This patent application is assigned to
The following quote was obtained by the news editors from the background information supplied by the inventors: "Primary and metastatic solid tumors comprise cancer cells, host cells such as endothelial cells and fibroblasts, and inflammatory immune cells. Yet, although individual cancer cells may exhibit a stable signature of protein marker expression (S. Ramaswamy et al., Nat. Genet. 33, 49 (2003);
"Within a clinical setting, cancer cells from primary tumors are typically obtained through image-guided intervention or open surgical procedures (W. Bruening et al., Ann. Intern. Med. 152, 238 (2010)). Percutaneous biopsy is a well-established clinical procedure that yields cells for cytology using 22-gauge (22G) fine-needle aspirates, and small tissue fragments for histology by core biopsies using 16G to 19G, needles. Samples are commonly processed using conventional histological stains before immunohistochemical evaluation if sufficient tissue remains (tissue sections contain billions of cells). However, immunohistochemical evaluation is only semiquantitative, time-consuming, and technically challenging."
In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "Described herein are methods for multiplexed analysis of protein expression in cancer cells. In some embodiments, the methods are practiced using a point-of-care molecular diagnostic system for rapid, quantitative, and multiplexed analysis of protein expression in cancer cells, e.g., cells obtained by fine-needle aspirates of patients' tumors, for real-time analysis within a clinical setting.
"Thus, in one aspect, the invention provides methods for diagnosing a tumor in a subject. The methods include obtaining a sample from the subject; detecting levels of MUC-1, HER2, EGFR, and EpCAM in the sample; and comparing the levels of MUC-1, HER2, EGFR, and EpCAM in the sample to reference levels; and diagnosing cancer in a subject who has levels of MUC-1, HER2, EGFR, and EpCAM above the reference levels.
"In a further aspect, the invention features methods for detecting the presence of tumor cells in a sample. The methods include detecting levels of MUC-1, HER2, EGFR, and EpCAM in the sample; and comparing the levels of MUC-1, HER2, EGFR, and EpCAM in the sample to reference levels; wherein the presence in a sample of levels of MUC-1, HER2, EGFR, and EpCAM above the reference levels indicates the presence of tumor cells in the sample.
"In some embodiments, detecting levels of MUC-1, HER2, EGFR, and EpCAM in the sample comprises contacting the sample with antibodies or antigen-binding fragments thereof that bind to MUC-1, HER2, EGFR, and EpCAM. In some embodiments, the antibodies are labeled, e.g., with magnetic nanoparticles, e.g., MIONs, e.g., CLIOs.
"In some embodiments, a single undivided sample is contacted with a mixture of antibodies, or antigen-binding fragments thereof, that bind to MUC-1, HER2, EGFR, and EpCAM, substantially simultaneously.
"In some embodiments, the sample is subdivided into at least four subparts, and each antibody, or antigen-binding fragment thereof, that binds to MUC-1, HER2, EGFR, or EpCAM is contacted with a single subpart.
"In some embodiments, the levels of each of the biomarkers MUC-1, HER2, EGFR, and EpCAM are weighted. In some embodiments, a quad biomarker value for a sample is determined using the following weighted equation:
"Quad Biomarker Value=4.90*Muc1+4.55*EGFR+1.54Her2+4.79EpCAM
"In some embodiments, the levels of MUC-1, HER2, EGFR, and EpCAM are detected using diagnostic magnetic resonance (DMR), e.g., using a portable relaxometer or MR imager; direct magnetic detection (e.g., using mass spectrometry or nanoparticle-based bio barcoding methods); optical detection methods (e.g., flow cytometry, fluorescence detection, e.g., with quantum dots); or electric measurements (e.g., using nanowires, or giant magnetosensor chips).
"In some embodiments, the sample comprises blood or a subfraction thereof, e.g., buffy coat. In some embodiments, the sample comprises a biopsy sample, e.g., a fine needle aspirate (FNA), endoscopic biopsy, or core needle biopsy. In some embodiments, the sample comprises cells from the pancreas, lung, breast, prostate, kidney, stomach, esophagus, bladder, endometrial, cervix, biliary, thyroid ovary or colon of the subject.
"In some embodiments, the tumor is a pancreas, lung, breast, prostate, kidney, stomach, esophagus, bladder, endometrial, cervix, biliary, thyroid ovary or colon tumor.
"In another aspect, the invention provides kits including reagents for detection of tumor cells, wherein the reagents comprise a panel of antigen-binding reagents consisting of: antibodies or antigen-binding fragments thereof that bind to MUC1, antibodies or antigen-binding fragments thereof that bind to EGFR, antibodies or antigen-binding fragments thereof that bind to HER2, and antibodies or antigen-binding fragments thereof that bind to EpCAM.
"In some embodiments, the antibodies or antigen-binding fragments thereof are linked to superparamagnetic nanoparticles, e.g., MIONs, e.g., CLIOs. In some embodiments, the antibodies or antigen-binding fragments thereof are linked to superparamagnetic nanoparticles via trans-cyclooctene (TCO)/tetrazine (Tz) chemistry.
"In an additional aspect, the invention provides methods for isolating tumor cells from a sample. The methods include providing a sample comprising or suspected of comprising tumor cells; contacting the sample with:
"antibodies or antigen-binding fragments thereof that bind to MUC 1,
"antibodies or antigen-binding fragments thereof that bind to EGFR,
"antibodies or antigen-binding fragments thereof that bind to HER2, and
"antibodies or antigen-binding fragments thereof that bind to EpCAM;
"under conditions sufficient for the antibodies or antigen-binding fragments thereof to bind to tumor cells in the sample; and removing the antibodies or antigen-binding fragments thereof that are bound to tumor cells from the sample, thereby isolating tumor cells from the sample.
"In some embodiments, the antibodies or antigen-binding fragments thereof are linked to superparamagnetic nanoparticles, and the antibodies or antigen-binding fragments thereof that are bound to tumor cells are removed from the sample by application of a magnetic field to the sample.
"In some embodiments, the sample comprises blood from a subject. In some embodiments, the method further comprises returning the blood to the subject after removal of the tumor cells.
"The terms 'quad biomarkers' or 'the biomarkers' as used herein refers to MUC1, EpCAM, HER2, and EGFR.
"An 'epithelial cancer,' as used herein is defined by the ICD-O (International Classification of Diseases-Oncology) code (revision 3), section (8010-8790), and can include tumors of the pancreas, lung, breast, prostate, kidney, ovary or colon.
"Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
"Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
DESCRIPTION OF DRAWINGS
"FIG. 1 is a set of three line graphs demonstrating validation of NMR measurements. The plots show the correlation of EGFR measurements obtained by .mu.NMR versus ELISA, FACS or immunohistochemistry (IHC) in clinical samples where sufficient cells were available for conventional proteomic techniques (typically 10.sup.5-6 for ELISA and FACS versus 10.sup.2 for DMR). Note the excellent correlation coefficients for the different methods.
"FIG. 2a is a set of calibration curves correlating .mu.NMR signals with molecular expression levels for cell lines.
"FIG. 2b is a set of correlative .mu.NMR and FACS measurements for 5 clinical patient samples.
"FIG. 3 is a set of 9 graphs illustrating biomarker expression level distribution. Waterfall plots showing the expression levels of each of the different biomarkers sorted from high (left) to low (right). Each column represents a different patient sample (lighter grey=malignant; darker grey=benign).
"FIG. 4 is a set of graphs showing expression levels of different biomarkers arranged by patient number. Patients 5, 12, 17, 18, 21, 42 had benign lesions.
"FIG. 5 is a graphical representation of the Spearman correlation coefficients (0: no correlation, 1: perfect correlation) between markers.
"FIG. 6 is a dot plot showing the variability of marker levels stratified by diagnosis and by global leukocyte versus non-leukocyte comparisons. Left: Individual marker expression levels for both malignant and benign samples. Right: Overall leukocyte and non-leukocyte cell counts.
"FIG. 7A is a graph showing receiver operating characteristic (ROC) curves for single markers, a dual marker set, as well as for triple and quadruple marker combinations to determine optimum DMR threshold values.
"FIG. 7B is a set of three bar graphs showing levels of the four markers measured individually (HER2, MUC1, EGFR, and EpCAM) and simultaneously (Quad) in three different cell lines.
"FIG. 8 shows images from a representative clinical case illustrating the potential role of .mu.NMR for enhancing diagnostic accuracy and influencing management. Patient (3) underwent computed tomography (CT)-guided biopsy for an enlarging (2.5.times.6.8 cm) pre-sacral lesion in the setting of active metastatic rectal adenocarcinoma. Both cytology and core biopsy assessed the lesion as benign (inflammatory tissue). The lesion was thus treated with a drainage catheter. As shown in the graph, .mu.NMR analysis, using the quadruple-marker combination (MUC-1+HER2+EGFR+EpCAM), unequivocally classified the lesion as malignant (.mu.NMR value: 11.25; malignancy threshold .gtoreq.1.6). Repeat chest and abdomen CT after two months noted a significant interval enlargement of the biopsied lesion, as well as new metastases.
"FIGS. 9A-C show the results of analysis of sample heterogeneity. (9A) Repeat measurement of the same samples (note the different scale compared to other graphs). (9B) Measurement of repeat FNA samples obtained via the same coaxial needle (see Table for variance component estimates for intra-subject variability). (9C) Measurement of repeat FNAs from different tumor sites.
"FIGS. 9D and 9E show the effect of prospective preservation treatments on extracellular and intracellular protein measurements. Live: live cells; FA: 2% formaldehyde; meth: 100% methanol; TX: triton X-100 0.05% in
"FIG. 9F shows the effect of time at 4.degree. C. before fixation (e.g. during transport to central laboratory facility) on protein measurements. Note the rapid change in expression levels in unfixed samples. The typical half-life of markers is
"FIG. 9G shows the effect of various means of fixation. With 2% paraformaldehyde (PFA), cellular proteins could be preserved (>12 hrs) at the level comparable to that of live cells.
"FIGS. 10A-B are tables presenting various methods of detection."
URL and more information on this patent application, see: Weissleder, Ralph;
Keywords for this news article include: Antibodies, Biopsy, Surgery, Cytology, Oncology, Pancreas, Immunology, Leukocytes, Proteomics, Blood Cells, Legal Issues, Nanoparticle, Immune System, Blood Proteins, Nanotechnology, Immunoglobulins, Gastroenterology, Protein Expression, Cancer Gene Therapy, Emerging Technologies, Operative Surgical Procedures.
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