Patent Application
20160334408
Cancer is treated with a collection of therapeutic agents that kill growing and dividing cells and that function in a variety of ways. A common collection of chemotherapeutic agents has been used for decades, either individually or in combinations, and this common collection of agents has become the traditional and routine cancer treatment in clinical oncology practice. These traditional chemotherapeutics agents act by killing all cells that divide rapidly, rapid cell division being one of the main properties of most cancer cells. However, these agents also kill growing normal cells and, accordingly, these agents are not considered to be “targeted” approaches to killing cancer cells. In recent years a large group of cancer therapeutic agents has been developed that selectively target only cancer cells where the therapeutic agent specifically attacks a protein that either is only expressed by the cancer cells and not by normal cells, or that is expressed in much higher amounts in cancer cells compared to normal cells. This approach is considered to be a “targeted” approach to cancer therapy. Most recently, another approach to killing cancer cells in a “targeted” fashion has been to specifically modulate the immune system to enhance that ability of the cancer patient's immune system to kill cancer cells.
Therapeutic agents that target the 6-O-methylguanine-DNA methyltransferase protein, which is also referred to as MGMT, have shown promise in early clinical trials. However, only those patients whose cancer cells express high amounts of the MGMT protein would be most likely to benefit from treatment with these MGMT-targeted therapeutic agents. The methods described below provide a quantitative proteomics-based assay that delivers a relevant measure of activation of the MGMT signal pathway as MGMT is not normally expressed in normal tissue and/or normal epithelial cells. In particular, the methods provide a mass spectrometry assay that quantifies MGMT in formalin fixed tissues from cancer patients and that enables improved treatment decisions for cancer therapy.
Specific peptides derived from subsequences of the 6-O-methylguanine-DNA methyltransferase protein, also referred to as MGMT, methylated-DNA-protein-cysteine methyltransferase, and referred to herein as MGMT, are provided. The peptide sequence and fragmentation/transition ions for each peptide are particularly useful in a mass spectrometry-based Selected Reaction Monitoring (SRM), which can also be referred to as a Multiple Reaction Monitoring (MRM) assay, and will be referred to as SRM/MRM. The use of peptides for SRM/MRM quantitative analysis of the MGMT protein is described.
This SRM/MRM assay can be used to measure relative or absolute quantitative levels of one or more of the specific peptides from the MGMT protein and therefore provide mass spectrometric methods of measuring by mass spectrometry the amount of the MGMT protein in a given protein preparation obtained from a biological sample.
More specifically, the 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. 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 references 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.).
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 are 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 (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. The most common way in which tissue is preserved is to soak whole tissue for extended periods of time (8 hours to 48 hours) 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. Thus molecular analytical methods to analyze formalin fixed cancer tissue will be the most accepted and heavily utilized methods for analysis of cancer patient tissue.
Results from the SRM/MRM assay can be used to correlate accurate and precise quantitative levels of the MGMT 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 and prognostic information about the cancer, but also permits a physician or other medical professional to more accurately determine appropriate therapy for the patient. Such an assay that provides diagnostically, prognostically, and therapeutically important information about levels of protein expression in a diseased tissue or other patient sample is termed a companion diagnostic assay. For example, such an assay can be designed to diagnose the stage or degree of a cancer and determine a therapeutic agent to which a patient is most likely to respond.
The assays described herein measure relative or absolute levels of specific unmodified peptides from the MGMT protein and also can measure absolute or relative levels of specific modified peptides from the MGMT protein. Examples of modifications include phosphorylated amino acid residues and glycosylated amino acid residues that may be present on the peptides.
Relative quantitative levels of the MGMT protein are determined by the SRM/MRM methodology for example by comparing SRM/MRM signature peak areas (e.g., signature peak area or integrated fragment ion intensity) of an individual MGMT peptide in different samples. Alternatively, it is possible to compare multiple SRM/MRM signature peak areas for multiple MGMT signature peptides, where each peptide has its own specific SRM/MRM signature peak, to determine the relative MGMT protein content in one biological sample with the MGMT protein content in one or more additional or different biological samples. In this way, the amount of a particular peptide, or peptides, from the MGMT protein, and therefore the amount of the MGMT protein, is determined relative to the same MGMT peptide, or peptides, across two or more biological samples under the same experimental conditions. In addition, relative quantitation can be determined for a given peptide, or peptides, from the MGMT protein within a single sample by comparing the signature peak area for that peptide 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. In this way, the amount of a particular peptide from the MGMT protein, and therefore the amount of the MGMT protein, is determined relative one to another within the same sample. These approaches permit quantitation of an individual peptide, or peptides, from the MGMT 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 the MGMT peptide 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 from multiple proteins and the MGMT protein simultaneously in a single sample and/or across many samples to gain insight into relative protein amounts of one peptide/protein with respect to other peptides/proteins.
Absolute quantitative levels of the MGMT protein are determined by, for example, the SRM/MRM methodology whereby the SRM/MRM signature peak area of an individual peptide from the MGMT protein in one biological sample is compared to the SRM/MRM signature peak area of a spiked internal standard. In one embodiment, the internal standard is a synthetic version of the same exact MGMT peptide that contains one or more amino acid residues labeled with one or more heavy isotopes. Such an isotope labeled internal standard is synthesized so that when analyzed by mass spectrometry it generates a predictable and consistent SRM/MRM signature peak that is different and distinct from the native MGMT peptide signature peak and which can be used as a comparator peak. Thus when the internal standard is spiked into a protein preparation from a biological sample in known amounts and analyzed by mass spectrometry, the SRM/MRM signature peak area of the native peptide is compared to the SRM/MRM 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, and/or the patient prognosis, for example, directly in patient-derived tissue, such as formalin fixed tissue, and to aid in determining which therapeutic agent 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. Moreover, the expression level of a protein, or multiple proteins, can be determined and compared to a “normal” or reference level found in healthy tissue. Normal or reference levels of proteins found in healthy tissue may be derived from, for example, the relevant tissues of one or more individuals that do not have cancer. Alternatively, normal or reference levels may be obtained for individuals with cancer by analysis of relevant tissues not affected by the cancer. Assays of protein levels (e.g., MGMT levels) can also be used to diagnose, and provide prognostic information about, the stage of cancer in a patient or subject diagnosed with cancer by employing the MGMT levels. The level of an individual MGMT peptide is defined as the molar amount of the peptide determined by the SRM/MRM assay per total amount of protein lysate analyzed. Information regarding MGMT can thus be used to aid in determining the stage or grade of a cancer by correlating the level of the MGMT protein (or fragment peptides of the MGMT protein) with levels observed in normal tissues. Once the quantitative amount of the MGMT protein has been determined in the cancer cells, that information can be matched to a list of therapeutic agents (chemical and biological) developed to specifically treat cancer tissue that is characterized by, for example, abnormal expression of the protein or protein(s) (e.g., MGMT) that were assayed. Matching information from an MGMT protein assay to a list of therapeutic agents that specifically targets, for example, the MGMT protein or cells/tissue expressing the protein, defines what has been termed a personalized medicine approach to treating disease. The assay methods 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.
In principle, any predicted peptide derived from the MGMT 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 MGMT protein in a sample using a mass spectrometry-based SRM/MRM assay. Similarly, any predicted peptide sequence containing an amino acid residue at a site that is known to be potentially modified in the MGMT protein also might potentially be used to assay the extent of modification of the MGMT protein in a sample.
MGMT fragment peptides may be generated by a variety of methods including by the use of the Liquid Tissue protocol provided in U.S. Pat. No. 7,473,532. 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. In the Liquid Tissue protocol 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). Following heat treatment 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 said biological sample and to liquefy the sample (e.g. a period of time from 30 minutes to 24 hours at a temperature from 37° to 65° C.). The result of the heating and proteolysis is a liquid, soluble, dilutable biomolecule lysate.
Surprisingly, it was found that many potential peptide sequences from the MGMT protein are unsuitable or ineffective for use in mass spectrometry-based SRM/MRM assays for reasons that are not immediately evident. This was especially true for peptides derived from formalin fixed tissue. As it was not possible to predict the most suitable peptides for MRM/SRM assay, it was necessary to experimentally identify modified and unmodified peptides in actual Liquid Tissue lysates to develop a reliable and accurate SRM/MRM assay for the MGMT protein. While not wishing to be bound by any theory, it is believed that some peptides might, for example, be difficult to detect by mass spectrometry because they do not ionize well or produce fragments distinct from other proteins. Peptides may also fail to resolve well in separation (e.g., liquid chromatography), or may adhere to glass or plastic ware.
MGMT peptides found in various embodiments of this disclosure (e.g., Tables 1 and 2) were derived from the MGMT 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 MGMT 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 typically employed. Ion trap mass spectrometers however may be the best type of mass spectrometer for conducting global profiling of peptides. Although an SRM/MRM assay can be developed and performed on any type of mass spectrometer, including a MALDI, ion trap, or triple quadrupole, advantageously the instrument platform for an SRM/MRM assay is a triple quadrupole instrument platform.
Once as many peptides as possible were identified in a single MS analysis of a single lysate under the conditions employed, then that list of peptides was collated and used to determine the proteins that were detected in that lysate. That process was repeated for multiple Liquid Tissue lysates, and the very large list of peptides was collated into a single dataset. 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 MGMT protein.
In one embodiment, the MGMT tryptic peptides identified as useful in the determination of absolute or relative amounts of the MGMT protein include one or more of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, each of which are listed in Table 1. Each of these peptides was detected by mass spectrometry in Liquid Tissue lysates prepared from formalin fixed, paraffin embedded tissue. Thus, each peptide is a candidate for use in developing a quantitative SRM/MRM assay for the MGMT protein in human biological samples, including directly in formalin fixed patient tissue.
The MGMT 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/MRM assay of the MGMT 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. Thus, each of these peptides is believed to be suitable for conducting SRM/MRM assays of the MGMT protein on a Liquid Tissue lysate from any formalin fixed tissue originating from any biological sample or from any organ site in the body.
An important consideration when conducting an SRM/MRM assay is the type of instrument that may be employed in the analysis of the peptides. Although SRM/MRM assays 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/MRM assay is presently most likely considered to be a triple quadrupole instrument platform. That type of a mass spectrometer is presently the most suitable instrument for putative analysis of 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.
In order to most efficiently implement an SRM/MRM assay for each peptide derived from the MGMT protein it is desirable to utilize information in addition to the peptide sequence in the analysis. That additional information may be used in directing and instructing the mass spectrometer (e.g. a triple quadrupole mass spectrometer) to perform the correct and focused analysis of specific targeted peptide(s), such that the assay may be effectively performed.
The additional information about target peptides in general, and about specific MGMT peptides, may include one or more of the mono isotopic mass of the peptide, its precursor charge state, the precursor m/z value, the m/z transition ions, and the ion type of each transition ion. Additional peptide information that may be used to develop an SRM/MRM assay for the MGMT protein is shown for these MGMT peptides in Table 1.
The method described below was used to: 1) identify candidate peptides from the MGMT protein that can be used for a mass spectrometry-based SRM/MRM assay for the MGMT protein, 2) develop an individual SRM/MRM assay, or assays, for target peptides from the MGMT protein in order to correlate and 3) apply quantitative assays to cancer diagnosis and/or choice of optimal therapy.
1. Identification of SRM/MRM candidate fragment peptides for the MGMT protein
2. Mass Spectrometry Assay for Fragment Peptides from the MGMT Protein
MGMT protein and demonstrate correlation with clinical outcomes from different treatment strategies, wherein this correlation has already been demonstrated in the field or can be demonstrated in the future through correlation studies across cohorts of patients and tissue from those patients. Once either previously established correlations or correlations derived in the future are confirmed by this assay then the assay method can be used to determine optimal treatment strategy
Specific and unique characteristics about specific MGMT peptides were developed by analysis of all MGMT peptides on both an ion trap and triple quadrupole mass spectrometers. That information includes the monoisotopic mass of the peptide, its precursor charge state, the precursor m/z value, the transition m/z values of the precursor, and the ion types of each of the identified transitions. That information must be determined experimentally for each and every candidate SRM/MRM peptide directly in Liquid Tissue lysates from formalin fixed samples/tissue; because, interestingly, not all peptides from the MGMT protein can be detected in such lysates using SRM/MRM as described herein, indicating that MGMT peptides not detected cannot be considered candidate peptides for developing an SRM/MRM assay for use in quantitating peptides/proteins directly in Liquid Tissue lysates from formalin fixed samples/tissue.
A particular SRM/MRM assay for a specific MGMT peptide is performed on a triple quadrupole mass spectrometer. An experimental sample analyzed by a particular MGMT SRM/MRM assay is for example a Liquid Tissue protein lysate prepared from a tissue that had been formalin fixed and paraffin embedded. Data from such as assay indicates the presence of the unique SRM/MRM signature peak for this MGMT peptide in the formalin fixed sample.
Specific transition ion characteristics for this peptide are used to quantitatively measure a particular MGMT peptide in formalin fixed biological samples. These data indicate absolute amounts of this MGMT peptide as a function of molar amount of the peptide per microgram of protein lysate analyzed. Assessment of MGMT protein levels in tissues based on analysis of formalin fixed patient-derived tissue can provide diagnostic, prognostic, and therapeutically-relevant information about each particular patient. In one embodiment, this disclosure describes a method for measuring the level of the 6-O-methylguanine-DNA methyltransferase protein (MGMT) in a biological sample, comprising detecting and/or quantifying the amount of one or more modified or unmodified MGMT fragment peptides in a protein digest prepared from the biological sample using mass spectrometry; and calculating the level of modified or unmodified MGMT protein in the sample; and wherein the level is a relative level or an absolute level. In a related embodiment, quantifying one or more MGMT fragment peptides comprises determining the amount of the each of the MGMT fragment peptides in a biological sample by comparison to an added internal standard peptide of known amount, wherein each of the MGMT fragment peptides in the biological sample is compared to an internal standard peptide having the same amino acid sequence. In some embodiments the internal standard is an isotopically labeled internal standard peptide comprises one or more heavy stable isotopes selected from 18O, 17O, 34S, 15N, 13C, 2H or combinations thereof.
The method for measuring the level of the MGMT protein in a biological sample described herein (or fragment peptides as surrogates thereof) may be used as a diagnostic and/or prognostic indicator of cancer in a patient or subject. In one embodiment, the results from measurements of the level of the MGMT protein may be employed to determine the diagnostic stage/grade/status of a cancer by correlating (e.g., comparing) the level of MGMT protein found in a tissue with the level of that protein found in normal and/or cancerous or precancerous tissues.
Because both nucleic acids and protein can be analyzed from the same Liquid Tissue™ biomolecular preparation it is possible to generate additional information about disease diagnosis and drug treatment decisions from the nucleic acids in same sample upon which proteins were analyzed. For example, if the MGMT protein is 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, potential drug resistance and the development of cancers can be obtained. At the same time, information about the status of the MGMT genes and/or the nucleic acids and proteins they encode (e.g., mRNA molecules and their expression levels or splice variations) can be obtained from nucleic acids present in the same Liquid Tissue™ biomolecular preparation can be assessed simultaneously to the SRM analysis of the MGMT protein. Any gene and/or nucleic acid not from the MGMT and which is present in the same biomolecular preparation can be assessed simultaneously to the SRM analysis of the MGMT protein. In one embodiment, information about the MGMT protein and/or one, two, three, four or more additional proteins may be assessed by examining the nucleic acids encoding those proteins. Those 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.
This application claims priority to provisional application Ser. No. 62/161,651, filed May 14, 2015, the contents of which are hereby incorporated by reference in their entirety. This application also contains a sequence listing submitted electronically via EFS-web, which serves as both the paper copy and the computer readable form (CRF) and consists of a file entitled “001152_8044 _US01 _SEQ_LISTING”, which was created on May 13, 2016, which is 849 bytes in size, and which is also incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62161651 | May 2015 | US |
