fragment peptide or the amount of CDK6 protein.
  • the treatment or the therapeutic agent may be directed to cancer cells expressing CDK6, and may be, for example, palbociclib or ribociclib.
  • a method of treating head and neck cancer including (a) detecting and quantifying the amount of one or more CDK6 fragment peptides in a protein digest prepared from a human biological sample of formalin-fixed tissue obtained from a patient suffering from the cancer, where the detecting and quantifying is carried out using mass spectrometry and calculating the level of CDK6 protein in the sample, and where the amount is a relative amount or an absolute amount; and (b) treating the patient with a regimen including an effective amount of a therapeutic agent that targets CDK6 when the detecting and quantifying indicates increased CDK6 expression, or (c) treating the patient with radiation and/or an effective amount of a chemotherapeutic that does not target CDK6 when the detecting and quantifying indicates no increased CDK6 expression.
  • the therapeutic agent that targets CDK6 may be palbociclib or ribociclib.
  • Figure 1 is a bar graph showing the relative expression level of the CDK6 protein in tumors cells procured directly from cancer patient tissue.
  • a specific CDK6 peptide was analyzed using the DIA mass spectrometry method in patients with ACC head and neck cancer and patients with SCC head and neck cancer.
  • CDK6 is highly upregulated in ACC head and neck tumors vs. SCC head and neck tumors.
  • Methods are provided for detecting and/or quantitating expression of the CDK6 protein directly in tumor cells procured from cancer patient tissue and for improved diagnosis and treatment of adenoid cystic carcinoma (ACC) of the head and neck.
  • ACC adenoid cystic carcinoma
  • CDK6 Cell division protein kinase 6
  • CDK6 is an enzyme encoded by the CDK6 gene. It is regulated by cyclin D and by cyclin-dependent kinase inhibitor proteins. The protein encoded by this gene is a member of the cyclin-dependent kinase (CDK) family, which also includes CDK4. CDK family members are involved in cell cycle progression at the point at Gl at which commitment occurs and the cell no longer requires growth factors to complete the cell cycle - known as the restriction (R) point.
  • CDK6 is a catalytic subunit of the protein kinase complex, important for the Gl phase progression and Gl/S transition of the cell cycle and the complex is composed also by an activating sub-unit; cyclin D.
  • CDK6 activity first appears in mid-Gl phase, which is controlled by the regulatory subunits including D-type cyclins and members of the INK4 family of CDK inhibitors.
  • CDK6, as well as CDK4 has been shown to regulate the activity the tumor suppressor Retinoblastoma (Rb) by
  • CDK6 an important protein in cancer development.
  • CDK6 is involved in an important point of restriction in the cell cycle
  • CDK6 and other regulators of the Gl phase of the cell cycle are known to be unbalanced in more than 80-90% of tumors.
  • CDK6 function has been shown to be altered indirectly by the pl6 inhibitor.
  • CDK6 is also overexpressed in tumors that exhibit drug resistance, for example glioma malignancies exhibit resistance to chemotherapy using temozolomide (TMZ) when they have a mutation overexpressing CDK6.
  • TMZ temozolomide
  • the overexpression of CDK6 is also associated with resistance to hormone therapy using the anti estrogen Fluvestrant in breast cancer.
  • Loss of normal cell cycle control is the first step to developing different hallmarks of cancer; alterations of CDK6 can directly or indirectly affect the following hallmarks:
  • CDK6 disregulated cell cellular energetics; sustaining of proliferative signaling; evading growth suppressors; and inducing angiogenesis.
  • Dysregulation of CDK6 has been shown to be important in lymphoid malignancies by increasing angiogenesis, a hallmark of cancer. These features are reached through upregulation of CDK6 due to chromosome alterations or epigenetic dysregulations. Additionally, CDK6 might be altered through genomic instability, a mechanism of downregulation of tumor suppressor genes, and this represents another evolving hallmark of cancer. The pharmaceutical industry has developed several inhibitors of CDK6 for cancer treatment.
  • Palbociclib and ribociclib are FDA approved inhibitors of CDK6 and the highly similar CDK4 protein. Ribociclib is approved in combination with letrozole for treatment of breast cancer in patients with a hormone receptor positive, HER2-negative advanced metastatic breast cancer. A phase three clinical trial found that ribociclib administered in combination with letrozole increased the likelihood of progression free survival to 63% in the first 18 months of therapy versus 42% for letrozole alone. Subsequent analysis demonstrated that patients treated with ribociclib and letrozole showed a median progression-free survival of 25.3 months.
  • Palbociclib also known as PD-0332991
  • Palbociclib is currently being assessed in more than 20 clinical trials, either as a single agent or as a coadjuvant of other therapies, and has shown promising results in the control of breast cancer in-vitro.
  • CDK6 and CDK4 inhibitors have been shown to be useful as coadjuvants in breast cancer chemotherapy.
  • CDK6 Specific peptides derived from subsequences of the CDK6 protein, which can also be referred to as CDKN6, Cyclin Dependent Kinase 6, Cell Division Protein Kinase 6, and which will be referred to as CDK6, are provided.
  • the amino acid sequences for these peptides are particularly useful in mass spectrometry -based Selected Reaction Monitoring (SRM) assays for absolute quantitation, which can also be referred to as a Multiple Reaction Monitoring (MRM) assays.
  • SRM Selected Reaction Monitoring
  • MRM Multiple Reaction Monitoring
  • the peptides are also particularly useful in Data Independent Acquisition (DIA) assays for detecting expression with relative quantitation.
  • DIA Data Independent Acquisition
  • An SRM assay can be used to measure relative and/or absolute quantitative levels of one or more of the specific peptides from the CDK6 protein and therefore provide a means of measuring the amount of the CDK6 protein in a given protein preparation obtained from a biological sample by mass spectrometry.
  • a DIA assay can be used to measure relative quantitative levels of one or more of the specific peptides from the CDK6 protein and therefore provide a means of detecting expression of the CDK6 protein in a given protein preparation obtained from a biological sample by mass spectrometry.
  • an SRM and/or DIA 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. Patent No. 7,473,532, the contents of which are hereby incorporated by references in their entirety.
  • the methods described in U.S. Patent No. 7,473,532 may conveniently be carried out using Liquid Tissue reagents and protocol available from Expression Pathology Inc. (Rockville, MD).
  • the Liquid Tissue protocol and reagents are capable of producing peptide samples suitable for mass spectroscopic analysis from formalin fixed paraffin embedded tissue by proteolytic digestion of the proteins in the tissue/biological sample.
  • the tissue/biological is heated in a buffer for an extended period of time (e.g., from about 80° C to about 100° C for a period of time from about 10 minutes to about 4 hours) to reverse or release protein cross-linking.
  • the buffer employed is a neutral buffer, (e.g., a Tris-based buffer, or a buffer containing a detergent).
  • the tissue/biological sample is treated with one or more proteases including, but not limited to, trypsin, chymotrypsin, pepsin, and endoproteinase Lys-C, for a time sufficient to disrupt the tissue and cellular structure of the biological sample.
  • the result of the heating and proteolysis is a liquid, soluble, dilutable biomolecule lysate.
  • formalin fixed, paraffin embedded tissue The most widely and advantageously available form of tissues from cancer patients tissue 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 for standard pathology practice.
  • Aqueous solutions of formaldehyde are referred to as formalin. "100%" formalin consists of a saturated solution of formaldehyde (this is about 40% by volume or 37% by mass) in water, with a small amount of stabilizer, usually methanol to limit oxidation and degree of polymerization.
  • tissue is preserved.
  • aqueous formaldehyde commonly termed 10% neutral buffered formalin
  • results from the SRM and/or DIA assay can be used to correlate accurate and precise expression levels of the CDK6 protein within the specific tissue samples (e.g., cancer tissue sample) of the patient or subject from whom the tissue (biological sample) was collected and preserved. 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.
  • Such an assay that provides diagnostically and therapeutically important information about levels of protein expression in a diseased tissue or other patient sample is termed a companion diagnostic assay.
  • a companion diagnostic assay can be designed to diagnose the precise histology of a cancer and determine a therapeutic agent to which a patient is most likely to respond.
  • Tumor tissue comprises many types of cells including tumor epithelial cells, normal epithelial cells, fibroblastic connective cells, and immune cells.
  • tumor cells are removed from the heterogeneous mixture of cells and studied. This can be achieved by tissue microdissection such as Laser Capture Microdissection (LCM) technology (see US Patent No. 6,867,038).
  • LCM Laser Capture Microdissection
  • LCM has improved the analysis of tissue samples by providing a means through which molecular profiling of cells derived from tissue samples can be placed in a pathologically relevant context.
  • tissue microdissection technologies besides LCM have been developed and patented which have led to the commercial availability of multiple tissue microdissection instruments including the PixCell systems (Arcturus), the PALM system (PALM Microlaser Technologies), the uCuT (Molecular Machines and Industries), the Leica AS LMD (Leica Microsystems), the LaserScissors (Cell Robotics), the MicroDissector (Eppendorf), xMD (xMDx), and the Clonis system (Bio-Rad).
  • PixCell systems Arcturus
  • PALM PALM Microlaser Technologies
  • uCuT Molecular Machines and Industries
  • Leica AS LMD Leica Microsystems
  • LaserScissors Cell Robotics
  • Eppendorf MicroDissector
  • xMDx xMDx
  • Clonis system Bio-Rad
  • any one of the above-mentioned tissue microdissection methods can be employed to collect tumor cells in the presently described methods and systems for mass spec analysis of neo-antigens present on the surface of the tumor cells within cancer patient tumor tissue.
  • the tissue microdissection method used for collecting tumor cells is the DIRECTOR technology.
  • the basis of this technology resides in the DIRECTOR slide which functions by utilizing laser induced forward transfer (LIFT) of tissue via the energy transfer interlayer coating as described in U.S. Pat. No. 7,381,440, the contents of which are hereby incorporated by reference in their entirety.
  • LIFT laser induced forward transfer
  • DIRECTOR slides comprise a standard glass slide that has been coated with an optically -translucent energy transfer layer that allows for tissue to be transferred to a collection vesicle using LIFT technology.
  • LIFT technology is defined as the movement of objects with laser energy via a thin energy transfer layer.
  • a tissue section from heterogeneous patient tumor tissue is sectioned onto a DIRECTOR slide, whereby specified and desired Areas of Interest (AOIs) are identified by standard histological methods and digitally marked to indicate to the microdissection instrument which AOIs to collect, which in this case are tumor cells to be microdissected for analysis.
  • AOIs Areas of Interest
  • Activation of the laser to transfer AOIs downward into a collection tube is induced by the presence of the digital markups that define the tumor cells for collection.
  • any predicted peptide derived from the CDK6 protein prepared for example by digesting with a protease of known specificity (e.g . trypsin), can be used as a surrogate reporter to determine the abundance of CDK6 protein in a sample using a mass spectrometry-based SRM and DIA assays.
  • a protease of known specificity e.g . trypsin
  • the CDK6 peptides described herein were derived from the CDK6 protein by protease digestion of all the proteins within a complex Liquid Tissue lysate prepared from cells procured from formalin fixed cancer tissue. Unless noted otherwise, in each instance the protease was trypsin. The Liquid Tissue lysate was then analyzed by mass spectrometry to determine those peptides derived from the CDK6 protein that are detected and analyzed by mass spectrometry.
  • Identification of a specific preferred subset of peptides for mass spectrometric analysis is based on; 1) experimental determination of which peptide or peptides from a protein ionize in mass spectrometry analyses of Liquid Tissue lysates, and 2) the ability of the peptide to survive the protocol and experimental conditions used in preparing a Liquid Tissue lysate. This latter property extends not only to the amino acid sequence of the peptide but also to the ability of a modified amino acid residue within a peptide to survive in modified form during the sample preparation.
  • Protein lysates from cells procured directly from formalin (formaldehyde) fixed tissue were prepared using the Liquid Tissue reagents and protocol that entails collecting cells into a sample tube via tissue microdissection followed by heating the cells in the Liquid Tissue buffer for an extended period of time. Once the formalin-induced cross linking has been negatively affected, the tissue/cells are then digested to completion in a predictable manner using a protease, as for example including but not limited to the protease trypsin. Each protein lysate is turned into a collection of peptides by digestion of intact polypeptides with the protease.
  • Each Liquid Tissue lysate was analyzed (e.g., by ion trap mass spectrometry) to perform multiple global proteomic surveys of the peptides where the data was presented as identification of as many peptides as could be identified by mass spectrometry from all cellular proteins present in each protein lysate.
  • An ion trap mass spectrometer or another form of a mass spectrometer that is capable of performing global profiling for identification of as many peptides as possible from a single complex protein/peptide lysate is employed.
  • Ion trap mass spectrometers are generally considered the best type of mass spectrometer for conducting global profiling of peptides for a DIA assay.
  • an SRM assay can be developed and performed on any type of mass spectrometer, including a MALDI, ion trap, or triple quadrupole, the most advantageous instrument platform for an SRM assay is often considered to be a triple quadrupole instrument platform.
  • That type of dataset can be considered to represent the peptides that can be detected in the type of biological sample that was analyzed (after protease digestion), and specifically in a Liquid Tissue lysate of the biological sample, and thus includes the peptides for specific proteins, such as for example the CDK6 protein.
  • each peptide was detected by mass spectrometry in Liquid Tissue lysates prepared from formalin fixed, paraffin embedded tissue.
  • each peptide is a candidate for use in developing a quantitative SRM assay and/or DIA assay for the CDK6 protein in human biological samples, including directly in formalin fixed patient tissue.
  • CDK6 tryptic peptides listed in Table 1 were detected from multiple Liquid Tissue lysates of multiple different formalin fixed tissues of different human organs including prostate, colon, and breast. Each of those peptides is considered useful for quantitative SRM and DIA assays of the CDK6 protein in formalin fixed tissue. Further data analysis of these experiments indicated no preference is observed for any of these peptides from any specific organ site.
  • Mass spectrometry proteomics can detect and identify peptides in a complex protein lysate either in a“global profiling” mode (also referred to as global proteomics or shotgun proteomics) which aims to detect and identify the presence of all peptides within a protein lysate, or in a“targeted” mode which aims to detect and identify only specified peptides suspected of being present in the complex protein lysate while ignoring all other peptides in the lysate.
  • a“global profiling” mode also referred to as global proteomics or shotgun proteomics
  • the presently described method may utilize both“global profiling” and“targeted” analysis of peptides in a complex protein lysate prepared from isolated tumor cells whereby “global profiling” is used to detect and identify presence of previously known and unknown peptides in a protein lysate while the“targeted” approach is used to detect and identify presence of previously discovered and known peptides that are suspected of being present in tumor cells from patient tumor tissue.
  • The“global” proteomic approach is most advantageously performed using an ion trap mass spectrometer, such as an Orbitrap®.
  • an ion trap mass spectrometer such as an Orbitrap®.
  • the optimal mass spectrometer for performing a global profile include but are not limited to ion trap, orbitrap, and hybrid ion trap instruments.
  • Such instruments are most often interfaced with liquid chromatography, which provides for an ion trap mass analyzer consisting of an outer barrel-like electrode and a coaxial inner spindle-like electrode that traps ions in an orbital motion around the spindle.
  • the image current from the trapped ions is detected and converted to a mass spectrum using the Fourier transform of the frequency signal.
  • This instrument is utilized most effectively, and is the most effective instrument, for detecting and identifying as many peptides from a complex protein lysate as possible without any prior knowledge of the peptide content of the complex protein lysate.
  • The“targeted” approach is most advantageously performed using a specific mass spectrometry instrument termed a triple quadrupole mass spectrometer.
  • the triple quadrupole instrument is most often interfaced with liquid chromatography and is a tandem mass spectrometer consisting of two quadrupole mass analyzers in series, with a (non-mass resolving) radio frequency (RF)-only quadrupole between them to act as a cell for collision- induced dissociation.
  • RF radio frequency
  • the triple quadrupole mass spectrometer follows the tandem-in-space arrangement, due to ionization, primary mass selection, collision induced dissociation (CID), mass analysis of fragments produced during CID, and detection occurring in separate segments of the instrument.
  • Ion trap instruments surpass the triple quadrupole mass spectrometer in mass resolution and mass range; however, the triple quadrupole has the advantage of being cheaper, easy to operate, and they are highly efficient.
  • SRM Selected Reaction Monitoring
  • the triple quadrupole mass spectrometer has superior detection sensitivity as well as quantification.
  • the triple quadrupole allows the study of low-energy low-molecule reactions, which is useful when peptides are being analyzed. This instrument is utilized most effectively, and is the most effective instrument, for quantitating known peptides from a complex protein lysate with some prior knowledge, or suspected prior knowledge, of the peptide content of the complex protein lysate.
  • the mass spectrometry instrumentation may include, but is not limited to, tandem mass spectrometry, Q-TOF 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), and/or Data Independent Acquisition (DIA). Any one of the above mentioned mass spectrometry instruments and mass spectrometry methods can be applied to presently described method.
  • LC-MS/MS Liquid chromatography interfaced to tandem mass spectrometers
  • LC-MS/MS tandem mass spectrometers
  • a mass spectrometry approach for obtaining a global proteomic/protein profile of a protein lysate from patient tissue is label-free shotgun proteomics where the mass spectrometer is operated in a Data Dependent Acquisition (DDA) mode.
  • DDA Data Dependent Acquisition
  • MS/MS or MS2 tandem mass spectra
  • Shotgun proteomics suffers from two major drawbacks, however; the frequent occurrence of under-sampling and analyzing the MS/MS spectra outside of the elution peak can lead to low reproducibility, whereby only a small fraction of detectable peptides can be reliably identified and sequenced.
  • SRM Selected Reaction Monitoring
  • DIA data-independent acquisition
  • MS/MS scans are collected systematically (independently without precursor information) throughout the acquisition process.
  • Multiple DIA formats are in use and/or currently under development and any number of the following can be applied in the presently described method.
  • DIA collects all MS/MS scans irrespective of precursor ion selections from a survey scan or full MS scan, which DDA necessitates.
  • the predefmition of target fragment peptides, which SRM/PRM requires, is unnecessary for a DIA experiment.
  • a broad range of precursors and corresponding transitions can be extracted after data procurement.
  • DIA aims at inclusive proteome-wide quantification using targeted data extraction strategy.
  • DIA-based targeted method in general provides lower sensitivity, specificity, and reproducibility, as well as smaller dynamic range in protein quantification.
  • the method of DIA was originally introduced using an LTQ-linear ion trap (LIT) mass spectrometer with application of a wide precursor isolation window (10 m/z) to perform sequential isolation and fragmentation of a predefined m/z range.
  • LIT LTQ-linear ion trap
  • S/N signal to noise ratio
  • the applicability of ion extractions in MS/MS level of DIA quantification was also demonstrated.
  • such low resolution DIA MS/MS with the wide precursor isolation window decreased the mass accuracy and the confidence in peptide identification, which potentially resulted in increasing the false positive discovery rate.
  • MS E was introduced along the way and can be effectively operated on a QqTOF instrument.
  • isolation windows 2.5u
  • a much faster scanning ion trap MS e.g., LTQ Orbitrap Velos MS
  • LTQ Orbitrap Velos MS reduced the whole data acquisition time to ⁇ 2 days.
  • Introduction of a bench top Exactive MS was used to demonstrate the application of all-ion fragmentation (AIF), in which peptides were injected to HCD collision cell for fragmentation without precursor selection and the fragments were returned back to C-trap and analyzed through Orbitrap mass analyzer.
  • AIF all-ion fragmentation
  • MS1 data i.e., HR/ AM precursor data with a longer fill time and enough precursor ion data points across the LC elution profile
  • MS/MS data from the fast ion trap MS/MS scan are used only for peptide identification/confirmation.
  • /iS ART and wiSIM-DIA can only provide a smaller number of MS/MS spectra for the detectable precursors with relatively easier data analysis because the majority of the duty cycle time is used for generating the high-quality MS1 data.
  • their sensitivity and precision are higher than those provided by the standard DIA methods, while their quantification accuracy (i.e., specificity or selectivity) could be somewhat lower than that from the standard DIA methods due to the increased chances for having co-eluting interference from MS1 than MS/MS.
  • HRM hyper reaction monitoring
  • DIA spectra are highly multiplexed and thus more elaborate data interpretation algorithms are needed compared to DDA or SRM/PRM.
  • DIA-Umpire starts with two dimensional (in z and retention time) feature-detection algorithm to discover all possible precursor and fragment ion signals in MS and MS/MS data. Fragment ions are grouped with a precursor ion that has a correlation of LC elution peak and retention time at a peak apex. Generated pseudo-MS/MS spectra for each precursor-fragment group are then processed with conventional database search engine including the abovementioned tools. See Tsou et al,“DIA-Umpire:
  • the other approach is to match multiplexed MS/MS to theoretical spectra of peptides (e.g., ProblDtree, Ion Accounting, M-SPLIT, MixDB, and FT-ARM).
  • the scoring algorithms are directly based on how many theoretical fragment ions of a peptide from sequence databases or spectral libraries are found on multiplexed spectra with a high mass accuracy.
  • the first two approaches have greatly tackled identification of peptides from DIA spectra prior to further quantification.
  • Tumor cells are procured directly from patient tumor tissue utilizing tissue microdissection and analyzed by mass spectrometry in order to detect, identify, and quantitate fragment peptides from the CDK6 protein.
  • Patient tumor tissue can take the form of either frozen tissue, ethanol fixed tissue, or formalin fixed paraffin embedded tissue. All forms of tissue are equally useful for this presently described method.
  • tissue section Prior to collecting tumor cells the tissue section is stained using standard staining methods such as hematoxylin/eosin and IHC to uniquely identify tumor cells from other tissue microenvironment features such as normal epithelial cells, normal endothelial cells, immunological cells, and intercellular spaces.
  • tissue microdissection instruments suitable for use in the methods include, but are not limited to, instruments from Ziess, Lieca, ThermoFisher Scientific, x Dx, and MMI.
  • a protein lysate suitable for analysis by mass spectrometry is prepared. This can be performed, for example, by using Liquid Tissue reagents and protocol. Mass spectrometry is used to detect, identify, discover, and quantitate CDK6 fragment peptides present within tumor cells following one or more of the modes previously described to achieve either a“global profile” using DIA methods and/or a “targeted” analysis using SRM methods.
  • Mass spectrometry data about detected CDK6 fragment peptides and expression levels of the CDK6 protein can be useful for indicating that the cancer patient from which the tumor cells were collected from said patient tissue may be treated with cancer therapeutic agents that inhibit the function of the CDK6 protein such as including but not limited to palbociclib and ribociclib.

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Abstract

L'invention concerne des peptides spécifiques de la protéine CDK6 particulièrement avantageux pour la détection et la quantification de la protéine CDK6 directement dans des échantillons biologiques ayant été fixés dans la formaline. Une spectrométrie de masse en mode suivi de réaction sélectionnée (SRM) et/ou acquisition indépendante des données (DIA) est utilisée pour la détection. Un échantillon de protéine est préparé à partir de l'échantillon biologique à l'aide des réactifs et du protocole de tissu liquide et un ou plusieurs des peptides de fragment décrits à partir de la protéine CDK6 sont détectés et/ou quantifiés dans l'échantillon de tissu liquide par l'intermédiaire des méthodes de spectrométrie de masse SRM et/ou DIA. La mesure de la CDK6 effectue une distinction entre un carcinome kystique adénoïde et un carcinome malpighien.
PCT/US2018/063292 2017-12-01 2018-11-30 Dosage srm/mrm destiné au sous-typage d'une histologie du cancer de la tête et du cou WO2019108922A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016141169A1 (fr) * 2015-03-03 2016-09-09 Caris Mpi, Inc. Profilage moléculaire du cancer
WO2016196522A1 (fr) * 2015-05-29 2016-12-08 Cedars-Sinai Medical Center Peptides corrélés pour une spectrométrie de masse quantitative
US20170168057A1 (en) * 2014-07-01 2017-06-15 Expression Pathology, Inc. Srm assays to chemotherapy targets

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170168057A1 (en) * 2014-07-01 2017-06-15 Expression Pathology, Inc. Srm assays to chemotherapy targets
WO2016141169A1 (fr) * 2015-03-03 2016-09-09 Caris Mpi, Inc. Profilage moléculaire du cancer
WO2016196522A1 (fr) * 2015-05-29 2016-12-08 Cedars-Sinai Medical Center Peptides corrélés pour une spectrométrie de masse quantitative

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
POOMSAWAT ET AL.: "Expression of cdk6 in head and neck squamous cell carcinoma", CLIN ORAL INVESTIG, vol. 20, no. 1, 1 May 2015 (2015-05-01), pages 57 - 63, XP035890149, doi:10.1007/s00784-015-1482-8 *
TADESSE ET AL.: "Targeting CDK6 in cancer: State of the art and new insights", CELL CYCLE, vol. 14, no. 20, 30 October 2015 (2015-10-30), pages 3220 - 3230, XP055618492 *

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