Patent Application
20100105087
Breast cancer is the most frequent neoplasm and the leading cause of cancer mortality in women worldwide. According to estimates, approximately 41,000 women in the United States and 130,000 women in the European Union die for breast cancer yearly. Mammographic screening has been widespread for the past twenty years and shown to reduce breast cancer mortality by 20-35% in women aged 40 to 69 years.
Mammography has a variety of short-comings, however. For example, according to the current guidelines, some women who develop breast cancer are “too young” to start regular mammograms. Also, less than half of eligible women get mammograms regularly, and the predictive value of mammography declines in cohorts of patients with denser breast tissue and smaller lesions. Furthermore, mammography is not effective in detecting early stages of breast cancer.
Needless to say, early detection is of paramount importance in reducing mortality from this major public health burden. Detection of breast cancer at the earliest stages results in a much greater favorable outcome, with 10-year disease-free survival rate as high as 98% in patients with pT1a,bN0M0 tumors (measuring 1 cm or less, with disease-free axillary lymph nodes and no distant metastasis). Thus, the potential for enhancing treatment by providing an early diagnosis has driven a search for better diagnostic tools.
Some biomarker genes and proteins, such as BRCA1, BRCA2 and Her-2/neu, have been identified and developed into tools for genetic screening. The advantages and limitations of these detection approaches have been discussed in the literature. See, e.g. Ponzone et al., Eur. J. Cancer 34(7): 966-967, 1998; Bradbury, Lancet Oncol. 3: 2, 2002; Ross et al., Expert Rev. Mol. Diagn. 3(5): 573-585, 2003.
A need exists, however, for additional biomarkers useful for detecting breast cancer, and in particular biomarkers that can detect early stages of the disease.
In one embodiment, a method for detecting breast cancer in a patient comprises obtaining a biological sample from the patient and evaluating the sample or a fraction of the sample for the presence of at least one biomarker selected from the group of peptides having the sequence of SEQ ID NOs: 1-217, wherein the presence of said at least one biomarker is indicative of breast cancer. In one aspect, the methods involve evaluating the sample for the presence of a biomarker selected from the group of peptides having the amino acid sequence of SEQ ID NOs: 132-217. In another, the methods comprise evaluating the sample for the presence of peptides having the amino acid sequence of SEQ ID NOs: 132, 139, 141 and 148. In one aspect, the breast cancer is in early stage, such as stage T1a. The biological sample can be, for example, blood, serum or plasma. In one embodiment, the evaluation step comprises assays such as mass spectrometry, an immunoassay such as ELISA, immunomass spectrometry or suspension bead array.
In another embodiment, the method further comprises, prior to the evaluation step, harvesting low molecular weight peptides from the biological sample to generate at least one fraction comprising the peptides. In one embodiment, the size of the low molecular weight peptides is less than 50 KDa, preferably less than 25 KDa, and more preferably less than 15 KDa. In another aspect, the method also comprises digesting the low molecular weight peptides. Such digestion can be accomplished using enzymatic or chemical means. In one example, trypsin can be used to digest the peptides.
In another aspect, a method for monitoring the progression of breast cancer in a patient comprises (i) obtaining a biological sample from the patient, (ii) evaluating the sample or a fraction of the sample for the presence of at least one biomarker selected from the group of peptides having the sequences of SEQ ID NOs: 1-217, wherein the presence of said at least one biomarker is indicative of breast cancer, and optionally, repeating steps (i) and (ii) as necessary. In one aspect, the methods involve evaluating the sample for the presence of a biomarker selected from the group of peptides having the amino acid sequence of SEQ ID NOs: 132-217. In another, the methods comprise evaluating the sample for the presence of peptides having the amino acid sequence of SEQ ID NOs: 132, 139, 141 and 148. In one embodiment, the method further comprises a step of harvesting low molecular weight peptides from the sample to generate at least one fraction comprising the peptides.
In other aspects, the invention relates to antibodies specific for identified biomarkers for breast cancer, as well as kits for detecting breast cancer in a patient, comprising at least one such antibody.
Other objects, features and advantages will become apparent from the following detailed description. The detailed description and specific examples are given for illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Further, the examples demonstrate the principle of the invention and cannot be expected to specifically illustrate the application of this invention to all the examples where it will be obviously useful to those skilled in the prior art.
Low molecular weight (LMW) peptides have been discovered that are indicative of breast cancer. Evaluating patient samples for the presence of such LMW peptides is an effective means of detecting breast cancer and monitoring the progression of the disease, for example during treatment. The LMW peptides are particularly useful in detecting breast cancer during its earliest stages, such as stage I.
The LMW peptides, or biomarkers, can be detected using a variety of methods known in the art. For example, antibodies can be utilized in immunoassays to detect the presence of a biomarker. Exemplary immunoassays include, e.g., ELISA, radioimmunoassay, immunofluorescent assay, “sandwich” immunoassay, western blot, immunoprecipitation assay and immunoelectrophoresis assays. Furthermore, methods involving beads, microbeads, arrays, microarrays, etc. can be applied in detecting the LMW peptides. Exemplary assays include, but are not limited to, suspension bead assays (Schwenk et al., “Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics,” Mol. Cell Proteomics, 6(1): 125-132 (2007)), antibody microarrays (Borrebaeck et al., “High-throughput proteomics using antibody microarrays: an update,” Expert Rev. Mol. Diagn. 7(5): 673-686 (2007)), aptamer arrays (Walter et al., “High-throughput protein arrays: prospects for molecular diagnostics,” Trends Mol. Med. 8(6): 250-253 (2002)), affybody arrays (Renberg et al., “Affibody molecules in protein capture microarrays: evaluation of multidomain ligands and different detection formats,” J. Proteome Res. 6(1): 171-179 (2007)), and reverse phase arrays (VanMeter et al., “Reverse-phase protein microarrays: application to biomarker discorvery and translational medicine,” Expert Rev. Mol. Diagn. 7(5): 625-633 (2007)). All these publications are incorporated herein by reference.
In another example, the inventive biomarkers can be detected using mass spectrometry (MS). One example of this approach is tandem mass spectrometry (MS/MS), which involves multiple steps of mass selection or analysis, usually separated by some form of fragmentation. Most such assays use electrospray ionization followed by two stages of mass selection: a first stage (MS 1) selecting the mass of the intact analyte (parent ion) and, after fragmentation of the parent by collision with gas atoms, a second stage (MS2) selecting a specific fragment of the parent, collectively generating a selected reaction monitoring assay. In one embodiment, collision-induced dissociation is used to generate a set of fragments from a specific peptide ion. The fragmentation process primarily gives rise to cleavage products that break along peptide bonds. Because of the simplicity in fragmentation, the observed fragment masses can be compared to a database of predicted masses for known peptide sequences. A number of different algorithmic approaches have been described to identify peptides and proteins from tandem mass spectrometry (MS/MS) data, including peptide fragment fingerprinting (SEQUEST, MASCOT, OMSSA and X!Tandem), peptide de novo sequencing (PEAKS, LuteFisk and Sherenga) and sequence tag based searching (SPIDER, GutenTAG).
10018] Likewise, multiple reaction monitoring (MRM) can be used to identify the inventive biomarkers in patient samples. This technique applies the MS/MS approach to, for example, tryptic digests of the input sample, followed by selected ion partitioning and sampling using MS to objectify and discreetize the analyte if interest by following the exact m/z ion of the tryptic fragment that represents the analyte. Such an approach can be performed in multiplex so that multiple ions can be measured at once, providing an antibody-free method for analyte measurement. See, e.g. Andersen et al., “Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins,” Molecular & Cellular Proteomics, 5.4: 573-588 (2006); Whiteaker et al., “Integrated pipeline for mass spectrometry-based discorvery and confirmation of biomarkers demonstrated in a mouse model of breast cancer,” J. Proteome Res. 6(10): 3962-75 (2007). Both publications are incorporated herein by reference in their entirety.
In another example, the inventive biomarkers can be detected using nanoflow reverse-phase liquid chromatography-tandem mass spectrometry. See, e.g., Domon B, Aebersold R. “Mass spectrometry and protein analysis.” Science, 312(5771):212-7(2006), which is incorporated herein by reference in its entirety. Using this approach, experimentalists obtain peptide fragments, usually by trypsin digest, and generate mass spectrograms of the fragments, which are then compared to a database, such as SEQUEST, for protein identification.
In another aspect, the inventive biomarkers can be detected using immuno-mass spectrometry. See, e.g., Liotta L et al. “Serum peptidome for cancer detection: spinning biologic trash into diagnostic gold.” J Clin Invest.,116(1):26-30 (2006); Nedelkov, “Mass spectrometry-based immunoassays for the next phase of clinical applications,” Expert Rev. Proteomics, 3(6): 631-640 (2006), which are incorporated herein by reference. Immuno-mass spectrometry provides a means for rapidly determining the exact size and identity of a peptide biomarker isoform present within a patient sample. When developed as a high throughput diagnostic assay, a drop of patient's blood, serum or plasma can be applied to a high density matrix of microcolumns or microwells filled with a composite substratum containing immobilized polyclonal antibodies, directed against the peptide marker. All isoforms of the peptide that contain the epitope are captured. The captured population of analytes including the analyte fragments are eluted and analyzed directly by a mass spectrometer such as MALDI-TOF MS. The presence of the specific peptide biomarker at its exact mass/charge (m/z) location would be used as a diagnostic test result. The analysis can be performed rapidly by simple software that determines if a series of ion peaks are present at defined m/z locations.
In yet another example, the inventive biomarkers can be detected using standard immunoassay-based approaches whereby fragment specific antibodies are used to measure and record the presence of the diagnostic fragments. See, e.g., Naya et al. “Evaluation of precursor prostate-specific antigen isoform ratios in the detection of prostate cancer.” Urol Oncol. 23(1):16-21 (2005). Moreover, additional immunoassays are well known to those skilled in the field, such as ELISAs (Maeda et al., “Blood tests for asbestos-related mesothelioma,” Oncology 71: 26-31 (2006)), microfluidic ELISA (Lee et al., “Microfluidic enzyme-linked immunosorbent assay technology,” Adv. Clin. Chem. 42: 255-259 (2006)), nanocantilever immunoassays (Kurosawa et al., “Quartz crystal microbalance immunosensors for environmental monitoring,” Biosens Bioelectron, 22(4): 473-481 (2006)), and plasmon resonance immunoassays (Nedelkov, “Development of surface Plasmon resonance mass spectrometry array platform,” Anal. Chem. 79(15): 5987-5990 (2007)). All of these publications are incorporated herein by reference.
In a further example, the biomarkers can be detected using electrochemical approaches. See, e.g., Lin et al., “Electrochemical immunosensor for carcinoembryonic antigen based on antigen immobilization in gold nanoparticles modified chitosan membrane,” Anal. Sci. 23(9): 1059-1063 (2007).
In one embodiment, the LMW peptides are harvested from a biological sample prior to the evaluation step. For example, 100 μl of serum can be mixed with 2× SDS-PAGE Laemmli Buffer (containing 200 mM DTT), boiled for 10 minutes, and loaded on Prep Cell (Model 491 Prep Cell, Bio-Rad Laboratories, CA) comprising a 5 cm length 10% acrylamide gel. Electrophoresis is performed under a constant voltage of 250V. Immediately after the bromophenol blue indicator dye is eluted from the system, LMW peptides and proteins migrate out of the gel and are trapped in a dialysis membrane in the elution chamber. These molecules can be eluted at a flow rate of 400 ml/l min by a buffer with the same composition of the Tris-Glycine running buffer and collected for 10 minutes in one fraction.
Alternatively, LMW peptides can be harvested using from a sample using a capture-particle that comprises a molecular sieve portion and an analyte binding portion as described in U.S. patent application Ser. No. 11/527,727, filed Sep. 27, 2006, which is incorporated herein by reference in its entirety. Briefly, either the molecular sieve portion or the analyte binding portion or both comprise a cross-linked region having modified porosity, or pore dimensions sufficient to exclude high molecular weight molecules.
In another embodiment, the LMW peptides are digested prior to detection, so as to reduce the size of the peptides. Such digestion can be carried out using standard methods well known in the field. Exemplary treatments, include but are not limited to, enzymatic and chemical treatments. Such treatments can yield partial as well as complete digestions. One example of an enzymatic treatment is a trypsin digestion.
The inventive biomarkers are particularly useful in detecting breast cancer during its early stages, i.e., prior to metastasis and large tumor volume (e.g. greater than 2 cm).
Antibodies specific for the inventive biomarkers can be produced readily using well known methods in the art. (See, J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning, a Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, pp. 18.7-18.18, 1989) For example, the inventive biomarkers can be prepared readily using an automated peptide synthesizer. Next, injection of an immunogen, such as (peptide)n-KLH (n=1-30) in complete Freund's adjuvant, followed by two subsequent injections of the same immunogen suspended in incomplete Freund's adjuvant into immunocompetent animals, is followed three days after an i.v. boost of antigen, by spleen cell harvesting. Harvested spleen cells are then fused with Sp2/0-Ag14 myeloma cells and culture supernatants of the resulting clones analyzed for anti-peptide reactivity using a direct-binding ELISA. Fine specificity of generated antibodies can be detected by using peptide fragments of the original immunogen.
In certain embodiments, one or more antibodies directed to the inventive biomarkers is provided in a kit, for use in a diagnostic method. Such kits also can comprise reagents, instructions and other products for performing the diagnostic method.
The detailed description of the present invention is provided below by the following example, which is illustrative only and not limiting the invention in any way.
Blood Collection and Serum Preparation
Blood samples were drawn from patients before the mammography screening under full Institutional Review Board approval and patient's consent. Specimens were collected in red-top Vacutainer Tubes and allowed to clot for 1 hour on ice, followed by centrifugation at 4° C. for 10 minutes at 2000 g. The serum supernatant was divided in aliquots and stored at −80° C. until needed. 10 serum samples with negative outcome were pooled in a single control group. 10 serum samples from patients with a diagnosed T1a stage breast cancer were pooled in a single disease group. Each experiment has been performed using 3 different aliquots from the same pool, both, for the control and for the disease group.
Low Molecular Weight (LMW) Protein Harvesting by Continuous Elution Electrophoresis
100 μl of serum was mixed with 2× SDS-PAGE Laemmli Buffer (containing 200 mM DTT), boiled for 10 minutes, and loaded on Prep Cell (Model 491 Prep Cell, Bio-Rad Laboratories, CA) in which 5 cm length 10% acrylamide gel was polymerized. Electrophoresis was performed under a constant voltage of 250V. Immediately after the bromophenol blue indicator dye was eluted from the system, LMW peptides and proteins migrate out of the gel and are trapped in a dialysis membrane in the elution chamber. These molecules were eluted at a flow rate of 400 μl min by a buffer with the same composition of the Tris-Glycine running buffer and collected for 10 minutes in one fraction.
SDS Removal from the Prep Cell Fractions
LMW fractions obtained by the Prep Cell were processed using a commercially available ion-exchange matrix (Proteo Spin Detergent Clean-Up Micro Kit, Norgen Biotek Corporation, Canada) following protocols outlined by the manufacturer for both acidic and basic proteins, resulting in a final volume of 55 μl.
Nanoflow Reversed-Phase Liquid Chromatography-Tandem MS (nanoRPLC-MS/MS)
The SDS-free LMW fractions obtained from the described procedure were analyzed by traditional bottom-up MS approaches. This was accomplished by treating the samples by reduction using 20 mM DTT, followed by alkylation using 100 mM iodoacetamide and lastly, trypsin digestion (Promega, WI) at 37° C. overnight in 50 mM ammonium bicarbonate in the presence of 1M urea in a final volume of 200 pl. Tryptic peptides were desalted by μC18 Zip Tip (Millipore, MA) and analyzed by reversed-phase liquid chromatography nanospray tandem mass spectrometry using a linear ion-trap mass spectrometer (LTQ, ThermoElectron, San Jose, Calif.). Reverse phase column was slurry-packed in-house with 5 μm, 200 Å pore size C18 resin (Michrom BioResources, CA) in 100 μm i.d.×10 cm long fused silica capillary (Polymicro Technologies, Phoenix, Ariz.) with a laser-pulled tip. After sample injection, the column was washed for 5 min with mobile phase A (0.4% acetic acid, 0.005% heptafluorobutyric acid) and peptides were eluted using a linear gradient of 0% mobile phase B (0.4% acetic acid, 0.005% heptafluorobutyric acid, 80% acetonitrile) to 50% mobile phase B in 30 min at 250 nl/min, then to 100% B in an additional 5 min. The LTQ mass spectrometer was operated in a data-dependent mode in which each full MS scan was followed by five MS/MS scans where the five most abundant molecular ions were dynamically selected and fragmented by collision-induced dissociation (CID) using a normalized collision energy of 35%.
Bioinformatic Analysis
Tandem mass spectra were matched against Swiss-Prot human protein database through SEQUEST algorithm incorporated in Bioworks software (version 3.2, Thermo Electron) using tryptic cleavage constraints and static cysteine alkylation by iodoacetamide. For a peptide to be considered legitimately identified, it had to achieve Delta Cn value above 0.1, cross correlation scores of 1.5 for [M+H]1+, 2.0 for [M+2H]2+, 2.5 for [M+3H]3+, and a probability cut-off for randomized identification of p<0.01.
The results are provided in Table 1. In short, 131 peptides were identified as biomarkers that correlate to the disease state. Thus, evaluating patient samples for the presence of one or more of these biomarkers will provide a useful method for detecting breast cancer.
In addition, the tandem mass spectra were analyzed using more stringent filtering criteria, with a goal of reducing false positives. In particular, the spectra were analyzed using the filtering algorithms of the Scalfold Software (Proteome Software Inc., Portland, Oreg.).
The results are provided in Table 2. In short, 86 peptides were identified as biomarkers that correlate to the disease state. Thus, evaluating patient samples for the presence of one or more of these biomarkers will provide a useful method for detecting breast cancer.
The above methods showed that a number of peptides previously known to be associated with breast cancer were not indicative of a disease state, and, thus, not useful as a biomarker. Examples include, RUN and FYVE domain-containing 1 variant (fragment), haptoglobin precursor that contains: haptoglobin alpha chain and haptoglobin beta chain, tetranectin precursor (TN) (Plasminogen-kringle 4 binding protein), vitamin D-binding protein, apolipoprotein C-IV precursor (Apo-CIV or ApoC-IV), VH1 protein precursor (fragment), Ig kappa chain V-III region SIE, hypothetical protein Q569I7, haptoglobin-related protein precursor, breast carcinoma amplified sequence 1 (novel amplified in breast cancer 1) (amplified and overexpressed in breast cancer), sodium-D-glucose cotransporter (regulatory solute carrier protein, family 1, member 1), complement component C8 gamma chain precursor, Ig heavy chain V-III region TIL, hypothetical protein DKFZp686I04196 (fragment), alpha-2-macroglobulin precursor (Alpha-2-M), hypothetical protein DKFZp761P18121, fibrinogen alpha chain precursor that contains fibrinopeptide A, IGHG1 protein, hypothetical protein MGC27016, seprase (EC 3.4.21.-) (fibroblast activation protein alpha) (integral membrane serine protease) (170-kDa melanoma membrane-bound gelatinase), PTPL1-associated RhoGAP, zinc finger protein 385 (hematopoietic zinc finger protein) (retinal zinc finger protein), hypothetical protein FLJ45950, hypothetical protein FLJ39462, smoothelin, latrophilin-3 precursor (calcium-independent alpha-latrotoxin receptor 3) (lectomedin-3), type III iodothyronine deiodinase (EC 1.97.1.11) (type-III 5′deiodinase) (DIOIII) (type 3 DI) (5DIII).
This application claims priority to U.S. Provisional Application No. 60/855,378, filed Oct. 31, 2006, which is hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/22967 | 10/31/2007 | WO | 00 | 12/17/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60855378 | Oct 2006 | US |
