Patent Application
20220291222
Not applicable.
This invention relates generally to a method to isolate proteins from biofluids, such as plasma and serum, for biomarker discovery or for clinical detection. More particularly, this invention relates to non-invasive early disease diagnosis, disease monitoring and disease classification. In one aspect, this invention relates to unique proteins capable of differentiating pancreatic cancer plasma or serum samples from healthy samples and non-cancer conditions control samples. In another aspect, this invention relates to early-stage detection of pancreatic cancer by plasma or serum test.
The rates of survival for pancreatic cancer are very low, primarily because the cancer is detected at a very late stage. It is critical to develop a non-invasive screening test for early detection of pancreatic cancer, which would significantly improve disease prognosis and survival rates.
Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all common tumors. The vast majority of patients are diagnosed when curative surgery is no longer possible (Rahib et al. 2014; Spanknebel and Conlon 2001). Earlier cancer diagnosis could increase survival rates by an estimated 5-fold and more reliable and real-time assessment of treatment effects in patients with cancer could improve quality of life and reduce healthcare costs (Ghatnekar et al. 2013).
Chronic pancreatitis and intraductal papillary mucinous neoplasm (IPMN) are established risk factors for PDAC accounting for up to 20% of the cases. Surveillance strategies for this heterogenous population is not standardized and patients with “worrisome” imaging features, particularly those with high risk IPMN, may undergo surgical resection. Unfortunately, the imaging criteria reflexing patients to surgery are imperfect, leading to both over- and under-treatment.
The gold standard for diagnosis of pancreatic cancer, particularly in those with underlying pancreatic diseases (cysts or pancreatitis) is the endoscopic ultrasound (EUS) with fine needle biopsy, which has a sensitivity of 90% and specificity of 72% (Li et al. 2014). The main limitations of the EUS are: (1) it is a relatively invasive procedure; (2) limited availability, needs to be performed in a center with high volume as the diagnostic accuracy is directly related to the operator's skills; (3) elevated costs, ranging from $2,152 to $2,605 per procure (Aadam et al. 2016).
The current blood-based biomarker that is clinically available (CA19-9) suffers from poor sensitivity (75.4%) and specificity for the diagnosis of pancreatic cancer (77.6%) (Ghatnekar et al. 2013). CA19-9 can be elevated in non-malignant etiologies such as jaundice and biliary obstruction. For patients with established diagnosis of pancreatic cancer, CA19-9 elevates late in the disease course making it unsuitable for detection of early-stage disease, when curative treatment options are available.
There are no credentialed biomarkers with high enough performance to assist in therapeutic stratification. Various approaches to achieving this are being explored, including circulating tumor cells (CTCs), metabolites, proteomics, cell-free DNA and circulating extracellular vesicles (EVs) (Duffy et al. 2010; Kelly et al. 2015; Nagrath et al. 2016).
Extracellular vesicles (exosomes and microvesicles) are nanosized particles released by most cell types and involved in intercellular communication, including transmission of oncogenic and inflammatory signals (Costa-Silva et al. 2015). Their exoDNA, exoRNA and protein profiles highly reflect parental cells, therefore offering an attractive strategy for diagnosing cancers using “liquid biopsies” (Melo et al. 2015; Costa-Silva et al. 2015). Cancer cells can shed a much higher concentration of EVs per cell, making them substantially more abundant than CTCs (Taylor and Gercel-Taylor 2008; Riches et al. 2014). EVs are also structurally more stable than soluble proteins, cell-free DNA and metabolites, underscoring their potential as serologic biomarkers (Thery 2015; Costa-Silva et al. 2015).
However, the application of EV biomarkers in clinic has been hampered by two practical challenges:
(1) Prior discovery EV proteomics have been done in vitro using cell lines, which are not representative of the heterogeneity of human PDAC and are unable to recapitulate the systemic/inflammatory response to cancer. Therefore, those prior EV signatures lacked performance to distinguish PDAC from high-risk chronic pancreatic diseases (Yang et al. 2017; Castillo et al. 2018; Madhavan et al. 2015) which is critical in clinical practice.
(2) Traditional workflow for EV isolation directly from blood samples require laborious techniques (such as ultracentrifugation) that are not scalable for clinical use.
There is a critical need for a new diagnostic test that would avoid these drawbacks and enable more effective non-invasive detection of pancreatic cancer at the earliest stage, which would significantly improve disease prognosis and survival rates, particularly in those with underlying diseases of the pancreas where detection of cancer is very challenging.
In one aspect, this disclosure is related to a robust method for the identification and detection of new biomarkers based on proteins for pancreatic cancer, for the purposes of disease diagnosis, prognosis, detection, monitoring, patient stratification, drug response analysis, therapy selection, or the like.
The proposed method introduces a novel platform technology to isolate proteins from biofluids, such as plasma and serum, for biomarker discovery or for clinical detection.
In another aspect, this disclosure is related to a method that successfully demonstrates the feasibility of developing biofluid-derived EV proteins for cancer profiling. It has tremendous transformative potential for early cancer diagnosis, monitoring and classification based on actual activated pathways using plasma and serum as the source.
Furthermore, once fully established, these new biomarkers can be employed either isolated or as part of a panel of biomarkers as a liquid biopsy in clinical scenarios: (1) as a surveillance test in high-risk patients, such as those with high-risk cystic diseases, hereditary risk of cancer, among others or (2) as a liquid biopsy for the longitudinal monitoring of treatment response in patients with already established cancer diagnosis.
In yet another aspect, this disclosure relates to a biomarker panel for detection and monitoring of pancreatic cancer.
Still further, it is envisioned to further apply this innovative procedure to validate and fully develop pre-determined biomarkers panels.
The invention now will be described more fully hereinafter with reference to the accompanying drawings, which are intended to be read in conjunction with both this summary, the detailed description and any preferred and/or particular embodiments specifically discussed or otherwise disclosed. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided by way of illustration only and so that this disclosure will be thorough, complete and will fully convey the full scope of the invention to those skilled in the art.
This disclosure is related to a method for the identification and detection of new biomarkers based on proteins—a true measure of dynamic activity and cellular signaling. The proposed method introduces a novel platform technology to isolate extracellular vesicles (EV) proteins from biofluids such as plasma and serum for biomarker discovery and clinical detection of pancreatic cancer.
The method for capture, enrichment or isolation of extracellular vesicles is selected from the group consisting of Extracellular Vesicles total recovery and purification (EVtrap™), ultracentrifugation (UC), filtrations, antibody-based purification, size-exclusion approach, polymer precipitation and affinity capture.
This disclosure is the first such method to successfully demonstrate the feasibility of developing plasma- and serum-derived EV proteins for pancreatic cancer detection and profiling. It is envisioned to further apply this innovative procedure to validate and fully develop the disclosed current biomarker panel in Table I for detection and monitoring of pancreatic cancer.
The above-mentioned and other features of this disclosure, and the manner of attaining them, will become more apparent and the disclosure itself will be better understood by reference to the following description of embodiments of the disclosure taken in conjunction with the accompanying drawings, wherein:
The following detailed description includes references to the accompanying drawings, which forms a part of the detailed description. The drawings show, by way of illustration, specific embodiments in which the invention may be practiced. These embodiments, which are also referred to herein as “examples,” are described in enough detail to enable those skilled in the art to practice the invention. The embodiments may be combined, other embodiments may be utilized, or structural, and logical changes may be made without departing from the scope of the present invention. The following detailed description is, therefore, not to be taken in a limiting sense.
Before the present invention of this disclosure is described in such detail, however, it is to be understood that this invention is not limited to particular variations set forth and may, of course, vary. Various changes may be made to the invention described and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process act(s) or step(s), to the objective(s), spirit or scope of the present invention. All such modifications are intended to be within the scope of the disclosure made herein.
Unless otherwise indicated, the words and phrases presented in this document have their ordinary meanings to one of skill in the art. Such ordinary meanings can be obtained by reference to their use in the art and by reference to general and scientific dictionaries.
References in the specification to “one embodiment” indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.
The following explanations of certain terms are meant to be illustrative rather than exhaustive. These terms have their ordinary meanings given by usage in the art and in addition include the following explanations.
Unless otherwise stated, a reference to a compound or component includes the compound or component by itself, as well as in combination with other compounds or components, such as mixtures of compounds.
As used herein, the term “and/or” refers to any one of the items, any combination of the items, or all of the items with which this term is associated.
As used herein, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.
As used herein, the terms “include,” “for example,” “such as,” and the like are used illustratively and are not intended to limit the present invention.
As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances.
Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.
It will be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first element could be termed a second element, and, similarly, a second element could be termed a first element without departing from the teachings of the disclosure.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.
While the invention has been described above in terms of specific embodiments, it is to be understood that the invention is not limited to these disclosed embodiments. Upon reading the teachings of this disclosure many modifications and other embodiments of the invention will come to mind of those skilled in the art to which this invention pertains, and which are intended to be and are covered by both this disclosure and the appended claims. It is indeed intended that the scope of the invention should be determined by proper interpretation and construction of the appended claims and their legal equivalents, as understood by those of skill in the art relying upon the disclosure in this specification and the attached drawings.
EV biomarkers discovery directly from blood samples:
The preferred method, EVtrap™, was applied for EV enrichment directly from blood samples to isolate EVs from plasma for subsequent liquid chromatography-mass spectrometry analysis. Processing and enrichment of EVs through this method enabled the removal of soluble proteins, retaining vesicle associated proteins which are more stable in circulation and have enhanced signals from cancer tissues. The protein profiles in EV concentrates are different from protein profiles naturally occurring in patient blood.
The research protocol was designed, and IRB approved (PI: Bockorny) allowing for recruitment of patients with established diagnosed of pancreatic cancer at all stages: patients with underlying non-malignant conditions of the pancreas such as chronic pancreatitis and cystic diseases of the pancreas; as well as healthy patients (controls). All patients provided written informed consent for this study. Clinical data were extracted through medical records and patient interview. Blood samples of 124 patients were collected by the research personnel and subsequently processed and stored in our laboratory. The blood samples were processed using the preferred method, EVtrap™, and subsequent liquid chromatography—mass spectrometry analysis.
In total, EV proteomics was conducted on a cohort of 124 patients including various stages of PDAC (N=93), chronic pancreatitis of different etiologies (N=12), IPMN (N=8) and healthy controls (N=11), wherein, on average, ˜1,000 unique EV proteins were identified from each individual case.
Different biostatistical analysis of the proteomics were performed, and it was observed that the EV secretome of PDAC patients had clear separation from chronic pancreatitis, IPMN and controls (
The differential expression of individual EV proteins was analyzed. Focusing on EV markers that were enriched in patients with pancreatic cancer as compared to patients with benign disease of the pancreas and normal controls (biomarkers for diagnosis of pancreatic cancer) as well as on the markers more enriched in advanced pancreatic cancer (stage IV) as compared to early stages (stages I/II) as those represent biomarkers that may serve for disease monitoring of tumor burden.
Summary of the EV Biomarkers Discovered is found in Table I. Table I lists a panel of EV proteins with significant overexpression and enrichment in advanced PDAC but not in early stages or controls, including novel biomarkers as well as proteins previously known to have functional or prognostic implications in PDAC (e.g. integrin B2, ezrin) (Yuan et al. 2015; Giulietti et al. 2018; Meng et al. 2010; Kocher et al. 2009).
The markers listed in Table I can be employed either isolated or as part of a panel of biomarkers as a liquid biopsy in different clinical scenarios: (1) as a surveillance test in high-risk patients, such as those with high-risk cystic diseases of the pancreas (e.g. IPMN), hereditary pancreatitis, chronic pancreatitis. (2) or as a liquid biopsy for the longitudinal monitoring of treatment response in patients with already established pancreatic cancer diagnosis.
It is envisioned that secondary validation of the candidate biomarkers listed in Table I might be performed in an independent cohort of patients (different from the patients from the proteomics) by employing an alternative method to identify such proteins of interest (ELISA, western blot, etc.).
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While this disclosure has been described as having an exemplary design, the present disclosure may be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the disclosure using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this disclosure pertains.
This U.S. patent application claims priority to U.S. Provisional Application No. 63/159,573 filed Mar. 11, 2021, to the above-named inventors, the disclosure of which is considered part of the disclosure of this application and is hereby incorporated by reference in its entirety.
This study was made with government support under Grant No. CA213863 awarded by the National Institutes of Health. The government has certain rights in the outcome of this study.
| Number | Date | Country | |
|---|---|---|---|
| 63159573 | Mar 2021 | US |
