COMPOSITIONS AND METHODS FOR DETECTING PANCREATIC CANCER
20260085360 ยท 2026-03-26
Inventors
Cpc classification
C07K14/4748
CHEMISTRY; METALLURGY
International classification
Abstract
Provided herein are methods of detecting pancreatic cancer in a subject, the method comprising measuring in a sample from the subject a level of CA19-9 polysaccharide relative to a reference, and a level of a polynucleotide or polypeptide of at least one marker selected from the group consisting of: OPN, MIA, CEACAM-1, MIC-1, SPON1, HSP27, POSTN, and LGALS3BP relative to a reference, wherein an increased level of the CA19-9 polysaccharide relative to a reference and an increased level of the polynucleotide or polypeptide relative to a reference indicates presence of pancreatic cancer in the subject.
Claims
1-28. (canceled)
29. A method of diagnosing and treating a subject having intraductal papillary mucinous neoplasm (IPMN), the method comprising: a) detecting in a sample from a subject an increased level relative to a healthy subject of two or more markers selected from the group consisting of OPN, MIA, CEACAM-1, MIC-1, SPON1, HSP27, POSTN, and LGALS3BP, and b) determining the subject has intraductal papillary mucinous neoplasm (IPMN) based on the increased level of markers; c) treating the subject for intraductal papillary mucinous neoplasm (IPMN).
30. The method of claim 29 wherein the at least two markers are selected from the group consisting of MIC1, OPN, CEACAM1 and MIA.
31. The method of claim 29 wherein the at least two markers are selected from the group consisting of HSP27 and SPON1.
32. The method of claim 29 wherein the at least two markers selected from the group consisting of HSP27, SPON1, POSTN and LGALS3BP.
33. The method of claim 29 wherein three or more markers are detected in the sample and are selected from the group consisting of OPN, MIA, CEACAM-1, MIC-1, SPON1, HSP27, POSTN, and LGALS3BP.
34. The method of claim 29 wherein four or more markers are detected in the sample and are selected from the group consisting of OPN, MIA, CEACAM-1, MIC-1, SPON1, HSP27, POSTN, and LGALS3BP.
35. The method of claim 29 wherein five or more markers are detected in the sample and are selected from the group consisting of OPN, MIA, CEACAM-1, MIC-1, SPON1, HSP27, POSTN, and LGALS3BP.
36. The method of claim 29 wherein the subject has an increased level of CA19-9 polysaccharide relative to a healthy subject.
37. The method of claim 29 wherein the subject is determined to have intraductal papillary mucinous neoplasm (IPMN) and not having pancreatic ductal adenocarcinoma (PDAC).
38. The method of claim 29 wherein treating the subject comprises surgery.
39. A method of diagnosing and treating a subject having pancreatic ductal adenocarcinoma (PDAC), the method comprising: a) detecting in a sample from a subject an increased level relative to a healthy subject of two or more markers selected from the group consisting of OPN, MIA, CEACAM-1, MIC-1, SPON1, HSP27, POSTN, and LGALS3BP, and b) determining the subject has pancreatic ductal adenocarcinoma (PDAC) based on the increased level of markers; c) treating the subject for pancreatic ductal adenocarcinoma (PDAC).
40. The method of claim 39 wherein the at least two markers are selected from the group consisting of MIC1, OPN, CEACAM1 and MIA.
41. The method of claim 39 wherein the at least two markers are selected from the group consisting of HSP27 and SPON1.
42. The method of claim 39 wherein the at least two markers are selected from the group consisting of HSP27, SPON1, POSTN and LGALS3BP.
43. The method of claim 39 wherein three or more markers are detected in the sample and are selected from the group consisting of OPN, MIA, CEACAM-1, MIC-1, SPON1, HSP27, POSTN, and LGALS3BP.
44. The method of claim 39 wherein four or more markers are detected in the sample and are selected from the group consisting of OPN, MIA, CEACAM-1, MIC-1, SPON1, HSP27, POSTN, and LGALS3BP.
45. The method of claim 39 wherein five or more markers are detected in the sample and are selected from the group consisting of OPN, MIA, CEACAM-1, MIC-1, SPON1, HSP27, POSTN, and LGALS3BP.
46. The method of claim 39 wherein the subject has an increased level of CA19-9 polysaccharide relative to a healthy subject.
47. The method of claim 39 wherein treating the subject comprises surgery.
48. The method of claim 35 wherein the subject is determined to have intraductal papillary mucinous neoplasm (IPMN) and not having pancreatic ductal adenocarcinoma (PDAC).
49. The method of claim 48 wherein treating the subject comprises surgery.
50. A method of detecting pancreatic cancer in a subject, the method comprising: (a) measuring in a sample from the subject a level of a polynucleotide or polypeptide of at least one marker selected from the group consisting of: OPN, MIA, CEACAM-1, MIC-1, SPON1, HSP27, POSTN, and LGALS3BP relative to a reference, wherein an increased level of said polynucleotide or polypeptide relative to a reference indicates presence of pancreatic cancer in the subject.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0095] The invention features compositions and methods that are useful for early detection of pancreatic cancer. The invention is based, at least in part, on the discovery of biomarkers (e.g., MIC-1, CEACAM-1, MIA, SPON1) that complement CA19-9, whereby the panel comprising CA19-9 and complementary biomarkers of the invention improve accuracy of detection of pancreatic cancer.
[0096] Pancreatic cancer is the 4th leading cause of cancer death in the United States. The majority of patients present with unresectable disease leading a median survival of 6 months and an overall 5-year survival of <5%. The early detection of this disease is critical because surgery at an early stage is the most promising therapy that could greatly improve the prognosis of patients.
Development and Validation of a 6-Plex Immunoassay
[0097] Customized magnetic bead-based multiplex immunoassays were developed for the selected candidate serum biomarkers using a Bio-Plex 200 suspension array system. Magnetic bead-based monoplex immunoassays were first developed for OPN, MIA, CEACAM-1, MIC-1, SPON1 and HSP27 using pooled normal human sera. The cross-reactivity studies through single-detection and multiplexed-detection antibody experiments indicated that the degree of cross-reactivity across the 6 immunoassays was generally <1%, based on the measurements in response to high concentrations of the recombinant proteins at first dilution point (except SPON1 at the third dilution because only 1.4% of sera with SPON1 exceed the third dilution) of the standard curve (Table 2). About 1.3-3.3% of nonspecific cross-reactions were observed in SPON1 antibody against other proteins. But, it should be noted that majority of these nonspecific cross-reactions were observed at recombinant protein concentrations that exceed physiological levels, thereby reducing the chance of cross-reactivity in physiological human serum samples.
[0098] By mixing the capture antibody-coupled beads and detection antibodies used in the monoplex immunoassays, a 6-plex immunoassay of OPN, MIA, CEACAM-1, MIC-1, SPON1 and HSP27 was developed and evaluated. The calibration curves of the 6-plex immunoassay generated using the 5PL logistic regression models are shown in
[0099] The analytical performance of the 6-plex immunoassay is shown in Table 3, with recovery of 89-104% (standard curve points and QCs), intra-assay precision of 2.1-15.4% (QCs) and inter-assay precision of 3.7-21.5% (QCs). The 6-plex immunoassay exhibited wide dynamic concentration ranges the calibration curves covered (median at 227-fold) defined by LLOQ and ULOQ, and low LOBs for target protein quantifications.
Application of the 6-Plex Immunoassay in the Detection of PDAC
[0100] The developed 6-plex immunoassay was applied to analyze the target protein levels in sera of 189 patients diagnosed with PDAC, 131 patients with benign pancreatic conditions, and 89 healthy controls (Table 1). The performances of the individual markers were compared to CA19-9 in discriminating PDAC versus healthy controls or benign conditions (
[0101] Serum levels of individual biomarkers were further analyzed in different subgroups consisting of 89 healthy controls, 68 chronic pancreatitis, 63 IPMN, 97 PDAC early stage, and 92 PDAC late stage patients (
[0102] In the present invention, the inventors identified a five-marker panel of CA19-9, MIC-1, CEACAM-1, MIA & OPN showing strong diagnostic performances and significant complementarities of these markers with CA19-9 in the detection of early stage PDAC from healthy controls and benign pancreatic conditions. These results provide an advanced validation on the utilities of these serum biomarkers in early detection of PDAC. MIC-1 belongs to transforming growth factor- superfamily, originally identified in activated macrophages and was found overexpressed in several cancer types. MIC-1 may have anticancer functions, as its promoter region is a target for p53. Koopmann et al reported that serum MIC-1 outperforms CA19-9 in the differention of patients with resectable pancreatic cancer from healthy controls with an AUC=0.99 (MIC-1) versue 0.78 (CA19-9) but not from chronic pancreatitis (0.81 versue 0.74). CEACAM-1 is a member of the human carcinoembryonic antigen (CEA) family. The CEACAM subgroup members belong to the immunoglobulin superfamily of adhesion molecules. CEACAM1 is expressed in a number of epithelia, granulocytes, and lymphocytes, and the expression of CEACAM-1 was also reported in different cancer types. CEACAM-1 plays an important role in the regulation of tumor growth, angiogenesis, and immune modulation. OPN is a glycophosphoprotein normally produced and secreted into most body fluids by osteoblasts, arterial smooth muscle cells, various epithelia, activated T cells and macrophages, and was often found overexpressed in different cancer types. OPN is most likely related to tumorigenesis, cancer cell proliferation and progression, migration and invasion, protection from apoptosis, and enhancement of metastatic ability. MIA is a small secreted protein coded by a single copy gene on chromosome 19q13.31-q13.33 and acts as an autocrine growth factor. MIA is strongly expressed by malignant melanoma cells and interacts with extracellular matrix proteins. Its overexpression promotes the metastatic behaviour of malignant melanoma. MIA was found overexpressed in pancreatic cancer and has the potential of promoting the invasiveness of pancreatic cancer cells, but its serum level were not significantly different between healthy donors and pancreatic cancer patients.
[0103] In the present invention, a 6-plex immunoassay of OPN, MIA, CEACAM-1, MIC-1, SPON1 and HSP27 was in-house developed, validated, and applied to a set of serum samples of PDAC patients, benign pancreatic conditions and healthy controls to evaluate their performances individually or in combination on their capacity to complement CA19-9 in early detection of pancreatic cancer. The assay was characterized by LOB/LLOQ, cross-reactivity, recovery, intra- and inter-assay precision; and demonstrated wide dynamic ranges for the target protein measurements that significantly correlated with their respective monoplex assays and/or commercial ELISAs. The assay shows advantages over traditional ELISA and other antibody-based approaches in both multiplexing and flexibility. It measures 6 candidate proteins in only 12.5 L of serum, and could include more candidate proteins into the panel as soon as appropriate pairs of capture and detection antibodies become available. It is important to note a few general considerations for the development of a multiplex immunoassay of human serum biomarkers. First, due to the different abundances of the candidate proteins in human serum, the effective biological range of each protein must be considered to ensure the fluorescence signal falling into the dynamic range of the assay. A more sensitive assay is needed for one protein with low abundance in the 6-plex immunoassay such as MIA, while a less sensitive assay may be required for another protein which may be of high abundance in the same multiplex immunoassay such as OPN. The sensitivity of each assay may be affected by the affinity/amount of the capture antibody and the amount of capture beads used for that protein. Second, antibody characteristics such as affinity and specificity are critical for the performance of a multiplex immunoassay. All pairs of capture and detection antibodies used in this study have been tested as compatible in the sandwich ELISA for human serum samples. The majority of the capture antibodies used in this study were monoclonal antibodies which are potentially more specific than polyclonal antibodies. All of the detection antibodies except SPON1 used in this study were commercially available biotinylated antibodies. Third, the performance of the multiplex immunoassays is more analyte and sample matrix dependent compared to monoplex immunoassays. Improper storage and non-optimal sample dilutions of serum samples can influence concentration measurements of some selected proteins in a complex sample matrix. It is vital to properly store serum samples at 80 C. prior to the analysis and avoid repeated freeze-thawing of serum samples.
[0104] In summary, a magnetic bead-based multiplex immunoassay was developed demonstrating sufficient analytical performance to evaluate serum biomarkers that may complement CA19-9 in early detection of PDAC. The biomarker panels identified in this study warrant additional clinical validation to determine their role in early detection of pancreatic cancer, which could lead to earlier intervention and better outcomes.
Pancreatic Cancer Treatment
[0105] The present invention provides methods of selecting a subject for pancreatic cancer treatment. Pancreatic cancer treatment includes, without limitation, surgery and/or administration of chemotherapeutic agent(s) to the subject. In one embodiment, the pancreatic cancer treatment is surgery. Chemotherapeutic agents suitable for treating pancreatic cancer include, without limitation, gemcitabine, 5-fluorouracil, irinotecan, oxaliplatin, paclitaxel, capecitabine, cisplatin, and docetaxel. Pancreatic cancer treatment comprising chemotherapeutic methods of (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of a chemotherapeutic agent to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human.
[0106] As used herein, the terms treat, treating, treatment, and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
[0107] As used herein, the terms prevent, preventing, prevention, prophylactic treatment and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
[0108] Such treatment (surgery and/or chemotherapy) will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for pancreatic cancer or disease, disorder, or symptom thereof. Determination of those subjects at risk can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, a marker (as defined herein), family history, and the like). In particular embodiments, determination of subjects susceptible to or having a pancreatic cancer is determined by measuring levels of at least one of the markers of the invention (e.g., CA19-9, MIA, MIC-1, CEACAM-1, OPN, SPON1, HSP27, POSTN, or LGALS3BP). In particular embodiments, a subject determined susceptible to or having a pancreatic cancer is selected for surgery.
Diagnostics
[0109] The present invention provides a number of diagnostic assays that are useful for early detection of pancreatic cancer in a subject. Current existing serum markers for pancreatic cancer such as CA19-9 lack the necessary sensitivity and specificity. Accordingly, the present invention provides other markers (e.g., MIA, MIC-1, CEACAM-1, OPN, SPON1, HSP27, POSTN, LGALS3BP) which are useful individually, in any combination with each other, or in any combination with CA19-9 for the detection of pancreatic cancer.
[0110] The presence or absence of the herein disclosed marker(s) is measured in a biological sample from a subject. Biological samples that are used to evaluate the presence or absence of the herein disclosed markers include without limitation blood, serum, plasma, urine. In one embodiment, the biological sample is serum.
[0111] While the examples provided below describe specific methods of detecting levels of these markers, the skilled artisan appreciates that the invention is not limited to such methods. The biomarkers of this invention can be detected by any suitable method. For example, marker levels are quantifiable by any standard method, such methods include, but are not limited to real-time PCR, Southern blot, PCR, mass spectroscopy, and/or antibody binding.
[0112] The methods described herein can be used individually or in combination for a more accurate detection of the biomarkers (e.g., immunoassay, mass spectrometry, and the like). The accuracy of a diagnostic assay can be characterized by a Receiver Operating Characteristic curve (ROC curve). An ROC is a plot of the true positive rate against the false positive rate for the different possible cutpoints of a diagnostic test. An ROC curve shows the relationship between sensitivity and specificity. That is, an increase in sensitivity will be accompanied by a decrease in specificity. The closer the curve follows the left axis and then the top edge of the ROC space, the more accurate the test. Conversely, the closer the curve comes to the 45-degree diagonal of the ROC graph, the less accurate the test. The area under the ROC is a measure of test accuracy. The accuracy of the test depends on how well the test separates the group being tested into those with and without the disease in question. An area under the curve (referred to as AUC) of 1 represents a perfect test, while an area of 0.5 represents a less useful test. In certain embodiments, biomarkers and diagnostic methods of the present invention have an AUC greater than 0.50. In other embodiments, biomarkers and diagnostic methods of the present invention have an AUC greater than 0.60. In other embodiments, biomarkers and diagnostic methods of the present invention have an AUC greater than 0.70. Exemplary combinations of markers (or panels of biomarkers) of the invention include, without limitation, the combination CA19-9 and MIA; the combination CA19-9 and SPON1; the combination CA19-9 and MIC-1; and, the combination CA19-9 and CEACAM-1. Exemplary combinations of markers (or panels of biomarkers) of the invention include, without limitation, the combination CA19-9, HSP27, and MIA1. Exemplary combinations of markers (or panels of biomarkers) of the invention include, without limitation, the combination CA19-9, CEACAM-1, MIC-1, SPON1 and MIA.
[0113] In particular embodiments, the biomarkers of the invention (e.g., CA19-9, MIA, MIC-1, CEACAM-1, OPN, SPON1, HSP27, POSTN, LGALS3BP) are measured by immunoassay. Immunoassay typically utilizes an antibody (or other agent that specifically binds the marker) to detect the presence or level of a biomarker in a sample. Antibodies can be produced by methods well known in the art, e.g., by immunizing animals with the biomarkers. Biomarkers can be isolated from samples based on their binding characteristics. Alternatively, if the amino acid sequence of a polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies by methods well known in the art.
[0114] This invention contemplates traditional immunoassays including, for example, Western blot, sandwich immunoassays including ELISA and other enzyme immunoassays, fluorescence-based immunoassays, and chemiluminescence. Other forms of immunoassay include magnetic immunoassay, radioimmunoassay, and real-time immunoquantitative PCR (iqPCR).
[0115] Immunoassays can be carried out on solid substrates (e.g., chips, beads, microfluidic platforms, membranes) or on any other forms that supports binding of the antibody to the marker and subsequent detection. A single marker may be detected at a time or a multiplex format may be used. Multiplex immunoanalysis may involve planar microarrays (protein chips) and bead based microarrays (suspension arrays).
[0116] In particular embodiments, the immunoassay is carried out using multiplexed bead assays. In particular embodiments, the immunoassay is carried out using magnetic bead-based multiplexed assays. Multiplexed bead assays use a series of spectrally discrete particles that are used to capture and quantitate soluble analytes. The analyte is then measured by detection of a fluorescence-based emission and flow cytometric analysis. Multiplexed bead assays generate data that is comparable to ELISA based assays, but in a multiplexed or simultaneous fashion. Concentration of unknowns is calculated for the cytometric bead array as with any sandwich format assay, i.e., through the use of known standards and by plotting unknowns against a standard curve. Further, multiplexed bead assays allow quantification of soluble analytes in samples never previously considered due to sample volume limitations. In addition to the quantitative data, powerful visual images are generated revealing unique profiles or signatures that provide the user with additional information at a glance.
[0117] In particular embodiments, subjects are characterized as having an increased level of CA19-9. In particular embodiments, subjects are characterized as having an increased level of MIA. In particular embodiments, subjects are characterized as having an increased level of MIC-1. In particular embodiments, subjects are characterized as having an increased level of CEACAM-1. In particular embodiments, subjects are characterized as having an increased level of OPN. In particular embodiments, subjects are characterized as having an increased level of SPON1.
[0118] In particular embodiments, subjects are characterized as having an increased level of CA19-9 and at least one of the markers selected from the group consisting of: MIA, MIC-1, CEACAM-1, OPN, SPON1, HSP27, POSTN, and LGALS3BP. In particular embodiments, subjects are characterized as having increased levels of CA19-9 and MIA. In particular embodiments, subjects are characterized as having increased levels of CA19-9 and MIC-1. In particular embodiments, subjects are characterized as having increased levels of CA19-9 and CEACAM-1. In particular embodiments, subjects are characterized as having increased levels of CA19-9 and SPON1.
[0119] In particular embodiments, subjects are characterized as having an increased level of the combination of markers CA19-9, HSP27, and MIA1. In particular embodiments, subjects are characterized as having an increased level of the combination of markers CA19-9, CEACAM-1, MIC-1, SPON1 and MIA.
[0120] In particular embodiments, the level of a marker is compared to a reference. In one embodiment, the reference is the level of marker present in a control sample obtained from a patient that does not have a pancreatic cancer. In some examples of the disclosed methods, when the level of expression of a biomarker(s) is assessed, the level is compared with the level of expression of the biomarker(s) in a reference standard. By reference standard is meant the level of expression of a particular biomarker(s) from a sample or subject lacking a pancreatic cancer, at a selected stage of pancreatic cancer or other pancreatic condition (e.g., pancreatitis, intraductal papillary mucinous neoplasm (IPMN), early stage or late stage pancreatic ductal adenocarcinoma (PDAC)) or in the absence of a particular variable such as a therapeutic agent. Alternatively, the reference standard comprises a known amount of biomarker. Such a known amount correlates with an average level of subjects lacking a cancer, at a selected stage of pancreatic cancer or pancreatic condition, or in the absence of a particular variable such as a therapeutic agent. A reference standard also includes the expression level of one or more biomarkers from one or more selected samples or subjects as described herein. For example, a reference standard includes an assessment of the expression level of one or more biomarkers in a sample from a subject that does not have a pancreatic cancer, is at a selected stage of progression of a pancreatic cancer, or has not received treatment for a pancreatic cancer. Another exemplary reference standard includes an assessment of the expression level of one or more biomarkers in samples taken from multiple subjects that do not have a pancreatic cancer, are at a selected stage of progression of a pancreatic cancer (e.g., pancreatitis, intraductal papillary mucinous neoplasm (IPMN), early stage or late stage pancreatic ductal adenocarcinoma (PDAC)), or have not received treatment for pancreatic cancer.
[0121] In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., CA19-9, MIA, MIC-1, CEACAM-1, OPN, SPON1, HSP27, POSTN, LGALS3BP) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with pancreatic cancer, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In some embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.
Kits
[0122] The invention provides kits for detecting a pancreatic cancer in a subject and/or characterizing a pancreatic cancer status in a subject. A diagnostic kit of the invention provides a reagent (e.g., an antibody or antigen binding fragment thereof that selectively bind a marker of the invention) for measuring relative expression of a marker (e.g., CA19-9, MIA, MIC-1, CEACAM-1, OPN, SPON1, HSP27, POSTN, LGALS3BP). In other embodiments, the kit further includes reagents suitable for CA19-9, MIA, MIC-1, CEACAM-1, OPN, SPON1, HSP27, POSTN, or LGALS3BP immunoassay.
[0123] In one embodiment, the kit includes a diagnostic composition comprising a capture reagent detecting a CA19-9 polysaccharide and a capture reagent detecting at least one marker selected from the group consisting of a MIA polynucleotide or polypeptide, a MIC-1 polynucleotide or polypeptide, a CEACAM-1 polynucleotide or polypeptide, a OPN polynucleotide or polypeptide, a SPON1 polynucleotide or polypeptide, a HSP27 polynucleotide or polypeptide, a POSTN polynucleotide or polypeptide, and a LGALS3BP polynucleotide or polypeptide. In one embodiment, the capture reagent detecting a CA19-9 polysaccharide is an anti-CA19-9 antibody or an antigen-binding fragment thereof. In one embodiment, the capture reagents are fixed to a substrate. In one embodiment, the substrate is a magnetic bead. In one embodiment, the kit includes a diagnostic composition comprising an anti-CA19-9 antibody or an antigen-binding fragment thereof and at least one antibody or antigen-binding fragment thereof selected from: an anti-MIC-1 antibody, an anti-CEACAM-1 antibody, an anti-MIA antibody, and an anti-SPON1 antibody. In one embodiment, the kit includes a diagnostic composition comprising an anti-CA19-9 antibody or an antigen-binding fragment thereof, an anti-HSP27 antibody or an antigen-binding fragment thereof, and an anti-MIA antibody or an antigen-binding fragment thereof. In one embodiment, the kit includes a diagnostic composition comprising an anti-CA19-9 antibody or an antigen-binding fragment thereof, an anti-CEACAM-1 antibody or an antigen-binding fragment thereof, an anti-MIC-1 antibody or an antigen-binding fragment thereof, an anti-SPON1 antibody or an antigen-binding fragment thereof, and an anti-MIA antibody or an antigen-binding fragment thereof.
[0124] The kits may be in combination with a therapeutic composition comprising an chemotherapeutic agent suitable for treating pancreatic cancer. In one embodiment, the kit includes a diagnostic composition and a therapeutic composition comprising a chemotherapeutic agent.
[0125] In some embodiments, the kit comprises a sterile container which contains a therapeutic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
[0126] If desired, the kit further comprises instructions for administering the therapeutic combinations of the invention. In particular embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for enhancing anti-tumor activity; precautions; warnings; indications; counter-indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
[0127] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, 1989); Oligonucleotide Synthesis (Gait, 1984); Animal Cell Culture (Freshney, 1987); Methods in Enzymology Handbook of Experimental Immunology (Weir, 1996); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, 1987); Current Protocols in Molecular Biology (Ausubel, 1987); PCR: The Polymerase Chain Reaction, (Mullis, 1994); Current Protocols in Immunology (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
[0128] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
EXAMPLES/METHODS
Example 1: Development of Magnetic Bead-Based Multiplex Immunoassay Comprising a Three Marker Panel of CA19-9, HSP27, and MIA to Evaluate Serum Biomarkers for the Early Detection of Pancreatic Cancer
[0129] Pancreatic cancer is the 4th leading cause of cancer death in the United States. The majority of patients present with unresectable disease leading a median survival of 6 months and an overall 5-year survival of <5%. The early detection of this disease is critical because surgery at an early stage is the most promising therapy that could greatly improve the prognosis of patients. The current existing serum markers such as CA19-9 lack the necessary sensitivity and specificity. Multiplex immunoassay simultaneously measuring multiple analytes in the same sample using minimum volume allows evaluation of serum biomarker panels that can potentially complement CA19-9 in early detection of pancreatic cancer. The study described herein is aimed at developing magnetic bead-based multiplex immunoassays to evaluate serum biomarkers for the early detection of pancreatic cancer.
[0130] Curated results from PUBMED database search using a combination of terms pancreatic cancer, pancreatic neoplasm, PANIN, pancreatic adenocarcinoma, sensitivity, and fold change were analyzed. Candidate biomarkers were selected using a weighted scoring system based on 1) fold changes and number of publications, or 2) sensitivity/specificity and study sample sizes. Magnetic bead-based multiplex immunoassays were developed for the selected candidate serum biomarkers using a Bio-Plex 200 suspension array system (Bio-Rad). Briefly, monoplex assays of individual candidates were first developed, cross-reactivity checked, and multiplex assays validated and optimized. All of these proteins plus HE4 (Roche) and CA19-9 (Tosoh) were analyzed in sera of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC: IB/IIA/IIB, n=10; IV, n=10), benign pancreatic conditions including intraductal papillary mucinous neoplasm (IPMN, n=10) and chronic pancreatitis (n=10), and healthy controls (n=19). The performances of these candidate markers were evaluated individually or in combination on their capacity to complement CA19-9 in early detection of pancreatic cancer.
[0131] The biomarkers evaluated included 1) a 5-plex assay of OPN, CEACAM-1, MIC-1, MIA, and SPON1; 2) a 2-plex assay of POSTN and HSP27; and 3) a monoplex assay of LGALS3BP. These assays were all in-house developed with negligible crossreactivity, recovery of 75-119%, and intra-assay or inter-assay precision of 0.3-9.6% or 0-18%, respectively. LOD or LLOQ was 0.179 ng/ml or 0.181 ng/mL (OPN), 0.101 ng/ml or 0.213 ng/ml (CEACAM-1), 0.001 ng/ml or 0.046 ng/ml (MIC-1), 0.009 ng/ml or 0.016 ng/ml (MIA), 0.041 ng/ml or 0.191 ng/ml (SPON1), 0.094 ng/ml or 0.767 ng/ml (POSTN), 0.005 ng/ml or 0.062 ng/ml (HSP27), and 0.035 ng/ml or 0.289 ng/mL (LGALS3BP). Individually, the best biomarkers (AUC in ROC analysis, 95% CI) to separate PDAC from benign pancreatic conditions were CA19-9 (0.9425, [0.85-1.00]), CEACAM (0.845, [0.71-0.98]), MIC (0.79, [0.65-0.93]), and SPON1 (0.68, [0.51-0.85]). However, stepwise backward logistic regression selected a three marker panel of CA19-9, HSP27, and MIA (p-values: <3E9, <0.03, <0.01, respectively) with an AUC=0.99 [0.97-1.00]. Probably due to the small sample size, the improvement over CA19-9 alone is not statistically significantly.
[0132] The multiplex immunoassay workflow provides sufficient analytical performance to evaluate serum biomarker panels that complement CA19-9 in early detection of pancreatic cancer.
Example 2: Development of Magnetic Bead-Based Multiplex Immunoassay Comprising a Five Marker Panel of (A19-9, MIA, SPON1, MIC-1 and CEACAM-1 to Evaluate Serum Biomarkers for the Early Detection of Pancreatic Cancer
[0133] Curated results from PUBMED database search using a combination of terms pancreatic cancer, pancreatic neoplasm, PANIN, pancreatic adenocarcinoma, sensitivity, and fold change were analyzed. Candidate biomarkers were selected using a weighted scoring system based on 1) fold changes and number of publications, or 2) sensitivity/specificity and study sample sizes. Magnetic bead-based multiplex immunoassays were developed for the selected candidate serum biomarkers using a Bio-Plex 200 suspension array system (Bio-Rad). Briefly, monoplex assays of individual candidates were first developed, cross-reactivity checked, and multiplex assays validated and optimized (
[0134] A 6-plex immunoassay of OPN, MIA, CEACAM-1, MIC-1, SPON1, and HSP27 was in-house developed with negligible cross-reactivity, recovery of 89-101%, and intra-assay or inter-assay precision of 3.5-11.6% or 6.1-17.3% for calibrators, respectively (Table 6). LOD or LLOQ was 0.053 ng/ml or 0.156 ng/ml (OPN), 0.054 ng/mL or 0.141 ng/ml (CEACAM-1), 0.002 ng/ml or 0.012 ng/ml (MIC-1), 0.002 ng/mL or 0.007 ng/mL (MIA), 0.011 ng/ml or 0.058 ng/ml (SPON1), and 0.004 ng/mL or 0.012 ng/mL (HSP27) (Table 3). The 6-plex assay demonstrated wide dynamic ranges for the target measurements, and was significant correlated with their respective monoplex assays (p<0.05) and/or commercial ELISAs (p<0.01) (
[0135] The multiplex immunoassay workflow provides sufficient analytical performance to evaluate serum biomarker panels that complement CA19-9 in early detection of pancreatic cancer. The biomarker panels identified in this study warrant further validation with a larger number of patient samples.
Patient Specimens
[0136] A total of 409 archived serum samples obtained from 189 patients with histologically diagnosed pancreatic ductal adenocarcinoma (PDAC) [mean (SD) age, 65 (10) years; M/F, 81/108] from January 2007 to October 2015, 131 patients with benign pancreatic conditions [57 (15) years; 71/60] from February 2007 to October 2015, and 89 healthy controls without a history of pancreatic diseases [35 (14) years; 45/44] from either April 2013 or August 2015 were collected at the Johns Hopkins Medical Institutions (JHMI) with institutional approval. Among 189 patients with PDAC, there are 97 early stage [IA/IB/IIA/IIB, 13/19/17/48; 65 (10) years; 34/63] and 92 late stage [III/IV, 19/73; 64 (10) years; 47/45] diseases. Among 131 patients with benign pancreatic conditions, there are 63 intraductal papillary mucinous neoplasm (IPMN) [64 (12) years; 24/39] and 68 chronic pancreatitis [51 (15) years; 47/21]. Detailed clinicopathologic characteristics of the study cohort, including diagnosis, age, sex and anatomic stage, were shown in Table 1. All serum samples were obtained before treatment and before surgery, and stored at 80 C. until analysis.
Reagents and Antibodies
[0137] All of the recombinant proteins and antibodies were purchased from R&D Systems (Minneapolis, MN)), except the detection antibody for SPON1 which was biotinylated in-house. Majority of the antibodies except those for OPN and SPON1 were from the DuoSet ELISA kits (R&D), which have been commercially tested as an appropriate pair of antibodies for the development of sandwich ELISAs to measure natural and recombinant human proteins in cell culture supernatants. Antibodies of OPN and SPON1 were also compatible for the ELISA applications. Detailed information for the recombinant proteins and antibodies are shown in Table 4. Magnetic COOH beads, amine coupling kits, and Bio-Plex Pro Reagent kits were purchased from Bio-Rad Laboratories (Hercules, CA). NHS and Sulfo-NHS, EDC, EZ-Link Sulfo-NHS-Biotin, and Zeba Spin Desalting Columns were purchased from Thermo Scientific (Rockford, IL). Human serum CA19-9 level was measured using a commercial kit from Tosoh Bioscience LLC (King of Prussia, PA). The human osteopontin ELISA kit (ABIN414433) and human heat shock protein 27 ELISA kit (ab113334) were purchased from Antibodies-Online (Atlanta, GA) or Abcam (Cambridge, MA), respectively.
Conjugation of Antibodies to Microspheres
[0138] The capture antibodies for OPN, MIA, CEACAM-1, MIC-1, SPON1 and HSP27 were respectively coupled to magnetic beads of different regions using the Bio-Rad amine coupling kit according to the manufacturer's instructions. The use of differentially detectable beads of the different regions enables the simultaneous identification and quantification of multiple analytes in the same sample and the individual immunoassays therefore could be multiplexed. The optimal amounts of capture antibodies for one coupling reaction were used at either 6 g for OPN, MIA, CEACAM-1, MIC-1 and HSP27 or 9 g for SPON1, after the titration. The coupled beads were counted using a Coulter Z2 counter, validated using biotinylated rabbit anti-mouse (B8520) or rabbit anti-goat (B7014) IgG antibodies (Sigma-Aldrich, St. Louis, MO), and stored in storage buffer at 4 C. in the dark.
Multiplex Immunoassay
[0139] The magnetic bead-based multiplex immunoassay was developed for the selected candidate serum biomarkers using a Bio-Plex 200 suspension array system (Bio-Rad, Hercules, CA). The general workflow of multiplex immunoassay is shown in
[0140] Before multiplexing the individual assays, assay specificity was examined by performing single-detection and multiplexed-detection antibody cross-reactivity studies to detect the fluorescence signals in response to high concentrations of the recombinant proteins at the first dilution point of the standard curve (except SPON1 at the third dilution). The single detection antibody study was conducted by testing an individual detection antibody in the presence of multiplexed capture beads and a single antigen, which evaluates the specificity of a capture antibody. The multiplexed-detection antibody study was conducted by testing multiplexed detection antibodys in the presence of multiplexed capture beads and a single antigen, which evaluates the specificity of a detection antibody and to some degree the specificity of the capture antibody. Cross-reactivity was defined as the percentage of nonspecific cross-reacting signal detected relative to the specific signal for that analyte.
[0141] For the multiplex immunoassay, the capture beads and the detection antibodies were prepared by mixing the 2500 coupled beads and the detection antibodies used in the monoplex assays. The final concentrations of the detection antibodies in the multiplex assay were used at 0.4 g/mL for OPN and CEACAM-1 or 2 g/mL for SPON1 or 0.2 g/mL for MIA and HSP27 or 0.0125 g/mL for MIC-1, respectively, after the titration. The calibration curve was established using 9 calibrators in 2-fold dilution series derived from a mixture of the highest standard points of 7 recombinant proteins. The highest standards of 7 recombinant proteins in the multiplex assay were used at 40, 1.5, 20, 3, 15 and 3 ng/ml for OPN, MIA, CEACAM-1, MIC-1, SPON1 and HSP27, respectively. To assess the correlations of the developed immunoassays in protein quantifications, the multiplex immunoassays were compared to the monoplex immunoassays by measuring 4 dilutions of individual recombinant proteins based on their respective calibration curves. The correlations of the developed multiplex immunoassays and commercial ELISA kits in serum OPN or HSP27 protein quantifications were also determined in 7 or 13 patient sera, respectively. The multiplex immunoassay was carried out using the Bio-Plex Pro Reagent kit in the same procedures as those in the monoplex assays described above. The serum samples were 4-fold diluted in the sample diluent in the multiplex immunoassay. Two quality controls (QC) were prepared by diluting the mixture of the highest standards of 6 recombinant proteins at either 3-fold (QC1) or 30-fold (QC2). Two pooled human sera with the known CA19-9 measurements at either high or low levels were used as the calibrators. The multiplex immunoassay was performed in duplicate on 1396-well Bio-Plex flat bottom plates with a calibration curve, 2 doses of QCs and 2 doses of calibrators in each plate. All samples were randomized with regard to their plate locations.
[0142] Data acquisition and primary data analysis were performed on the Bio-Plex 200 system in combination with Bio-Plex Manager Software version 6.1.1 by use of a 5-parametric (5-PL) nonlinear logistic regression curve fitting model (Bio-Rad). According to Bio-Rad Bio-Plex multiplex immunoassay handout (bio-rad.com/en-us/applications-technologies/bio-plex-multiplex-immunoassays), in this study, the assay sensitivity (limit of black, LOB) was defined as the concentration of analyte corresponding to the median fluorescent intensity (MFI) of the background plus two standard deviations (SD) of the mean background MFI. The assay reproducibility was assessed in both intra- and inter-assay precisions. Intra-assay precision was calculated as the coefficient of variance (% CV) on the duplicates of two QCs or two calibrators on a single assay plate. Inter-assay precision was calculated as the % CV from 6 independent assays. The assay accuracy (recovery percentage) was calculated as the percentage of the observed concentration relative to the expected concentration of each standard point or QC. The assay working dynamic range was defined as the range between the lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ) in which an assay is both precise (intra-assay % CV10% and inter-assay % CV15%) and accurate (80-120% recovery).
Data Analysis
[0143] The nonparametric Mann-Whitney U test was used to compare serum biomarker levels between PDAC patients, benign pancreatic conditions and healthy controls, with a p-value less than 0.05 considered significant. Receiver operator characteristic (ROC) analysis was performed and the area under the curve (AUC) was calculated separately for each of 7 biomarkers and the combinations of biomarkers. Delong test was used to compare the AUCs. Pearson correlation coefficients were determined to assess correlation of the measurements between the multiplex and monoplex immunoassays or commercial ELISA kits. Logistic regression analysis (both backward stepwise and forward stepwise) was performed to select the panels of biomarkers with the highest performance. The Statistica 12 (StatSoft) and GraphPad Prism 6 (GraphPa Software) were used for statistical analysis.
TABLE-US-00017 TABLE 1 Clinicopathologic characteristics of the study cohort. Variables Number (%) Total 409 Healthy control 89 (21.8) Age (year) Mean SD 35 14 Range 21-67 Gender Male 45 (50.6) Female 44 (49.4) Benign conditions 131 (32) Age (year) Mean SD 57 15 Range 13-89 Gender Male 71 (54.2) Female 60 (45.8) Chronic pancreatitis 68 (51.9) IPMN 63 (48.1) PDAC 189 (46.2) Age (year) Mean SD 65 10 Range 30-92 Gender Male 81 (42.9) Female 108 (57.1) Early stage 97 (51.3) IA/IB/IIA/IIB 13/19/17/48 Late stage 92 (48.7) III/IV 19/73 NOTE: IPMN, intraductal papillary mucinous neoplasm.
TABLE-US-00018 TABLE 2 Assay specificity of the 6-plex immunoassay. Percentage of cross-reactivity (single-detection/multiplexed-detection antibody) was calculated based on fluorescence signals detected in response to high concentrations of the recombinant proteins at the 1.sup.st dilution point (at 3.sup.rd for SPON1) of the standard curve in single- detection and multiplexed-detection antibody cross-reactivity studies (both with multiplexed beads and single antigen). Target OPN MIA CEACAM-1 MIC-1 SPON1 HSP27 OPN 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 MIA 0.3/0.1 0.4/0.0 0.1/0.1 0.3/0.0 0.1/0.1 CEACAM- 0.0/0.3 0.2/0.0 0.0/0.2 0.0/0.0 0.0/0.0 1 MIC-1 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 SPON1 2.4/2.1 1.7/1.3 2.0/2.0 2.7/2.4 3.3/3.3 HSP27 0.1/0.1 0.2/0.1 0.2/0.1 0.2/0.1 0.7/0.7
TABLE-US-00019 TABLE 3 Analytical performance of the 6-plex immunoassay. QC1, high control. QC2, low control. LOD, limit of detection. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantification. The correlation of 6-plex vs monoplex was examined on 4 doses of individual recombinant proteins. The correlation of 6-plex vs the commercial ELISA kit was examined on 7 (OPN) or 13 (HSP27) patient sera. Intra-assay Inter-assay 6-plex vs 6-plex vs Mean Precision Precision Monoplex, ELISA, (pg/mL) (% CV) (% CV) LOD LLOQ ULOQ Replicates* Pearson Pearson QC1 QC2 QC1 QC2 QC1 QC2 (pg/mL) (pg/mL) (pg/mL) (% CV) R/p value R/p value OPN 13448.3 1228.3 2.1 5.1 3.7 4.2 52.5 155.7 34728.7 3.4 0.9987/0.0013 0.8945/0.0066 MIA 481.7 48.3 8.6 10.0 4.4 8.4 2.3 6.7 1541.1 9.2 0.9888/0.0112 ND** CEACAM-1 6141.7 658.3 9.3 6.6 8.6 16.3 53.7 140.7 20118.4 7.5 0.9715/0.0285 ND MIC-1 963.3 93.3 8.1 10.3 21.5 18.8 1.5 11.6 2327.0 5.1 0.9996/0.0004 ND SPON1 4746.7 473.3 4.3 2.7 5.0 6.8 10.7 58.1 15032.7 6.3 0.9675/0.0325 ND HSP27 933.3 88.3 15.4 12.6 14.5 13.2 4.4 11.7 3000.6 9.0 0.9997/0.0003 0.9254/ <0.00001 *mean of % CV for replicates in all samples for each protein. **ND, not determined.
TABLE-US-00020 TABLE 4 6-plex immunoassay recombinant proteins and antibodies. Recombinant Capture Antibody Detection Antibody Target Protein Cat. # Cat. # Host Cat. # Host OPN 1433-OP-050 MAB14332 Mouse BAF1433 Goat MIA DY2050 DY2050 Mouse DY2050 Goat CEACAM-1 DY2244 DY2244 Mouse DY2244 Goat MIC-1 DY957 DY957 Mouse DY957 Goat SPON1 3135-SP/CF AF3135 Goat AF3135* Goat HSP27 DY1580 DY1580 Goat DY1580 Rabbit NOTE: *all of the recombinant proteins and antibodies were purchased from R&D Systems, except detection antibody of SPON1 was biotinylated in-house.
TABLE-US-00021 TABLE 5 Statistics of individual biomarkers in healthy controls, benign conditions and PDAC patients. Biomarker Subgroup Number Min Max Median Mean OPN Healthy Control 89 2.14 21.66 6.88 7.93 Chronic 68 0.76 103.56 13.59 20.68 Pancreatitis IPMN 63 2.28 84.75 8.51 12.05 PDAC early stage 97 2.03 135.26 14.19 18.99 PDAC late stage 92 2.31 154.05 14.53 21.48 MIA Healthy Control 89 0.12 1.50 0.63 0.69 Chronic 68 0.12 1.24 0.48 0.51 Pancreatitis IPMN 62 0.05 1.48 0.62 0.64 PDAC early stage 97 0.14 1.40 0.51 0.59 PDAC late stage 92 0.24 2.05 0.58 0.66 CEACAM-1 Healthy Control 89 2.33 26.86 12.66 13.01 Chronic 68 5.15 93.21 16.83 22.88 Pancreatitis IPMN 63 5.65 35.40 16.06 17.01 PDAC early stage 97 4.82 120.85 21.19 31.16 PDAC late stage 92 4.58 117.79 22.63 29.74 MIC-1 Healthy Control 89 0.11 0.77 0.26 0.32 Chronic 68 0.22 3.35 0.86 1.02 Pancreatitis IPMN 63 0.25 3.01 0.69 0.83 PDAC early stage 96 0.35 7.93 1.14 1.51 PDAC late stage 92 0.20 7.05 1.01 1.30 SPON1 Healthy Control 89 1.87 14.90 4.70 5.02 Chronic 68 1.00 17.46 5.92 6.42 Pancreatitis IPMN 63 0.60 21.82 5.15 5.87 PDAC early stage 97 1.95 42.76 5.81 7.24 PDAC late stage 92 2.09 21.14 5.85 6.46 HSP27 Healthy Control 89 0.22 4.20 0.86 1.16 Chronic 68 0.22 4.83 0.97 1.26 Pancreatitis IPMN 63 0.10 8.62 0.90 1.34 PDAC early stage 97 0.15 7.10 1.20 1.51 PDAC late stage 92 0.15 5.22 1.13 1.33 CA19-9 Healthy Control 89 1.00 71.60 11.00 15.55 Chronic 68 1.00 203.20 20.10 32.24 Pancreatitis IPMN 63 1.00 386.90 16.80 26.85 PDAC early stage 97 1.00 27027.80 90.60 824.71 PDAC late stage 92 1.00 25110.70 354.75 1638.68 (NOTE: all biomarkers are at ng/ml, except CA19-9 at U/ml.
TABLE-US-00022 TABLE 6 Assay specificity of the 6-plex immunoassay. Percentage of cross-reactivity (single-detection/multiplexed-detection antibody) was calculated based on fluorescence signals detected in response to high concentrations of the recombinant proteins at the 1.sup.st dilution point (except SPON1 at 3.sup.rd because only 1.4% of sera with SPON1 exceed STD3) of the standard curve in single-detection and multiplexed-detection antibody cross-reactivity studies (both with multiplexed beads and single antigen). Target OPN MIA CEACAM-1 MIC-1 SPON1 HSP27 OPN 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 MIA 0.3/0.1 0.4/0.0 0.1/0.1 0.3/0.0 0.1/0.1 CEACAM- 0.0/0.3 0.2/0.0 0.0/0.2 0.0/0.0 0.0/0.0 1 MIC-1 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 0.0/0.0 SPON1 2.4/2.1 1.7/1.3 2.0/2.0 2.7/2.4 3.3/3.3 HSP27 0.1/0.1 0.2/0.1 0.2/0.1 0.2/0.1 0.7/0.7
OTHER EMBODIMENTS
[0144] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
[0145] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
[0146] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.