EP2659272A2 - Biomarqueurs protéiques de cancer du sein récurrent - Google Patents

Biomarqueurs protéiques de cancer du sein récurrent

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Publication number
EP2659272A2
EP2659272A2 EP11853746.3A EP11853746A EP2659272A2 EP 2659272 A2 EP2659272 A2 EP 2659272A2 EP 11853746 A EP11853746 A EP 11853746A EP 2659272 A2 EP2659272 A2 EP 2659272A2
Authority
EP
European Patent Office
Prior art keywords
proteins
breast cancer
peptides
protein
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11853746.3A
Other languages
German (de)
English (en)
Other versions
EP2659272A4 (fr
Inventor
David B. Krizman
Marlene M. Darfler
Thomas P. Conrads
Brian L. Hood
Nicholas W. BATEMAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Expression Pathology Inc
Original Assignee
Expression Pathology Inc
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Filing date
Publication date
Application filed by Expression Pathology Inc filed Critical Expression Pathology Inc
Publication of EP2659272A2 publication Critical patent/EP2659272A2/fr
Publication of EP2659272A4 publication Critical patent/EP2659272A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • stage 3 and 4 providing the ability to identify those patients at greater risk of having later stage cancer (stage 3 and 4) that may at first appear to be early stage cancer (stage 0-2) is paramount to increasing survival rates. This is because enhanced, more informed treatment decisions can be made based on identifying, at an earlier point, those patients that harbor more aggressive disease, which will ultimately save lives.
  • a typical initial treatment is to remove the tumor by surgery followed secondarily by chemotherapy treatment designed to kill any residual cancer cells not removed by surgery.
  • Knowledge of the stage of the breast cancer is critical to patient treatment because different stages/grades of breast cancer respond differently to different treatment strategics. Determining the stage, grade, and or aggressiveness of breast cancer is best determined by analyzing the actual breast tumor tissue after removal from the patient. Tumor cells within the breast tumor tissue can be histologicaly and molecularly analyzed in order to determine grade, stage, and/or extent of breast cancer as well as identify which therapeutic agent is best to use against any tumor cells that remain in the patient.
  • the most widely and advantageously available form of cancer patient tissue is formalin fixed, paraffin embedded tissue.
  • Formaldehyde/formalin fixation of surgically removed tissue is by far and away the most common method of preserving cancer tissue worldwide and is the accepted convention for standard pathology practice.
  • Aqueous solutions of formaldehyde are referred to as formalin.
  • Formaldehyde/formalin fixation typically employs aqueous solutions of formaldehyde referred to as formalin.
  • " 100%" formalin consists of a saturated solution of formaldehyde (about 40% formaldehyde by volume or 37% by mass) in water, with a small amount of stabilizer, usually methanol to limit oxidation and degree of polymerization.
  • ⁇ critical issue for determining breast cancer treatment is to identify those patients who at first appear to harbor non-aggressive localized disease (stage 0-2) that may actually harbor more aggressive disease (stage 3-4) that will more than likely recur despite surgery and first-line chemotherapy treatment. If patients can be better identified whose disease will more than likely recur because it is actually a more aggressive form of breast cancer than my appear from histopathology or other measures, then more aggressive surgical (e.g., radical mastectomy as opposed to tylectomy aka "lumpectomy”), first line chemotherapy, or an additional second line of therapy can be performed on those patients.
  • stage 02 non-aggressive localized disease
  • stage 3-4 more aggressive disease
  • Tests that utilize proteins as indicators of aggressive forms of breast cancer are more advantageous because it is the proteins, not the nucleic acids, that principally do the work of the cell, and it is the aberrantly expressed proteins that cause a cell to become cancerous.
  • aberrantly expressed proteins can be targeted by drugs to selectively or specifically attack the cancer cells.
  • diagnostic tests that analyze proteins, and proteomic technologies to perform analysis are advantageous.
  • proteomics strives to establish the identities, quantities, structures, and biochemical and cellular functions of all proteins in an organism.
  • Application of proteomics has historically proceeded mostly on a one-protein-at-a-time basis.
  • the human protcome contains hundreds of thousands of proteins, and using recently developed proteomic techniques, changes in proteins that are over expressed in cells within solid tissue as well as proteins that are shed into body fluids throughout disease progression can now be examined.
  • Specific proteins, and patterns of proteins, that are found to be differentially expressed in diseased cells vs. normal cells can be reflective and diagnostic of a given disease state.
  • LC-MS/MS liquid-chromatography-tandem mass spectroscopy
  • the present disclosure provides, among other things, a method of diagnosing the presence of recurring breast cancer disease that is masked by its histological appearance as a less-aggressive, nonrecurrent form of the disease.
  • a sample is obtained from a patient.
  • the sample is breast cancer tissue, breast cancer cells, or a bodily fluid such as serum or fluid aspirate that may contain cells/proteins derived from a patient's cancerous tissue.
  • the presence and level of expression of at least one, two, three, four, five, six, seven, eight or more of the proteins listed in Table 1 are determined in the sample.
  • the level of expression of the detected proteins in early stage, recurrent breast cancer tissue is compared to the level of expression of the same proteins in early stage, nonrecurrent breast cancer.
  • the differential expression of at least one or more proteins, or combinations of multiple proteins indicates the presence of breast cancer disease that will likely recur in the patient, irrespective of any current or prior treatment. In this way a prognosis can be made, which is to predict if a breast cancer (e.g., an early stage breast cancer) may likely recur after initial treatment.
  • a breast cancer e.g., an early stage breast cancer
  • proteins, or peptide fragments thereof, arc detected by mass spectroscopy, and the level of expression of at least one or more than one of the proteins is determined by a spectral count quantization mass spectrometry or by Selected Reaction Monitoring (SRM) mass spectrometry; which can also be referred to as a Multiple Reaction Monitoring (MRM) mass spectrometry, alternatively referred to hereinafter referred to as SR.M/MRM assay(s).
  • SRM Selected Reaction Monitoring
  • MRM Multiple Reaction Monitoring
  • the proteins are detected and their levels of expression are determined by a protein microarray or by an immunoassay.
  • This disclosure also provides a method of identifying protein targets for therapeutic intervention in breast cancer.
  • the presence and level of expression of one, two, three, four, five, six, seven, eight or more of the proteins listed in Table 1 are detected in the sample.
  • the level of expression of the detected proteins in early stage, recurrent breast cancer tissue is compared to the level of expression of the same proteins in early stage, non-recurrent breast cancer.
  • the differential expression of one, two, three, four, five, six, seven, eight or more proteins may indicate choice of therapy and define specific targets for therapeutic intervention in breast cancer.
  • sample for assessing protein expression includes solid tissue (normal or diseased) and bodily fluids derived from the patient through surgical means including biopsy and aspiration. Protein expression is most advantageously detected and measured in cells or tissue samples from solid tumor tissue because these are the actual cells that are growing and causing the disease. However, it is sometimes less invasive and more comfortable for the patient to collect a bodily fluid such as blood, lymph fluid and/or ascites fluid that surrounds the tumor itself. These fluid sources may contain a number of the proteins listed in Table I because they can be secreted by the tumor cells into the surrounding fluid or the tumor cells themselves become dislodged from the solid tumor and can now be found in the fluid, and which in many cases is an easier sample to collect from a breast cancer patient. The proteins listed in Table 1 can be detected and levels measured in either solid tissue or a bodily fluid from the breast cancer patient.
  • one, two, three, four, five, six, seven, eight or more of the proteins listed in Table 1 as undergoing a decrease are examined.
  • one, two, three, four, five, six, seven, eight or more of the proteins listed in Table 1 as undergoing an increase, in combination with one, two, three, four, five, six, seven, eight or more of the proteins listed in Table 1 as undergoing a decrease arc examined.
  • the greater the number of biomarker proteins found in Table 1 to be increased (over-expressed) and/or decreased (under-expressed ) in a sample obtained from a patient, the higher the probability that a primary breast cancer will recur in that patient. Certain embodiments of the invention arc described below.
  • a method of prognosing that an early stage primary breast cancer may recur in a patient after initial treatment comprising the steps of:
  • bodily fluids include but are not limited to fractionated or unfractionated blood, serum, plasma, lymphatic fluid, or fluid collected by pleural effusion.
  • immunoassay is selected from the group consisting of immunohistochemistry, Western blot, dot blot, and FLISA.
  • ⁇ metliod of indicating choice of therapy of primary breast cancer that is most likely to recur after initial treatment comprising the steps of:
  • a method comprising quantifying the amount of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more of the proteins in Table 1 or peptide fragments thereof.
  • composition comprising one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or ten or more of the proteins in Table 1 , peptides thereof, or antibodies thereto.
  • composition of embodiment 1 8 comprising one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more peptides of proteins in Tabic 1 , wherein each peptide is derived from a different protein.
  • composition of any of any of embodiments 19-2 1 comprising one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more peptides of proteins in Table I that are decreased in tissues from primary tumors that recurred in two years.
  • nucleic acids have a length selected independently from greater than about 1 5. 20, 25, 30, 35, 40, 50, 60, 75, or 100 nucleotides in length.
  • nucleic acids have a length selected independently from less than about 150, 200, 250, 300, 350, 400, 500, 600, 750, 1 ,000, 2,000, 4,000, 5,000, 7,500, 10,000, 15,000, or 20,000 nucleotides in length.
  • assessing and/or determining the level (amount) or sequence comprises, determining either the sequence of nucleotides in the nucleic acids and or a characteristic of the nucleic acids by any one or more of: nucleic acid sequencing, conducting restriction fragment polymorphism analysis, conducting hybridization with another nucleic acid, identification of one or more deletions and/or insertions, and/or determining the presence of mutations, including but not limited to, single base pair polymorphisms, transitions and/or transversions.
  • nucleic acids encode for proteins of SEQ ID os: 1 -20, 21 -41 , 1 - 10, 1 1 -20, 21 -30, 3 1 -41 , or fragments thereof Table 1.
  • these proteins may be readily assayed in bodily fluids that derive from breast cancer cells, such as ascites fluid or fluids derived from blood such as plasma and scrum. It is either breast-derived tissue, breast epithelial cells, or bodily fluids that would be assayed for diagnostic evaluation of breast cancer by assaying for specific protein expression from the list described herein. Also, one or more of the same proteins form the basis for a targeted therapeutic approach whereby a drug would be directed towards these proteins.
  • Identification of these proteins provides for the ability to detect early stage breast cancer that is most likely to recur at a later time following the initial treatment in a broad variety of biological samples collected from a subject, including fixed and frozen tissue, and bodily fluid samples derived from both blood and ascites fluids.
  • the diagnostic and prognostic endpoint for disease analysis includes both single analytes and proteomic patterns.
  • Proteomic patterns may be composed of many individual proteins, each of which may not individually identify breast cancers that are likely to recur, but collectively identify breast cancers with an increased probability of recurrence.
  • individual proteins, patterns of proteins, and/or collections of multiple proteins to be utilized for diagnosis, prognosis, and therapy of recurrent breast cancer.
  • the methods provided herein make possible the evaluation of the likelihood of recurrence for a primary breast cancer and treatment strategies for a subject (patient) with breast cancer.
  • the methods are useful for determining if a breast cancer that appears to be early stage, non-aggressive by visual histological methods is likely a more aggressive advanced stage of breast cancer that potentially recur after first line evaluation of the presence, absence, nature and/or extent of breast cancer.
  • By measuring one, two, three, four, five, six, seven, eight or more of the proteins from the list of proteins in Table 1 breast cancer can be diagnosed in a subject, the prognosis of that subject can be determined, and the specific drug for that subject's disease can be chosen.
  • a sample of tissue such as that which is surgically procured or biopsied from a subject and frozen or chemically fixed, or a bodily fluid, such as blood, serum, plasma, lymph fluid, and/or ascites fluid is examined to evaluate and measure protein expression.
  • the difference between the recurrent breast cancer protein profile and the reference non-recurrent breast cancer protein profile comprises a difference in the amount of one, two, three, four, five, six, seven, eight or more biomarker proteins from the list in Table 1.
  • the method for evaluating breast cancer pathology in a subject includes discriminating between different disease states or between a disease state and normal state. Such a profile is also used to determine prognosis, which aims to monitor the extent and expectations of the progression or regression of breast cancer in a given subject.
  • the recurrent breast cancer protein profile can be derived from a biological sample previously obtained from the subject, for example a biological sample obtained prior to treatment or as part of a general health screening.
  • the method is also well-suited to evaluate the efficacy of treatment decisions, such as drugs or surgeries.
  • one or more of the proteins within the breast cancer protein profile can serve as a target for drug treatment.
  • the drug specifically interacts with individual and specific proteins from the list of proteins in Table 1 .
  • the drug interacts with a binding partner of a protein from the list in Table 1 , thereby altering the ability of the protein in Table 1 to interact with its binding partner or to carry out its biological function.
  • the expression profile of one, two, three, four, five, six, seven, eight or more of the proteins may be used to select the drug therapy, and/or the duration/regimen
  • the method further comprises a classification model or algorithm, based on one or more protein differences from the protein list of Table 1 between the test protein profile of a biological sample from a subject suspected of having recurrent breast cancer and the reference protein profile from a biological sample from a subject not having recurrent breast cancer.
  • recurrent or non-recurrent breast cancer protein profiles or both arc generated using mass spectrometry.
  • the methods of mass spectrometry employed may advantageously use ion trap instruments or triple quadrupole instruments.
  • a proteolytic enzyme e.g. trypsin, papain, chymotrypsin, and others, thus rendering a complex protein sample preparation to a complex lysate consisting of peptides.
  • Such peptide lysates are the preferred form of sample for analysis of proteins from a biological sample by mass spectrometry, where the quantitative presence of specific and individual peptides is indicative of the quantitative presence of the ull length intact proteins from which the peptides derive.
  • analysis of all peptides simultaneously in a global fashion may advantageously be performed on an ion trap mass spectrometry instrument.
  • analysis of targeted peptides that specifically focus assays on individual and specific peptides, and thus the proteins from which they derive is conducted on a triple quadrapole mass spectrometry instrument. Performing targeted quantitative protein analysis by triple quadrupole mass spectrometry may be accomplished using SRM/MRM methodology.
  • That methodology can be used to generate a protein profile to investigate the likelihood of recurrent breast cancer in a subject from which a biological sample was obtained.
  • peptides in the lysates may be subject to a variety of techniques that facilitate their analysis and measurement by mass spectrometry.
  • the peptides may be separated by an affinity technique, such as immunologically-based purification (e.g., immunoaffinity chromatography), chromatography on ion selective media, or if the peptides are modified, by separation using appropriate media, such as lectins for separation of carbohydrate modified peptides.
  • the SISCAPA method which employs immunological separation of peptides prior to mass spectrometric analysis is employed.
  • the SISCAPA technique is described, for example, in U.S. Patent No. 7,632,686.
  • lectin affinity methods e.g., affinity purification and/or chromatography may be used to separate peptides from a lysate prior to analysis by mass spectrometry.
  • Methods for separation of groups of peptides, including lectin- based methods are described, for example, in Geng et al. , J. Chromatography B, 752 :293-306 (2001 ).
  • Immunoaffinity chromatography techniques may be used in any suitable combination to facilitate the analysis of peptides by mass spectrometry.
  • Another assay method includes immobilizing the proteins and/or peptides from the proteins, on a microarray (e.g., using immobilized antibodies) prior to detecting the proteins using antibody- based methods including sandwich-type assays.
  • Other assay methods include immunohistochemical analysis utilizing antibody-based protein detection methods on thin tissue sections, where the proteins arc maintained in full length (not subject to proteolysis) within the tissue section.
  • Still other assay methods include antibody-based Western blot and ELISA protein detection methods, where the protein preparations interrogated arc full length intact proteins and/or derivative peptides.
  • All of these described protein detection methods may be used to detect individual polypeptides that derive from whole intact proteins, and thus these methods do not necessarily require the detection of whole intact proteins, but can involve the detection of peptides derived from the whole intact proteins. These methods may be used alone or in any combination, including in combination with mass spectroscopy based methods. Any suitable report/detection system known in the art may be employed with such assays including, but not limited to, fluorescence, UV/Vis chromatophore development, plasmon resonance, metal staining, and the like.
  • a useful method for detecting proteins from the protein list in Table 1 and polypeptides derived from these proteins.
  • the presence, absence, nature or extent of breast cancer pathology indicating recurrent breast cancer disease in a patient can be evaluated in view of the expression of one or more expressed biomarker proteins from the list, and/or a derivative peptide or peptides from the same proteins.
  • a method is provided for screening a patient or population of patients for breast cancer by assaying for the presence of one or more proteins found in Table 1 , or their derivative peptides.
  • the assay(s) employed may include mass spectrometric assays, immunologic assays, such as a Western blot, enzyme linked immunosorbent assay (EI, ISA), or immunohistochemical methods on intact tissue sections, or any combination thereof.
  • immunologic assays such as a Western blot, enzyme linked immunosorbent assay (EI, ISA), or immunohistochemical methods on intact tissue sections, or any combination thereof.
  • EI enzyme linked immunosorbent assay
  • ISA enzyme linked immunosorbent assay
  • one, two, three, four, five, six, seven, eight or more of the proteins listed in Table 1 as undergoing an increase, in combination with one, two, three, four, five, six, seven, eight or more of the proteins listed in Table 1 as undergoing a decrease are examined.
  • the protein biomarkers (e.g., the proteins in Table 1 ) were selected based on their differential patterns of expression observed in breast cancer epithelial cells obtained from primary tumors that gave rise to recurrent breast cancer after surgery' an breast cancer epithelial cells obtained from primary tumors that did not give rise to recurrent breast cancer post surgery, irrespective of current or prior treatment. Levels of some proteins were increased in cancerous cells obtained from recurrent breast cancer tissue while levels of other proteins decreased in recurrent cancer tissue cells.
  • Protein lysates obtained from the cells of the two patient populations contain all the necessary information about differential protein expression. Protein lysates from the cells of those patient populations were prepared using the Liquid TissueTM protocol and reagents. The preparation method included collecting cells (tissue sample) into a tube via tissue microdissection followed by maintaining the cells (tissue sample) at an elevated temperature 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.
  • 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
  • the buffer employed is a neutral buffer, (e.g., a Tris-based buffer, or a buffer containing a detergent) and advantageously is a buffer that does not interfere with mass spectrometric analysis.
  • a neutral buffer e.g., a Tris-based buffer, or a buffer containing a detergent
  • the cells are then digested to completion in a predictable manner using a protease (e.g., trypsin).
  • a protease e.g., trypsin
  • the prepared lysates were then analyzed by global proteomic mass spectrometry and the data is initially presented as identification of the total number of peptides in each protein lysatc. Once as many peptides as possible were identified in a single MS analysis of a single lysate, then that list of peptides was compared to the list of peptides identified across all lysates in a study set. Thus, the starting point for determining differential protein expression by mass spectrometry was a list of peptides found to be expressed in one sample and/or group of similar samples and compared to the list of peptides found expressed in a second sample and/or group of similar samples.
  • the first group of four (4) Liquid TissueTM samples were derived from early stage primary breast cancer tissue from patients whose cancer did not recur after at least 2 years post initial treatment while the second group of five (5) Liquid TissueTM samples were derived from early stage primary breast cancer tissue from patients whose cancer recurred within 2 years post initial treatment.
  • the comparison of those proteins that were differentially expressed between these two groups of patients, recurrent early stage breast cancer vs. non-recurrent early stage breast cancer formed the initial study set of proteins set forth in Table 1.
  • the classification of differential protein expression from the lists of peptides found in patients that suffered recurrent and those that did not suffer from recurrent breast cancer was accomplished by first determining which proteins were represented by a given list of peptides, and then to count the total number of peptides identified for each protein. That method of data collating is known as the Spectral Count method (SC).
  • SC Spectral Count method
  • the spectral count for a given protein is thus based on the total number of peptides identified for that protein in a single lysate, which is a relative indicator for the abundance of that protein in the lysate that was analyzed by MS.
  • Spectral count is a mathematical method that provides the ability to compare relative protein abundances for a given protein from one sample and/or group of similar samples to the next sample and/or group of similar samples. This approach can also be uses to distinguish protein abundance between individual proteins within a given sample.
  • Spectral counts between thousands of individual proteins arc compared for samples obtained from breast cancer epithelial cells obtained from multiple primary patient-derived tumors that gave rise to recurrent breast cancer and breast cancer epithelial cells obtained from multiple primary patient-derived tumors that did not give rise to recurrent breast cancer.
  • the protein abundance was thus derived by mass spectrometry analysis of protein lysates from multiple breast cancer tissues using spectral counting (SC) of peptides.
  • SC spectral counting
  • peptides whose sequences mapped to multiple protein isoforms were grouped as per the principle of parsimony.
  • a hierarchical supervised cluster analysis of peptides identified from stage 11 nonrecurrenct (Stage II NR) versus stage M with disease recurrence (Stage II R) patient samples was performed in which the variance in total spectral count peptides identified was determined utilizing the Mann-Whitney rank-sum test (significance level p ⁇ 0.05, Fisher's exact test) paired with the filter criteria requiring that 60% of the samples in a supervised group had a minimum peptide count of two (2) or greater for a given protein.
  • Table 1 shows the names of 41 proteins, 3 1 that were increased and 1 1 decreased in abundance, which significantly differentiate stage II NR versus 11 R patients.
  • the method of prognosis will employ at least one or more proteins that have increased levels, another embodiment that employs decreased levels, and yet another embodiment that employs a combination of both increased and decreased levels, in addition, the method of prognosis may involve specific combinations of decreased expression and/or increased expression across multiple proteins in a single assay to give a pattern of protein expression changes indicative of and prognostic for early stage recurrent breast cancer.
  • protcasome prosome, macropain
  • the present methods encompass not only methods of diagnosis, prognosis, therapeutic treatment, and compositions that employ the proteins recited in Table 1 , but also those that employ related proteins.
  • the related proteins encompass proteins/polypeptides that share at least some amino acid sequence with the proteins in Table 1 , and which are produced by translation of alternate transcripts (or alternately processed transcripts) from the genes encoding the proteins in Table 1 .
  • related proteins encompasses proteins/polypeptides that share at least some amino acid sequence with the proteins in Table 1 produced by changes at the translational or post-translation level (e.g., post translational modifications).
  • related proteins may comprise a sequence of greater than five, six, seven, eight, ten, twelve, fifteen, eighteen, or twenty contiguous amino acids that is identical to a sequence found in a protein in Table 1.
  • compositions comprising one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more of the proteins in Table 1 , or polypeptide fragments thereof.
  • the compositions comprise two or more, three or more, four or more, five or more, six or more, or seven or more antibodies that bind specifically to proteins found in Table l or peptide fragments of those proteins.
  • Compositions comprising peptides may include one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more peptides that are isotopically labeled.
  • compositions comprising peptides from the any of the proteins in Table 1 do not need to contain all of the peptides from any given protein (e.g., a complete set of tryptic peptides). In some embodiments the compositions will comprise only one, two, three, four, five, six, or seven peptides for two, three, four, five, six, seven, eight, nine, ten, or more of the proteins appearing in Table 1 or Table 2.
  • Compositions comprising peptides may be in the form of dried or lyophilized materials, liquid (e.g., aqueous) solutions or suspensions, arrays, or blots.
  • the protein biomarkers described herein can be advantageous employed to improve the treatment of patients with breast cancer.
  • the over-expression and/or under-expression of one or more proteins in recurrent breast cancer vs. non-recurrent breast cancer and the ability lo assay for this over-expression and/or under-expression in a biological sample can be used to determine whether or not a person with breast cancer has a type of cancer that will likely recur.
  • the results may also indicate choices for therapy and/or treatment regimens which are different from those that would be used for a non-recurrent breast cancers.
  • determinations bases upon the altered expression of multiple proteins are more likely to be effective as indicators of recurrent breast cancer than assessment of one or two proteins individually.
  • the present methods include and provide for assessment and correlation of multiple proteins simultaneously in a single biological sample from an individual suspected of being afflicted with a recurrent form of breast cancer.
  • Over-expression and/or under-expression of one or more proteins in recurrent breast cancer vs. non-recurrent breast cancer and the ability to assay for this over-expression and/or under- expression in a biological sample can be used to help determine which therapeutic agent is chosen to achieve the best course of disease treatment.
  • One or more of the proteins indicated herein can be targeted directly with a drug so that breast cancer cells can be killed preferentially instead of the normal cells in the tissue that arc not expressing one or more of these proteins.
  • the type of biological sample assayed using one or more of these proteins as biomarkers of recurrent breast cancer include biopsied tissue or tissue removed surgically.
  • the tissue can be fresh, frozen, and/or chemically fixed such as that which is preserved in formalin and other chemical fixatives of the like.
  • Another form the biological sample can lake is fractionated or unfractionated biofluid samples such as serum, plasma, whole blood, and ascites fluids. All of these forms of a patient-derived biological sample can be assayed for expression of one or more of the proteins in Table 1.
  • nucleic acids and protein can be analyzed from the same biomolecular lysate preparations employed herein (e.g. as U.S. Patent No. 7,473,532) it is possible to generate additional information about disease diagnosis and drug treatment decisions from the same sample. For example, additional information about the state of the cells and their potential for uncontrolled growth, potential drug resistance, and the development of cancers can be obtained by analyzing nucleic acids from those lysate preparations.
  • iysate preparations for both protein/peptide analysis and nucleic acid analysis it is possible to obtain information about the status of any one, two, three, four, five or more genes and/or the nucleic acids, and/or the proteins they encode (e.g., mRNA molecules and their expression levels or splice variations) from the same biomolecular lysate preparation. For example information about any one, two, three, four, five or more peptides in Table 1 , and or the proteins from which they were derived or the nucleic acids encoding those proteins may be assessed.
  • the nucleic acids can be examined, for example, by: one or more sequencing methods- conducting restriction fragment polymorphism analysis, conducting hybridization with another nucleic acid, identify deletion, insertions, and/or detennining the presence of mutations, including but not limited to, single base pair polymorphisms, transitions and/or transversions.
  • Such tests may be conducted in any suitable format including, but not limited to, arrays, microarrays, on blots, or in solution (e.g., by polymerase chain reaction "PCR” or ligase chain reaction "LCR”).
  • the assay or test may be conducted in any suitable format (e.g., arrays/mi croarrays, blots, and the like) by contacting nucleic acids under conditions of suitable stringency to obtain specific binding.
  • suitable format e.g., arrays/mi croarrays, blots, and the like
  • the required "stringency" of hybridization reactions determinable by one of ordinary skill in the art, and generally involves an empirical calculation dependent upon probe length, washing temperature, and salt concentration, in general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures.
  • Hybridization generally depends on the ability of denatured DNA to anneal when complementary strands are present in an environment below their melting temperature, with.
  • “Stringent conditions” typically employ low ionic strength with or without a denaturant (e.g., formamide) and high temperature for washing, for example, 0.015 M sodium chloride/0.0015 M sodium citrate/0.1 % sodium dodecyl sulfate at 50° C.
  • a denaturant e.g., formamide
  • Modely stringent conditions may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above.
  • moderately stringent conditions is overnight incubation at 37° C in a solution comprising: 20% formamide, 5X SSC ( 150 mM NaCl, 1 5 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing in 1 XSSC at about 37-50° C.
  • 5X SSC 150 mM NaCl, 1 5 mM trisodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5X Denhardt's solution 10% dextran sulfate
  • 20 mg/ml denatured sheared salmon sperm DNA followed by washing in 1 XSSC at about 37-50° C.
  • samples are analyzed for one, two, three, four, five, six.
  • samples are analyzed for two, three, four, five, six, seven, eight, nine or more peptides produced from the proteins in Table 1 , and/or two, three, four, five, six, seven, eight, nine or more nucleic acids encoding proteins from Table 1, where the proteins from Table 1 are selected from any range of proteins represented by SEQ ID Nos: 1 -20, 21 -41 , 1 -10, 1 1 -20, 21-30, 3 1 -41 .
  • Tissue sections were prepared from each tissue for histologic analysis and procurement of epithelial cancer cells was performed by tissue microdissection.
  • Soluble protein lysates were prepared from microdissected breast cancer tissue samples using the Liquid Tissue 1 M MS Protein Prep Kit (Expression Pathology, Inc.). Each lysate consisted of the total protein content of the microdissected cells digested into predictable peptide fragments by the protease trypsin. In this form each and every protein lysatc can be evaluated by the technology of mass spectrometry for identification and quantification of the proteins present in each lysate. In addition, the total mass spectrometry data across all samples is used to determine differential protein expression between individual samples and between primary tumors from non-recurrent breast cancer patients and primary tumors from patients with recurrent breast cancer.
  • Mass spectrometry analysis of each trypsin-digested protein lysate was performed according to the following. Liquid chromatography (LC) was performed using a Dioncx Ultimate 3000 system coupled on-line to a ThermoFisher linear ion trap mass spectrometer (MS). Separation of the sample was performed using a 75 ⁇ ID x 360 ⁇ ⁇ ⁇ OD x 10-cm-Iong fused silica capillary column 5 ⁇ , 300 A pore size Jupiter C- 18 stationary phase.
  • LC Liquid chromatography
  • MS ThermoFisher linear ion trap mass spectrometer
  • LIT-MS Linear Ion Trap Mass Spectrometer
  • C1D relative collision-induced dissociation
  • peptide identifications were filtered according to specific SEQUEST scoring criteria: delta correlation (AC,,) > 0.08 and charge state dependent cross correlation (Xcorr) > 1.9 for [ +H]' ⁇ 2.2 for [M+2H] 2* , and > 3.5 for [M+3H] 3* (Supplemental Table 1 ).
  • FDR false discovery rate
  • stage II NR stage II nonrecurrent disease
  • Stage II R stage II with disease recurrence
  • Spectral Count Quantitation is the process of counting the number of unique peptides associated with each protein. ⁇ value of 4 beside a protein name reflects that there were 4 unique peptides that were associated with that particular protein.
  • the SRM/MRM assays described herein can measure relative or absolute quantitative levels of one or more specific peptides derived from one or more of the proteins listed in Table 1 .
  • the method is utilized to provide a means of measuring the amount of a given peptide, peptides, protein, or proteins, by mass spectrometry in a given peptide/protein preparation obtained from a patient's biological sample such as bodily fluid or a Liquid Tissue I M lysate from formalin fixed paraffin embedded tissue.
  • SRM/MRM assay can measure peptides directly in complex protein lysates prepared from cells procured from patient tissue samples, such as formalin fixed cancer patient tissue.
  • Results from the SRM MRM assay can be used to correlate accurate and precise quantitative levels of a given peptide, peptides, protein, or proteins, with the specific cancer of the patient from whom the biological sample was collected. 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, such as bodily fluids, is termed a companion diagnostic assay.
  • a companion diagnostic assay can be designed to diagnose the stage or degree of a cancer and determine which therapeutic agent, or course of therapy, to which a patient is most likely to respond with a positive outcome.
  • An SRM/MRM assay measures relative or absolute levels of specific unmodified peptides from a given protein, or protein, and also can measure absolute or relative levels of specific modified peptides from proteins. Examples of modifications include phosphorylated amino acid residues and glycosylated amino acid residues that are present on the peptides.
  • Relative quantitative levels of a given peptide, peptides, protein, or proteins are determined by the SRM/MRM methodology, whereby the mass spectrometry-derived signature peak area (or the peak height if the peaks arc sufficiently resolved) of an individual peptide, or multiple peptides, from a given protein, or proteins, in one biological sample is compared to the signature peak area determined for the same identical peptide, or peptides, from the same protein, or proteins, using the same methodology in one or more additional and different biological samples.
  • the amount of a particular peptide, or peptides, from a given protein, or proteins is determined relative to the same peptide, or peptides, from the same protein, or proteins, across 2 or more biological samples under the same experimental conditions.
  • relative quantitation can be determined for a given peptide, or peptides, from a single protein within a single sample by comparing the signature peak area for that peptide for that given protein by SRM/MRM methodology to the signature peak area for another and different peptide, or peptides, from a different protein, or proteins, within the same protein preparation from the biological sample.
  • the amount of a particular peptide from a given protein is determined relative one to another within the same sample.
  • These approaches generate quantitation of an individual peptide, or peptides, from a given protein to the amount of another peptide, or peptides, between samples and within samples wherein the amounts as determined by signature peak area are relative one to another, regardless of the absolute weight to volume or weight to weight amounts of peptides in the protein preparation from the biological sample.
  • Relative quantitative data about individual signature peak areas between different samples are normalized to the amount of protein analyzed per sample.
  • Relative quantitation can be performed across many peptides simultaneously in a single sample and/or across many samples to gain insight into relative protein amounts, one pcpiidc/protcin with respect to other peptides/protcins.
  • Absolute quantitative levels of a given protein, or proteins are determined by the SRM/M RM methodology whereby the SRM/MRM signature peak area of an individual peptide from a given protein in one biological sample is compared to the SRM/MRM signature peak area of a known amount of a "spiked" internal standard.
  • the internal standard is a synthetic version of the same exact peptide that contains one or more amino acid residues labeled with one or more heavy isotopes.
  • isotope labeled internal standards arc synthesized so that mass spectrometry analysis generates a predictable and consistent SRM/MRM signature peak thai is different and distinct from the native peptide signature peak, and which can be used as a comparator peak.
  • the signature peak area of the native peptide is compared to the signature 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.
  • Absolute quantitative data for fragment peptides are displayed according to the amount of protein analyzed per sample. Absolute quantitation can be performed across many peptides, and thus proteins, simultaneously in a single sample and/or across many samples to gain insight into absolute protein amounts in individual biological samples and in entire cohorts of individual samples.
  • the SRM/MRM assay method can be used to aid diagnosis of the stage of cancer, for example, directly in patient-derived tissue, such as formalin fixed tissue, and to aid in determining which therapeutic agent and/or treatment strategy would be most advantageous for use in treating that patient.
  • Cancer tissue that is removed from a patient either through surgery, such as for therapeutic removal of partial or entire tumors, or through biopsy procedures conducted to determine the presence or absence of suspected disease is analyzed to determine whether or not a specific protein, or proteins, and which forms of proteins, are present in that patient tissue.
  • the expression level of one or more proteins can be determined and compared to a "normal" or reference level found in healthy tissue or tissue that shows a different stage/grade of cancer.
  • This information can then be used to assign a stage or grade to a specific cancer and can be matched to a strategy for treating the patient based on the determined levels of specific proteins.
  • Matching specific information about levels of a given protein, or proteins, as determined by an SRM/MRM assay, to a treatment strategy that is based on levels of these proteins in cancer cells derived from the patient defines what has been termed a personalized medicine approach to treating disease.
  • the SRM/MRM assay method described herein form the foundation of a personalized medicine approach by using analysis of proteins from the patient's own tissue as a source for diagnostic and treatment decisions.
  • the SRM/MRM method described herein can be used to specifically assay proteins in Table 1 .

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Abstract

Cette invention concerne des protéines qui se sont révélées d'être exprimées de façon différentielle entre des cellules de cancer du sein à tumeur primaire, qui n'ont pas donné naissance à une maladie du cancer du sein récurrent après un diagnostic initial, et des cellules de cancer du sein primaire, qui ont donné naissance à une maladie du cancer du sein récurrent après un diagnostic initial. Ces protéines peuvent être utilisées soit individuellement, soit dans des combinaisons spécifiques dans des dosages protéiques de diagnostic et de pronostic sur divers échantillons biologiques provenant de patients atteints d'un cancer du sein pour indiquer la susceptibilité qu'un cancer chez des patients atteints d'un cancer du sein réapparaîtra après le diagnostic initial et le traitement. Une détermination d'une expression différentielle de ces protéines peut également être utile pour l'indication de thérapies supplémentaires pour combattre la susceptibilité d'un cancer du sein récurrent. Les protéines intactes, de longueur totale, peuvent être dosées ou des peptides issus de ces protéines peuvent être dosés en tant que rapporteurs pour ces protéines. Ces protéines peuvent également être identifiées en tant que protéines «compagnon de diagnostic », les protéines exprimées de façon différentielle, qui sont utilisées en tant qu'indicateurs de diagnostic et de pronostic, pouvant également être utilisées en tant que cibles pour une intervention thérapeutique du cancer du sein. La présente invention concerne également des versions marquées par un isotope de peptides provenant des protéines.
EP11853746.3A 2010-12-29 2011-12-29 Biomarqueurs protéiques de cancer du sein récurrent Withdrawn EP2659272A4 (fr)

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