Analysis of exosomal proteins from biological fluids for cancer detection and treatment response monitoring

Abstract

Methods for cancer detection and monitoring are provided that analyze exosomal proteins isolated from biological fluids. The methods comprise obtaining a biological fluid sample from a subject, separating exosomes from the sample, and generating a mass spectrum of exosomal proteins using an analytical method such as MALDI-TOF mass spectrometry, ESI-MS, LC-MS, or other mass spectrometry techniques. Biological fluids include plasma, serum, whole blood, urine, pancreatic cyst fluid, cerebrospinal fluid, and saliva. The mass spectrum is compared to reference spectra to detect cancer presence, monitor treatment response, identify cancer recurrence or progression, or determine cancer type in cases of unknown primary origin. The methods are applicable to solid tumors including pancreatic, breast, lung, prostate, and colorectal cancers, as well as hematologic malignancies. In certain embodiments, a personalized cancer spectrum approach compares a patient's sample to their own baseline spectrum for enhanced sensitivity and specificity.

Description

Atty. Dkt. No.: 059304-0502ANALYSIS OF EXOSOMAL PROTEINS FROM BIOLOGICAL FLUIDS FOR CANCER DETECTION AND TREATMENT RESPONSE MONITORING CROSS-REFERENCE TO RELATED PATENT APPLICATION

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Serial No. 63 / 789972, filed April 16, 2025, the contents of which is incorporated herein in its entirety.BACKGROUND

[0002] The American Cancer Society estimates about 37,370 cases of cancer of unknown primary will be diagnosed in 2025 in the United States. This number represents about 2% of all cancers.

[0003] In addition, current liquid biopsy methods, when no protein biomarker exists, rely on detection of circulating tumor DNA in blood, which is relatively expensive and may take two to three weeks or longer to obtain results. In addition, many cancers do not have specific biomarkers that are used clinically in the monitoring of patients for potential early signs of cancer recurrence (some cancers are associated with protein or glycoprotein biomarkers, e.g., CEA, CA 19-9, PSA, Chromogranin A or CA-125).

[0004] A biomarker is needed that can be used to detect early response or resistance to treatment before changes in tumor size can be seen on radiologic imaging studies. Currently, no definitive blood biomarkers exist for detecting many types of cancer.

[0005] Moreover, even where a disease-associated biomarker is known, there are very few blood tests that can be used for screening to detect early cancers. Most notable is PSA but even its use in screening is controversial (https: / / pmc.ncbi.nlm.nih.gov / articles / PMC9875204 / ) PMCID: PMC9875204.Thus, a need exists in the art for a fast and cost-effective test that can be used for early detection of cancer as well as to identify cancers of unknown primary origin. This disclosure furthers a solution to this need and provides related advantages as well.-1- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502SUMMARY OF THE DISCLOSURE

[0006] Biomarkers are useful clinical tools for monitoring patients with various diseases, including cancer. Cancer biomarkers in current clinical use include prostate specific antigen (PSA) for prostate cancer, carcinoembryonic antigen (CEA) for colon cancer, CAI 9-9 for pancreatic adenocarcinoma, CA-125 for ovarian cancer, and thyroglobulin (TG) for thyroid cancer. Many cancers do not have a biomarker and each of the ones in use have limitations in their utility. Papillary thyroid cancer (PTC) is the most common type of thyroid cancer and is usually cured by surgery. However, 8-28% of patients will have recurrence of their cancers.1Assays for TG have several limitations. First, measuring TG is not recommended prior to surgery.2Thyroid lobectomy, rather than total thyroidectomy, is increasingly used for differentiated thyroid cancer.3The presence of residual normal thyroid makes interpretation of postoperative levels problematic.4Furthermore, the presence of antithyroglobulin antibodies in up to 25% of patients also confounds use of TG levels to assess patients for recurrence of thyroid cancer after surgery.5For these reasons, an improved biomarker is needed for PTC.

[0007] Recently, biomarker development has focused on tests to detect minute quantities of circulating DNA (ctDNA) released into the blood stream by cancer cells.6'8Several commercial ctDNA assays are now available for physicians to use in the care of their patients the such as the Grail Galleri™ test which detects abnormalities in the methylation patterns of cell-free DNA (cfDNA)9and the Guardant 360™ test for ctDNA.6,10’11It may be possible to increase the sensitivity and accuracy of ctDNA sequencing by designing the assay based on the DNA sequencing results of the individual patient’s tumor.7Such tumor informed circulating DNA assays are more costly than tumor naive ctDNA approaches and require access to the original tumor specimen. Genomic assays are promising but are relatively expensive and turnaround times are generally two to three weeks for clinical results.

[0008] Thus, the present disclosure provides methods for detecting and monitoring cancer using analysis of exosomal proteins isolated from biological fluids.In one aspect, the disclosure provides a method of treating or managing cancer in a subject, the method comprising, or consisting essentially of, or consisting of: (a) generating a first mass spectrum using an analytical method from protein cargo of exosomes isolated from a subject's first biological fluid sample; (b) administering an anti-cancer therapy to the subject; and (c) -2- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502generating a second mass spectrum using the analytical method from protein cargo of exosomes isolated from the subject's second biological fluid sample after administering the anti-cancer therapy. In certain embodiments, the method further comprises, or consists essentially of, or consists of comparing the first mass spectrum and the second mass spectrum to determine whether one or more cancer-associated exosomal protein peaks are reduced or absent in the second mass spectrum relative to the first mass spectrum, optionally wherein the comparison is used to treat or manage the subject. In some embodiments, the biological fluid sample has a volume from about 25 pl to about 200 pl, or from about 25 pl to about 150 pl, or about 25 pl to about 100 pl, or from about 30 pl to about 75 pl, or from about 35 pl to about 50 pl, to about 40 pl. Alternatively, the fluid sample has a volume from about 400pl to about 2.5mL, or from about 450 pl to about 2.5mL, or from about 500 pl to about 2.0mL, or from about 150 pl, to about 250pl, ImL to about 2mL, or about 500 pl to about 2mL, or about 200 pl. In one aspect, the biological sample is plasma and the fluid volume is from about 150 pl to about 250pl. In another aspect, the fluid sample is pancreatic cyst fluid and the sample volume is from about ImL to about 2mL, or from about 500 pl to about 2mL.

[0009] In some embodiments, cancer is a solid tumor and the anti-cancer therapy comprises, or consists essentially of, or consists of tumor resection. In some embodiments, isolating exosomes comprises, or consists essentially of, or consists of immunoaffinity capture using one or more exosome markers selected from Flotillin, TSG101, CD9, CD63, and CD81.

[0010] In another aspect, the disclosure provides a method of detecting cancer recurrence or minimal residual disease in a subject after anti-cancer therapy, the method comprising, or consisting essentially of, or consisting of: generating a post-treatment mass spectrum using an analytical method from protein cargo of exosomes isolated from a biological fluid sample of the subject; comparing the post-treatment mass spectrum to a reference spectrum, wherein the reference spectrum comprises, or consists essentially of, or consists of one or more cancer-associated exosomal protein peaks; and determining that cancer recurrence or minimal residual disease is present when the one or more cancer-associated exosomal protein peaks are present in the post-treatment mass spectrum. In certain embodiments, the reference spectrum is a pretreatment mass spectrum generated using the analytical method from protein cargo of exosomes isolated from the subject's first biological fluid sample and the post-treatment spectrum is a post--3- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502treatment mass spectrum generated using the analytical method from protein cargo of exosomes isolated from the subject's second biological fluid sample after administering the anti-cancer therapy. In some embodiments, the cancer is selected from ileocecal junction carcinoma, thyroid cancer, melanoma, rectal cancer, ovarian cancer, pancreatic cancer, breast cancer, lung cancer, prostate cancer, renal cancer, colon cancer, follicular thyroid carcinoma, medullary thyroid cancer, angiosarcoma of the thyroid, papillary thyroid carcinoma (PTC), leukemia, lymphoma, or multiple myeloma. In some embodiments, the cancer is PTC and the one or more cancer-associated exosomal protein peaks comprise, or consist essentially of, or consist of peaks at about 13.9 kDa, about 15.1 kDa, and about 15.9 kDa.

[0011] In a further aspect, the disclosure provides a method of preparing a personalized cancer spectrum for a subject, the method comprising, or consisting essentially of, or consisting of: (a) obtaining biological fluid samples from (i) a subject or subjects having the cancer and (ii) control subjects not having the cancer and / or the subject or subjects after anti-cancer therapy; (b) isolating exosomes from the biological fluid samples; (c) generating mass spectra using an analytical method from protein cargo of isolated exosomes; (d) identifying one or more peaks that distinguish the subject having the cancer from the control subjects and / or that decrease or disappear after anti-cancer therapy; and optionally (e) designating the one or more peaks, or a peptide or protein corresponding to the one or more peaks, as a biomarker candidate for the cancer or patient. In certain embodiments, the method further comprises, or consists essentially of, or consists of sequencing a peptide corresponding to at least one of the one or more peaks. In some embodiments, isolating exosomes comprises, or consists essentially of, or consists of alternating current electrokinesis.

[0012] In some embodiments of the methods described herein, the analytical method comprises, or consists essentially of, or consists of MALDI-TOF mass spectrometry, and generating the mass spectra comprises, or consists essentially of, or consists of spotting an exosome preparation mixed with a sinapinic acid matrix on a MALDI-TOF target plate and acquiring spectra in positive linear mode. In some embodiments, the spectra are acquired over a mass range of about 4 kDa to about 300 kDa, or from about 4 kDa to about 200 kDa, or from about 4 kDa to aboutlOO kDa, or from about 4kDa to about 50kDa, or from about 4kDa to about 20kDa, or from about 4kDa to about lOkDa. In other embodiments, the analytical method is selected from-4- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESI-MS), liquid chromatography-mass spectrometry (LC-MS), tandem mass spectrometry (MS / MS), quadrupole time-of-flight mass spectrometry (Q-TOF), or Orbitrap mass spectrometry. In some embodiments, the biological fluid sample is selected from whole blood, plasma, serum, urine, pancreatic cyst fluid, cerebrospinal fluid, or saliva. In certain embodiments, the biological fluid sample is pancreatic cyst fluid and the cancer is pancreatic cancer.BRIEF DESCRIPTION OF THE FIGURES

[0013] FIGS. 1A - IB: MALDI-TOF generated spectra of preoperative (FIG. 1A) and postoperative (FIG. IB) samples from patients with thyroid cancer. The disappearance of certain peptide peaks following surgery (x-axis is the mass to charge ratio, or m / z, which helps identify the peptide(s) of interest), designated by the orange arrows. The difference between the mass spectra before and after surgery implies a change in the composition of the protein cargo which the patients’ exosomes are carrying in their plasma. Proteins present before surgery to remove a tumor that disappear after removal or treatment of the tumor are candidate biomarkers that may be associated with the tumor.

[0014] FIGS. 2A - 2B: MALDI-TOF generated spectra of preoperative (FIG. 2A) and postoperative (FIG. 2B) samples from patients with ileocecal junction carcinoma. The appearance of new peptide peaks (FIG. 2B) (x-axis is the mass to charge ratio, or m / z, which assists in identifying the peptide(s) of interest), designated by the arrows. Different types of cancer exhibit distinct characteristic protein spectra (FIG. 5A). This supports the hypothesis that different cancer types produce distinct spectra, as further illustrated in FIGs. 5A-5F and methods have the ability to distinguish patients by cancer type by analyzing the cargo of exosomes found in plasma and other biological fluid.

[0015] FIGS. 3A-3C: Images showing the use of NTA to validate particles extracted from plasma are of the expected size range for exosomes (100-150nm). (FIG. 3A) is an image of nanoparticles. (FIG. 3B) shows the size distribution of the particles. (FIG. 3C) shows a peak analysis of the EVs.

[0016] FIGS. 4A - 4B: Demographics of patients enrolled in study who have had plasma collected and exosomes extracted. (FIG. 4A) is a table summarizing the demographics. (FIG.4B) is a graphical summary of the demographics.-5- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502

[0017] FIGS. 5A- 5F: MALDI-TOF spectra of exosomes isolated from plasma of patients with cancer are unique by cancer type. (FIG. 5A) Melanoma. (FIG. 5B) Adrenal cancer. (FIG. 5C) GYN Cancer (Ovarian (FIG. 5D) GI Cancer (Rectal). (FIG. 5E) Thyroid (Papillary) (FIG. 5F) Pancreatic adenocarcinoma. A protein spectrum of a patient with a cancer of unknown primary can be matched to spectra of a known cancer type to identify the site of origin.

[0018] FIGS. 6A- 6C: Distinct MALDI-TOF spectra depending on patient status (i.e., before (top line) vs. after surgery (bottom line)) (FIG. 6A) Patients with papillary thyroid cancer who had tumor resected (top and bottom panels). Peaks at approximately 14, 15, and 16 kDa disappear upon successful removal of thyroid tumor. (FIG. 6B) Patient with papillary thyroid cancer and a secondary metastasis to cervical lymph nodes. Peaks at the 14-16 kDa range which disappeared in patients without secondary metastases are persistent despite having surgery for thyroid tumor resection. (FIG. 6C) Persistent peaks may be a harbinger of persistent or recurrent tumor. Patients with the same cancer types have distinct spectrum depending on the subtype of cancer. For example, patients with minimally invasive follicular thyroid cancer (shown below) have different mass spectra even from those with papillary thyroid cancers (see FIG. 6A)

[0019] FIGS. 7A - 7D: Preoperative spectra: from papillary thyroid carcinoma (FIG. 7A), follicular thyroid carcinoma (FIG. 7B), medullary thyroid cancer (FIG. 7C) and a rare angiosarcoma of the thyroid gland (FIG. 7D). Only PTC has the candidate triplet (below bar in graph).

[0020] FIGS. 8A - 8B: Spectrum of a patient’s sample: prior to thyroidectomy for PTC (FIG.8A) and after surgery (FIG. 8B) where the preop triplet disappears

[0021] FIGS. 9A- 9C: Patient spectra: from preoperative sample (FIG. 9A), initial post operative plasma sample (FIG. 9B), and after lateral dissection (FIG. 9C).

[0022] FIGS. 10A-10B show representative MALDI-TOF mass spectra comparing plasma (FIG. 10A) and pancreatic cyst fluid (FIG. 10B) obtained from the same patient. The paired analysis demonstrates distinct spectral profiles between the two biological fluid types, with the pancreatic cyst fluid exhibiting characteristic peaks in the 4-10 kDa range that differ from the plasma signature. This comparison supports the personalized cancer spectrum approach wherein-6- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502a patient's own samples from different biological fluids or time points are compared to establish individualized baselines and detect cancer-associated changes.

[0023] FIGS. 11A-11D show MALDI-TOF mass spectra of pancreatic cyst fluid samples demonstrating characteristic spectral features associated with pancreatic cyst analysis. FIG. 11A shows plasma and FIG. 11B shows a representative spectrum with peaks at approximately 4.5, 5.4, 6.3, 7.0, 8.2, 8.7, 9.2, and 9.9 kDa. FIGS. 11C and 11D show spectral comparison data supporting the use of pancreatic cyst fluid analysis for distinguishing benign pancreatic cysts from those with malignant potential, thereby informing clinical decisions regarding surgical intervention.DETAILED DESCRIPTION OF THE DISCLOSURE

[0024] Definitions

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are presented in the 5' to 3' direction.Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, particular, non-limiting exemplary methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. In some respects, the reference to a technical publication is identified by an author name and date, or an Arabic number. The full bibliographic citation for these technical publications is found immediately preceding the claims. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

[0026] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell culture, tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Green and Sambrook eds. (2012) Molecular Cloning: A Laboratory Manual, 4thedition; the series Ausubel et al. eds. (2015) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (2015) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical -7- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502Approach; McPherson et al. (2006) PCR: The Basics (Garland Science); Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Greenfield ed. (2014) Antibodies, A Laboratory Manual; Freshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 6thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Herdewijn ed. (2005) Oligonucleotide Synthesis: Methods and Applications; Hames and Higgins eds. (1984) Transcription and Translation; Buzdin and Lukyanov ed. (2007) Nucleic Acids Hybridization: Modem Applications; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi ed. (2007) In Vitro Transcription and Translation Protocols, 2ndedition; Guisan ed. (2006) Immobilization of Enzymes and Cells; Perbal (1988) A Practical Guide to Molecular Cloning, 2ndedition; Miller and Calos eds, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Lundblad and Macdonald eds. (2010) Handbook of Biochemistry and Molecular Biology, 4thedition; and Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology, 5thedition.

[0027] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1.0 or 0.1, as appropriate or alternatively by a variation of + / - 15%, or alternatively 10% or alternatively 5% or alternatively 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0028] As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a polypeptide” includes a plurality of polypeptides, including mixtures thereof.

[0029] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the intended use. Thus, a composition consisting -8- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0030] As used herein, the term “optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0031] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0032] As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The term “about” when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by (+) or (-) 15%, 10%, 5%, 3%, 2%, or 1 %.

[0033] The term “substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0034] The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.-9- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502

[0035] The terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment disclosed herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0036] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. The term “isolated” or “recombinant” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule as well as polypeptides. The term “isolated or recombinant nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polynucleotides, polypeptides and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated or recombinant” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in -10- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502nature. For example, an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype. An isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart.

[0037] It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide or antibody, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof’ is intended to be synonymous with “equivalent thereof’ when referring to a reference protein, antibody, fragment, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. In one aspect, an equivalent polynucleotide is one that hybridizes under stringent conditions to the polynucleotide or complement of the polynucleotide as described herein for use in the described methods. In another aspect, an equivalent antibody or antigen binding polypeptide intends one that binds with at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% affinity or higher affinity to a reference antibody or antigen binding fragment. In another aspect, the equivalent thereof competes with the binding of the antibody or antigen binding fragment to its antigen tinder a competitive ELISA assay. In another aspect, an equivalent intends at least about 80% homology or identity and alternatively, at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively 98% percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.

[0038] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those-11- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In certain embodiments, default parameters are used for alignment. A non-limiting exemplary alignment program is BLAST, using default parameters. In particular, exemplary programs include BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity were determined by incorporating them into clustalW (available at the web address: align.genome.jp, last accessed on Mar. 7, 2011.

[0039] As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.

[0040] The term “higher” intends a value greater than a control or reference value. In one aspect, the reference value of the expression level of the protein in a patient sample or population of patients not suffering from the disease or condition, or a measured value from the same patient taken at a different time point.

[0041] The term “lower” intends a value less than a control or reference value. In one aspect, the reference value of the expression level of the protein in a patient sample or population of patients not suffering from the disease or condition, or a measured value from the same patient taken at a different time point.

[0042] As used herein, the term “subject” includes a human patient and, in some embodiments, a mammal under veterinary care. In preferred embodiments, the subject is a human suspected of having cancer, diagnosed with cancer, undergoing cancer therapy, or previously treated for cancer.

[0043] As used herein, the term "cancer" refers to any malignant neoplasm, including solid tumors and hematologic cancers. Solid tumors include, without limitation, carcinomas, sarcomas, and melanomas arising in any tissue or organ, including but not limited to thyroid cancer (including papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid cancer,-12- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502and anaplastic thyroid cancer), endometrial cancer, pancreatic cancer (including pancreatic adenocarcinoma), ovarian cancer, rectal cancer, colon cancer, breast cancer, lung cancer, prostate cancer, renal cancer, hepatocellular carcinoma, melanoma, angiosarcoma, ileocecal junction carcinoma, and cancer of unknown primary. Hematologic cancers include, without limitation, leukemias (including acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, and chronic myeloid leukemia), lymphomas (including Hodgkin lymphoma and non-Hodgkin lymphoma), and multiple myeloma. The term "cancer" as used herein encompasses primary tumors, metastatic disease, cancer of unknown primary, and minimal residual disease.

[0044] “ Solid cancer” or “solid tumor” intends an abnormal mass of tissue that does not contain cysts or liquid areas, including but not limited to carcinomas, sarcomas, and adenocarcinomas arising in organs such as the pancreas, breast, lung, prostate, colon, ovary, stomach, liver, kidney, and skin.

[0045] As used herein, the term “blood sample” refers to whole blood or plasma obtained from a subject by any suitable collection technique (e.g., venipuncture, fingerstick, capillary collection, or collection from an indwelling line). A blood sample includes anti coagulated blood (e.g., EDTA, citrate, heparin) unless the context indicates otherwise.

[0046] As used herein, the term “plasma” refers to the cell-free or cell-reduced liquid component of blood obtained after fractionation or separation of cellular components from whole blood, including by centrifugation, filtration, sedimentation, or combinations thereof.

[0047] As used herein, the term "biological fluid" (or "biological fluid sample") refers to any fluid obtained from or produced by a subject that may contain exosomes or other extracellular vesicles. Biological fluids include, without limitation, whole blood, plasma, serum, urine, pancreatic cyst fluid, cerebrospinal fluid, saliva, pleural fluid, peritoneal fluid (ascites), synovial fluid, amniotic fluid, seminal fluid, and breast milk. In preferred embodiments, the biological fluid is blood, plasma, serum, or pancreatic cyst fluid. A biological fluid sample may be processed (e.g., fractionated, centrifuged, filtered, or diluted) prior to exosome isolation.

[0048] As used herein, the term “exosome” refers to a membrane-bound extracellular vesicle of endosomal origin. Exosomes typically have a diameter of about 30 to about 150 nm and can contain protein, peptide, lipid, DNA, and / or RNA cargo. Unless otherwise indicated, the term -13- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502“exosomes” as used in this disclosure excludes larger microvesicles and apoptotic bodies.Exosomes may be identified, enriched, or captured using exosome-associated markers, including but not limited to Flotillin, TSG-101, CD9, CD63, and CD81.

[0049] As used herein, the term “isolating” (or “isolation”) with respect to exosomes refers to any process that enriches, separates, concentrates, captures, or purifies exosomes from a biological sample (e.g., plasma) relative to at least one non-exosomal component. Isolation may be partial or substantially complete and includes, without limitation, immunoaffmity capture, alternating current electrokinesis, precipitation, ultracentrifugation, size-exclusion chromatography, filtration, microfluidic separation, or combinations thereof.

[0050] As used herein, the term “immunoaffmity capture” refers to isolating exosomes by binding exosomes to one or more affinity reagents (e.g., antibodies or antigen-binding fragments) that specifically bind an exosome-associated surface marker, including but not limited to Flotillin 1, TSG101, CD9, CD63, and / or CD81, optionally immobilized on a solid support (e.g., beads, columns, plates, membranes, or microfluidic surfaces).

[0051] As used herein, “Flotillin” (including Flotillin- 1 (FLOT1) and / or Flotillin-2 (FLOT2)) refers to membrane-associated proteins enriched in lipid rafts and commonly detected in extracellular vesicles, including exosomes. Flotillin proteins may be present on or associated with exosomal membranes and are used in the art as exosome-associated markers for identification, enrichment, and / or capture of exosomes, including by immunoaffmity methods using anti-Flotillin antibodies or antigen-binding fragments.

[0052] As used herein, “TSG101” refers to tumor susceptibility gene 101 protein, a component of the ESCRT (endosomal sorting complexes required for transport) machinery involved in multivesicular body formation and exosome biogenesis. TSG101 is commonly enriched in exosomes and is used in the art as an exosome-associated marker for identification, enrichment, and / or capture of exosomes, including by immunoaffmity methods using anti-TSGlOl antibodies or antigen-binding fragments.

[0053] As used herein, “CD9” refers to cluster of differentiation 9, a tetraspanin (also known as TSPAN29) that is enriched on extracellular vesicles, including exosomes. CD9 is a commonly used exosome-associated surface marker for identifying, enriching, and / or capturing exosomes,-14- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502for example by immunoaffinity binding using an anti-CD9 antibody or antigen-binding fragment.

[0054] As used herein, “CD63” refers to cluster of differentiation 63, a tetraspanin (also known as LAMP-3 or TSPAN30) that is enriched on extracellular vesicles, including exosomes. CD63 is a commonly used exosome-associated surface marker for identifying, enriching, and / or capturing exosomes, for example by immunoaffinity binding using an anti-CD63 antibody or antigen-binding fragment.

[0055] As used herein, “CD81” refers to cluster of differentiation 81, a tetraspanin (also known as TAPA-1 or TSPAN28) that is enriched on extracellular vesicles, including exosomes. CD81 is a commonly used exosome-associated surface marker for identifying, enriching, and / or capturing exosomes, for example by immunoaffinity binding using an anti-CD81 antibody or antigenbinding fragment.

[0056] As used herein, the term “alternating current electrokinesis” (also referred to as AC electrokinetics) refers to isolating or enriching exosomes using an applied alternating electric field that induces motion of particles and / or fluids (including, for example, dielectrophoresis and / or electrothermal flow) to concentrate or separate exosomes from other components of a biological sample.

[0057] As used herein, the term “protein cargo” (or “exosomal protein cargo”) refers to proteins and / or peptides present in, on, or associated with isolated exosomes, including intact proteins, fragments, and post-translationally modified forms thereof.

[0058] As used herein, the term “MALDI-TOF” refers to matrix-assisted laser desorption / ionization time-of-flight mass spectrometry and includes instruments and methods capable of generating a mass spectrum from analytes, including proteins and peptides, using a matrix material. In some embodiments, MALDI-TOF analysis is performed in positive linear mode and / or over a mass range of about 4kDA to about 25kDa, although other modes and mass ranges are encompassed.

[0059] As used herein, the term "analytical method" refers to any technique capable of generating a mass spectrum or proteomic profile from protein cargo of exosomes. Analytical methods include, without limitation, matrix-assisted laser desorption / ionization time-of-flight-15- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502mass spectrometry (MALDI-TOF), electrospray ionization mass spectrometry (ESI-MS), liquid chromatography-mass spectrometry (LC-MS), tandem mass spectrometry (MS / MS), quadrupole time-of-flight mass spectrometry (Q-TOF), ion trap mass spectrometry, Orbitrap mass spectrometry, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICRMS), and surface-enhanced laser desorption / ionization mass spectrometry (SELDI). In preferred embodiments, the analytical method is MALDI-TOF mass spectrometry.

[0060] As used herein, the term “matrix” refers to a material combined with an analyte to facilitate desorption / ionization in MALDI mass spectrometry. In some embodiments, the matrix comprises sinapinic acid, including saturated sinapinic acid preparations, optionally in a solvent comprising acetonitrile and water and optionally further comprising an acid (e.g., trifluoroacetic acid).

[0061] As used herein, the term “mass spectrum” refers to data output from a mass spectrometer, including MALDI-TOF, representing detected ions as a function of mass-to-charge ratio (m / z) and / or an apparent molecular mass (e.g., expressed in kDa). A mass spectrum may be raw or processed (e.g., baseline corrected, smoothed, normalized, and / or calibrated).

[0062] As used herein, the term “peak” refers to a feature in a mass spectrum corresponding to one or more detected ions, typically characterized by an m / z (or apparent molecular mass) and an intensity. A peak can be defined by its apex, centroid, area, height, full width at half maximum, and / or a tolerance window around the peak location. The term “peak” includes isotopic clusters and / or adduct-related peaks unless the context indicates otherwise.

[0063] In one non-limiting example, a peak is evaluated after external calibration, baseline subtraction, smoothing, peak picking, and total ion current (TIC) normalization, using a tolerance window of ±0.2 kDa around the target peak position; the peak is deemed “absent” if no signal within the window meets a minimum signal-to-noise ratio (S / N) of 5 (i.e., S / N < 5), “reduced” if the normalized peak height (or integrated peak area) is decreased by at least 30% relative to the reference spectrum, and “elevated” if the normalized peak height (or integrated peak area) is increased by at least 30% relative to the reference spectrum.

[0064] As used herein, the term “cancer-associated exosomal protein peak” refers to a peak (or set of peaks) in a mass spectrum generated from exosomal protein cargo using an analytical method that is associated with presence of a cancer, a cancer type, tumor burden, treatment -16- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502response, minimal residual disease, and / or recurrence. In some embodiments, a cancer-associated exosomal protein peak is a peak that is (i) present in a pre-treatment spectrum and reduced or absent following successful therapy, and / or (ii) enriched or present in subjects having the cancer as compared to control subjects without the cancer. Association may be determined statistically and / or by predetermined rules or thresholds.

[0065] As used herein, the term “comparing” refers to any qualitative or quantitative evaluation of two or more spectra, including determining the presence or absence of a peak, a change in peak intensity, a change in peak area, a change in peak ratios, similarity scoring, correlation, classification by a rules-based algorithm, or classification using a machine-learning model. Comparison may include use of calibration, normalization, alignment, and / or peak picking.

[0066] As used herein, the term “reference spectrum” refers to a spectrum (or spectral representation) used for comparison to a subject’s spectrum. A reference spectrum may be generated from the same subject at a different time point (e.g., pre-treatment), or from one or more other subjects (e.g., a cohort with a known cancer type, benign tumors or healthy controls), or from a database of spectra. A reference spectrum may be a single spectrum, an average / consensus spectrum, a set of peaks, or a model derived from spectra.

[0067] As used herein, the term “treating or managing” cancer includes administering, recommending, selecting, initiating, continuing, modifying, or discontinuing an anti-cancer therapy based on information obtained by the methods described herein, including information indicating treatment response, lack of response, minimal residual disease, or recurrence.

[0068] As used herein, the term "personalized cancer spectrum" refers to a mass spectrum, or a set of spectral features derived therefrom, that is generated from the protein cargo of exosomes isolated from a biological fluid, e.g., blood, of an individual subject and that characterizes the subject's cancer-associated exosomal protein profile at a given point in time. A personalized cancer spectrum may be used as a baseline for longitudinal monitoring, for comparison with post-treatment spectra, or for identifying cancer-associated exosomal protein peaks unique to the subject. The term encompasses spectra associated with any cancer type, including solid tumors, hematologic cancers, and cancers of unknown or undefined primary origin. A personalized cancer spectrum is not limited to a single acquisition and may comprise or be derived from one or more replicate spectra, averaged spectra, or processed spectral data.-17- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502

[0069] As used herein, the term “anti-cancer therapy” includes surgical resection (tumor resection), radiation therapy, chemotherapy, targeted therapy, hormonal therapy, immunotherapy, ablation, and combinations thereof.

[0070] As used herein, the term “treatment response” refers to a reduction in tumor burden and / or reduction in cancer activity following therapy. In some embodiments, treatment response is indicated by reduction or disappearance of one or more cancer-associated exosomal protein peaks in a post-treatment spectrum relative to a pre-treatment spectrum.

[0071] As used herein, the term “minimal residual disease” (MRD) refers to persistence of cancer cells and / or tumor-derived material in a subject after therapy at a level not readily detectable by standard imaging and / or clinical examination. In some embodiments, MRD is indicated by persistence of one or more cancer-associated exosomal protein peaks after therapy.

[0072] As used herein, the term “recurrence” refers to return or reappearance of cancer following an interval of response or remission. In some embodiments, recurrence is indicated by persistence and / or reappearance of one or more cancer-associated exosomal protein peaks in a spectrum obtained after completion of therapy.

[0073] As used herein, the term “about” when used with respect to a peak location (e.g., “about 13.9 kDa”) includes measurement variability inherent in sample preparation, calibration, and instrument resolution, and in some embodiments encompasses a tolerance of ±0.1 kDa, ±0.2 kDa, ±0.5 kDa, or another predefined tolerance suitable for MALDI-TOF analysis in the disclosed mass range.

[0074] A “composition” is intended to mean a combination of polynucleotides, active agent or another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.

[0075] A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0076] “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum -18- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, poly acrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, microspheres, microparticles, or nanoparticles (comprising e.g., biodegradable polymers such as Poly(Lactic Acid-co-Glycolic Acid)), and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.Modes For Carrying Out the Disclosure

[0077] Cancer cells communicate with each other through various means to accomplish their goals of rapid growth, immune evasion (or treatment resistance), and invasion of distant tissue sites, or metastasis. One of these means of intercellular communication is through the exchange of extracellular vesicles (EV’s) which are sent out into circulation through body fluids where they are transported to the recipient cells. One type of EV’s are membrane-bound, nano-sized biovesicles called exosomes which carry RNA, DNA, or proteins through the body’s fluids, including blood, urine, saliva, and cerebral spinal fluid.

[0078] Methods of Treating or Managing CancerIn one aspect, the disclosure provides methods of treating or managing cancer in a subject, the methods comprising, or consisting essentially of, or consisting of: (a) generating a first mass spectrum using an analytical method from protein cargo of exosomes isolated from a subject's first biological fluid sample; (b) administering an anti-cancer therapy to the subject; and (c) generating a second mass spectrum using the analytical method from protein cargo of exosomes isolated from the subject's second biological fluid sample after administering the anti-cancer therapy. In one aspect, the methods further include comparing the first mass spectrum and the second mass spectrum to determine whether one or more cancer-associated exosomal protein peaks are reduced or absent in the second mass spectrum relative to the first mass spectrum, -19- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502optionally wherein the comparison is used to treat or manage the subject. The biological fluid sample can have a volume from about 25 pl to about 200 pl, or from about 25 pl to about 150 pl, or about 25 pl to about 100 pl, or from about 30 pl to about 75 pl, or from about 35 pl to about 50 pl, to about 40 pl. Alternatively, the fluid sample has a volume from about 400pl to about 2.5mL, or from about 450 pl to about 2.5mL, or from about 500 pl to about 2.0mL, or from about 150 pl, to about 250pl, or from aboutlmL to about 2mL, or from about 500 pl to about 2mL, or about 200 pl. In one aspect, the biological sample is plasma and the volume is from about 150 pl to about 250pl. In another aspect, the fluid sample is pancreatic cyst fluid and the sample volume is from about ImL to about 2mL, or about 500 pl to about 2mL.

[0079] Where the cancer is a solid tumor, the anti-cancer therapy comprises, or consists essentially of, or consists of tumor resection.

[0080] In one embodiment, the disclosed methods provide that isolating exosomes comprises, or consists essentially of, or consists of immunoaffinity capture using one or more exosome markers selected from Flotillin, TSG101, CD9, CD63, and CD81. The method can further include selecting, continuing, discontinuing, or changing the anticancer therapy based on the comparing of the first and second mass spectra.

[0081] For the disclosed methods the cancer can be selected from ileocecal junction carcinoma, thyroid cancer, melanoma, rectal cancer, ovarian cancer, pancreatic cancer, breast cancer, lung cancer, prostate cancer, renal cancer, colon cancer, follicular thyroid carcinoma, medullary thyroid cancer, angiosarcoma of the thyroid, papillary thyroid carcinoma (PTC), leukemia, lymphoma, or multiple myeloma. The cancer can also be a hematologic cancer selected from leukemia, lymphoma, or multiple myeloma.Specifically, the cancer can be PTC, and the one or more cancer-associated exosomal protein peaks comprise, or consist essentially of, or consist of peaks at about 13.9 kDa, about 15.1 kDa, and about 15.9 kDa.

[0082] Alternatively, isolating exosomes comprises, or consists essentially of, or consists of alternating current electrokinesis. The analytical method comprises, or consists essentially of, or consists of MALDI-TOF mass spectrometry, and generating the first mass spectrum and generating the second mass spectrum comprises, or consists essentially of, or consists of spotting an exosome preparation mixed with a sinapinic acid matrix on a -20- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502MALDI-TOF target plate and acquiring spectra in positive linear mode. The spectra can be acquired over a mass range of about 4 kDa to about 300 kDa, or from about 4 kDa to about 200 kDa, or from about 4 kDa to about 100 kDa, or from about 4kDa to about 50kDa, or from about 4kDa to about 20kDa, or from about 4kDa to about lOkDa. The exosome preparation can be mixed with a solvent comprising, or consisting essentially of, or consisting of acetonitrile and water and further comprising, or consisting essentially of, or consisting of trifluoroacetic acid. The spectra can be acquired using an external calibrant. Non-liming examples of a biological fluid sample includes selected from whole blood, plasma, serum, urine, pancreatic cyst fluid, cerebrospinal fluid, or saliva. The biological fluid sample can be pancreatic cyst fluid, and the cancer can be pancreatic cancer. Alternatively, the analytical method is selected from MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESI-MS), liquid chromatography-mass spectrometry (LC-MS), tandem mass spectrometry (MS / MS), quadrupole time-of-flight mass spectrometry (Q-TOF), or Orbitrap mass spectrometry.

[0083] Methods of Detecting Cancer Recurrence or Minimal Residual Disease

[0084] Further provided are methods of detecting cancer recurrence or minimal residual disease in a subject after anti-cancer therapy, the methods comprising, or consisting essentially of, or consisting of: generating a post-treatment mass spectrum using an analytical method from protein cargo of exosomes isolated from a biological fluid sample of the subject; comparing the post-treatment mass spectrum to a reference spectrum, wherein the reference spectrum comprises, or consists essentially of, or consists of one or more cancer-associated exosomal protein peaks; and determining that cancer recurrence or minimal residual disease is present when the one or more cancer-associated exosomal protein peaks are present in the post-treatment mass spectrum.

[0085] The reference spectrum can be a pre-treatment mass spectrum generated using the analytical method from protein cargo of exosomes isolated from the subject's first biological fluid sample, and the post-treatment spectrum can be a post-treatment mass spectrum generated using the analytical method from protein cargo of exosomes isolated from the subject's second biological fluid sample after administering the anti-cancer therapy. The biological fluid sample can have a volume from about 25 pl to about 200 pl,-21- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502or from about 25 pl to about 150 pl, or about 25 pl to about 100 pl, or from about 30 pl to about 75 pl, or from about 35 pl to about 50 pl, to about 40 pl. Alternatively, the fluid sample has a volume from about 400pl to about 2.5mL, or from about 450 pl to about 2.5mL, or from about 500 pl to about 2.0mL, or from about 150 pl, to about 250pl, or from aboutlmL to about 2mL, or from about 500 pl to about 2mL, or about 200 pl. In one aspect, the biological sample is plasma and the volume is from about 150 pl to about 250pl. In another aspect, the fluid sample is pancreatic cyst fluid and the sample volume is from about ImL to about 2mL, or about 500 pl to about 2mL.

[0086] The cancer can be a solid tumor, and the anti-cancer therapy comprises, or consists essentially of, or consists of tumor resection. The method includes isolating exosomes, which comprises, or consists essentially of, or consists of immunoaffinity capture using one or more exosome markers selected from Flotillin, TSG101, CD9, CD63, and CD81. The method further includes selecting, continuing, discontinuing, or changing the anticancer therapy when the one or more cancer-associated exosomal protein peaks are present in the post-treatment mass spectrum.

[0087] The cancer can be selected from ileocecal junction carcinoma, thyroid cancer, melanoma, rectal cancer, endometrial cancer, ovarian cancer, pancreatic cancer, breast cancer, lung cancer, prostate cancer, renal cancer, colon cancer, follicular thyroid carcinoma, medullary thyroid cancer, angiosarcoma of the thyroid, papillary thyroid carcinoma (PTC), leukemia, lymphoma, or multiple myeloma. In particular embodiments, the cancer is PTC and the one or more cancer-associated exosomal protein peaks comprise, or consist essentially of, or consist of peaks at about 13.9 kDa, about 15.1 kDa, and about 15.9 kDa.

[0088] Isolating exosomes can alternatively comprise, or consist essentially of, or consist of alternating current electrokinesis. The analytical method comprises, or consists essentially of, or consists of MALDI-TOF mass spectrometry, and generating the posttreatment mass spectrum and the reference spectrum comprises, or consists essentially of, or consists of spotting an exosome preparation mixed with a sinapinic acid matrix on a MALDI-TOF target plate and acquiring spectra in positive linear mode. The spectra can be acquired over a mass range of about 4 kDa to about 300 kDa, or from about 4 kDa to -22- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502about 200 kDa, or from about 4 kDa to about 100 kDa, or from about 4kDa to about 50kDa, or from about 4kDa to about 20kDa, or from about 4kDa to about lOkDa. The exosome preparation can be mixed with a solvent comprising, or consisting essentially of, or consisting of acetonitrile and water and further comprising, or consisting essentially of, or consisting of trifluoroacetic acid. The spectra can be acquired using an external calibrant.

[0089] The biological fluid sample is selected from whole blood, plasma, serum, urine, pancreatic cyst fluid, cerebrospinal fluid, or saliva. Alternatively, the analytical method is selected from MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESI-MS), liquid chromatography-mass spectrometry (LC-MS), tandem mass spectrometry (MS / MS), quadrupole time-of-flight mass spectrometry (Q-TOF), or Orbitrap mass spectrometry. The cancer can also be a hematologic cancer selected from leukemia, lymphoma, or multiple myeloma.

[0090] Methods of Preparing a Personalized Cancer Spectrum

[0091] Further provided is a method of preparing a personalized cancer spectrum for a subject, the method comprising, or consisting essentially of, or consisting of: (a) obtaining biological fluid samples from (i) a subject or subjects having the cancer and (ii) control subjects not having the cancer and / or the subject or subjects after anti-cancer therapy; (b) isolating exosomes from the biological fluid samples; (c) generating mass spectra using an analytical method from protein cargo of isolated exosomes; (d) identifying one or more peaks that distinguish the subject having the cancer from the control subjects and / or that decrease or disappear after anti-cancer therapy; and optionally (e) designating the one or more peaks, or a peptide or protein corresponding to the one or more peaks, as a biomarker candidate for the cancer or patient.

[0092] The method further comprises, or consists essentially of, or consists of sequencing a peptide corresponding to at least one of the one or more peaks. Isolating exosomes comprises, or consists essentially of, or consists of alternating current electrokinesis.

[0093] The analytical method comprises, or consists essentially of, or consists of MALDI-TOF mass spectrometry, and generating the mass spectra comprises, or consists essentially of, or consists of spotting an exosome preparation mixed with a sinapinic acid -23- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502matrix on a MALDI target plate and acquiring spectra in positive linear mode over a mass range of about 4 kDa to about 25 kDa. The biological fluid sample can be selected from whole blood, plasma, serum, urine, pancreatic cyst fluid, cerebrospinal fluid, or saliva. The analytical method can alternatively be selected from MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESI-MS), liquid chromatography-mass spectrometry (LC-MS), tandem mass spectrometry (MS / MS), quadrupole time-of-flight mass spectrometry (Q-TOF), or Orbitrap mass spectrometry.

[0094] The analytical method can be MALDI-TOF mass spectrometry and the spectra are acquired over a mass range of about 4 kDa to about 300 kDa, or from about 4 kDa to about 200 kDa, or from about 4 kDa to about 100 kDa, or from about 4kDa to about 50kDa, or from about 4kDa to about 20kDa, or from about 4kDa to about lOkDa. The biological fluid sample can comprise, or consist essentially of, or consist of blood, and the method further comprises, or consists essentially of, or consists of separating plasma from the blood prior to isolating exosomes.

[0095] General Methods

[0096] The disclosure further provides that the exosomal protein cargo can be analyzed to detect early cancer, detect cancer recurrence or progression prior to evidence of cancer on radiologic imaging, detect cancer type in a setting of unknown primary origin, or assist pathologists in classifying tissue of origin in cases where patients have cancer of unknown primary. The methods require mass spectral analysis of a biological fluid sample from the patient to identify possible markers present in exosomes from the patient for one or more of the above.

[0097] The mass spectral analysis can be performed before or after clinical intervention, such as surgery, to determine if the intervention has reduced tumor burden or the presence of cancer cells in the patient. Changes in the composition of the cargo in exosomes of the sample, wherein a change in the peaks of the analysis, indicates a change in the health of the patient, such as no minimal residual disease or a reduction in tumor burden.

[0098] Exosomes can be isolated from blood plasma using as little as a single tube of blood. Quality control of extracted exosomes can be performed using nanoparticle tracking analysis to validate particles are of the expected size range for exosomes (30- -24- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502150nm), with an average diameter of 100-150nm. An exosome preparation can be deemed to meet quality control when nanoparticle tracking analysis shows a particle population having a modal diameter of about 80 nm to about 150 nm, optionally about 90 nm to about 140 nm, and wherein the majority of counted particles (e.g., at least about 70%, 80%, or 90%) fall within about 30 nm to about 200 nm.

[0099] The MALDI-TOF analysis can comprise, or consist essentially of, or consist of combining samples in equal volumes (IpL) with a 30:70 acetonitrile:MilliQ H2O with 0.1% trifluoroacetic acid, saturated with 30mg / mL sinapinic acid matrix and allowing cocrystallization for 5-10 minutes. Approximately 1.5pL of the sample and matrix mixture can be spotted on a ground steel MALDI target plate and allowed to air dry. Protein Standard I can be used as an external calibrant, and a MicroFlex LRF MALDI-TOF mass spectrometer can be used to acquire mass spectra in positive linear mode with a mass range of 4 to 25 kDa using a laser power of 70%. About 300 laser shots can be accumulated for each sample, with a frequency of 60 shot increments.

[0100] Papillary thyroid carcinoma exhibits a recurrent triplet of peptides at approximately 13.9, 15.1, and 15.9 kDa, and thus peptides in the 13.9, 15.1, and 15.9 kDa triplet are potential biomarkers for papillary thyroid carcinoma. After surgical removal of PTC by partial or total thyroidectomy such that the patient should no longer have cancer, the characteristic peptide triplet spectra observed prior to operation disappears after surgery to remove the cancer. These peptides are candidates for protein sequencing and potential biomarker development.

[0101] Pancreatic cyst fluid represents a preferred embodiment of the biological fluid for use in the disclosed methods, particularly for applications involving early detection of pancreatic cancer, risk stratification of pancreatic cysts, and personalized monitoring of patients with known pancreatic lesions. Pancreatic cyst fluid can exhibit distinct spectral profiles compared to plasma from the same patient, with characteristic peaks observed in the 4-10 kDa range, including peaks at approximately 4.5, 5.4, 6.3, 7.0, 8.2, 8.7, 9.2, and 9.9 kDa. The paired analysis of pancreatic cyst fluid and plasma from the same patient exemplifies the personalized cancer spectrum approach disclosed herein, wherein by establishing an individualized baseline spectrum from one biological fluid, changes -25- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502detected in another biological fluid or in subsequent samples over time can be identified with greater sensitivity and specificity than comparison to population-based reference spectra alone.

[0102] The following examples are provided to illustrate but not limit the scope of this disclosure.

[0103] Experimental

[0104] Applicant provides herein methods that use analytical methods including, but not limited to, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze exosomes isolated from biological fluid samples of patients (such as plasma, serum, urine, pancreatic cyst fluid, cerebrospinal fluid, or saliva) to identify personalized protein “signatures” associated with specific cancer types, including solid tumors and hematologic cancers, to use as biomarkers by analyzing exosomes from patient biological fluid samples prior to, during, and after cancer therapy.

[0105] In one aspect, Applicant compared pre-surgery samples to patient-matched postsurgery samples to identify protein increases or decreases, which Applicant found indicates cancer recurrence or growth sooner than traditional methods. In another aspect, this disclosure provides methods that can:a) detect early cancer (e.g. PSA for prostate cancer).b) detect cancer recurrence or progression prior to evidence of cancer on radiologic imaging.c) detect cancer type in a setting of unknown primary origin.d) assist pathologists in classifying tissue of origin in cases where patients have cancer of unknown primary.

[0106] The methods require mass spectral analysis of a biological fluid sample from the patient to identify possible markers present in exosomes from the patient for one or more of the above. In one aspect, the mass spectral analysis is performed before or after clinical intervention, such as surgery, to determine if the intervention has reduced tumor burden or the presence of cancer cells in the patient. For example, changes in the composition of the-26- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502cargo in exosomes of the sample, wherein a change in the peaks of the analysis indicates a change in the health of the patient, e.g., no minimal residual disease or a reduction in tumor burden.

[0107] Briefly, the process is as follows:

[0108] Isolation of exosomes from blood plasma (as little as a single tube). Patients with cancer had blood specimen collected before and after their tumor resection surgeries. Blood specimens were fractionated, and plasma was stored at -80°C until further processing. Exosomes were isolated from plasma using either alternating current electrokinesis or magnetic immunoaffinity assays specific for exosomal markers (e.g., CD9, CD63, CD81).

[0109] Quality control of extracted exosomes. Particles were validated using the Particle Metrix ZetaView QU ATT tracking microscope for Nanoparticle Tracking Analysis.Particles were analyzed using real time visualization of Brownian motion and electrophoretic mobility for measuring size, concentration, and Zeta potential of all biological particles. Exosomes range in size from 30-150 nm in diameter and extracted particles were confirmed to be in this range, with an average diameter of 100-150nm.

[0110] Quality control (QC) and size acceptance criteria. Isolated extracellular vesicles were confirmed to be consistent with exosomes by nanoparticle tracking analysis (NTA). For purposes of this disclosure, exosomes are vesicles typically having a diameter of about 30 nm to about 150 nm. In certain embodiments, an exosome preparation is deemed to meet QC when NTA shows a particle population having a modal diameter of about 80 nm to about 150 nm (optionally about 90 nm to about 140 nm), and wherein the majority of counted particles (e.g., at least about 70%, 80%, or 90%) fall within about 30 nm to about 200 nm. Preparations that do not meet the QC criteria may be re-isolated, further purified, or excluded from downstream MALDI-TOF analysis.

[0111] MALDI-TOF analysis of exosome samples. All samples were combined in equal volumes (IpL) with a 30:70 acetonitrile:MilliQ H2O with 0.1% trifluoroacetic acid, saturated with 30mg / mL sinapinic acid matrix and allowed to co-crystallize for 5-10min.1.5pL of this sample and matrix mixture was spotted on a ground steel MALDI target plate (Bruker Daltonics) and allowed to air dry. Protein Standard I (Bruker Daltonics) was -27- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502used as an external calibrant. A MicroFlex LRF MALDI-TOF mass spectrometer (Bruker Daltonics) was used to acquire mass spectra in positive linear mode with a mass range of 4 to 25 kDa using a laser power of 70%, in flexControl v3.4 (Bruker Daltonics). About 300 laser shots were accumulated for each sample, with a frequency of 60 shot increments.

[0112] The methods described herein are performed ex vivo and / or in vitro on a biological fluid sample (e.g., whole blood, plasma, serum, pancreatic cyst fluid, cerebrospinal fluid, saliva, or a fraction thereof) that has been obtained from a subject. Following collection, exosomes are isolated from the sample and the exosomal protein cargo is analyzed by an analytical method such as MALDI-TOF mass spectrometry. Accordingly, the analytical steps (including exosome isolation, sample preparation, mass spectral data acquisition, and spectral processing and comparison) are performed on the sample outside thesubject’s body, and any clinical action (e.g., administering, selecting, modifying, continuing, or discontinuing an anti-cancer therapy) may be based on the in vitro results.

[0113] The following examples are intended to illustrate the present disclosure.

[0114] Methods

[0115] Isolation of exosomes from blood plasma

[0116] Patients with cancer had blood specimen collected before and after their tumor resection surgeries. Blood specimens were fractionated, and plasma was stored at -80 until further processing, where exosomes were extracted from plasma.

[0117] Quality control of extracted exosomes

[0118] Particles were validated using the Particle Metrix ZetaView QU ATT tracking microscope for Nanoparticle Tracking Analysis to measure size, concentration, and Zeta potential of all biological particles. Exosomes range in size from 30-150nm in diameter, and extracted particles were confirmed to be in this range, with an average diameter of 100-200nm.

[0119] MALDI-TOF analysis of exosome samples

[0120] Protein Standard I (Bruker Daltonics) was used as an external calibrant. A MicroFlex LRF MALDI-TOF mass spectrometer (Bruker Daltonics) was used to acquire mass spectra in positive-28- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502linear mode with a mass range of 4 to 25 kDa using a laser power of 70%, in fl exControl v3.4 (Bruker Daltonics).

[0121] FIGs 1 - 9 summarize the results and show the sensitivity of the approach and its universal application.

[0122] As described herein as an example only, Applicant used a PCR based assay of miRNAs within exosomes to develop a signature for early pancreatic adenocarcinoma that could be foundational for an early detection screening test for cancer.18

[0123] Applicant utilized MALDI-TOF which involves vaporization of a protein sample in a matrix such as cinnamic acid that absorbs energy when pulsed with a nitrogen (usually) laser resulting in the transfer of protons to the peptides and formation of gas-phase positively charged molecular ions.25,26The peptides are separated on a high-voltage column as particles travel toward a detector.27Spectra are generated by the detector based on the frequency of individual peptides separated based on mass and charge. MALDI-TOF approaches have been effective in early detection of melanoma, an aggressive skin cancer using murine xenograft models.28Melanoma lacks clinically tractable biomarkers. In their study, Zhu and colleagues demonstrated that exosomal peptides from melanoma tumors could be detected in the plasma of host mice before tumor implants became visible28Furthermore, the process required only 40 pL blood and was completed in less than 3 hours. Follow-up experiments in 3 human patients with melanoma demonstrated the assay was clinically feasible.28Taken together, these findings suggest that the detection of exosomal peptides by MALDI-TOF has the potential to be more sensitive than assays for ctDNA while also being less expensive, more rapid, and requires less blood.

[0124] Blood samples were collected from patients undergoing surgery for a variety of cancers (FIGs. 4A and 4B). Applicant shows herein that exosomal proteins analyzed by MALDI-TOF generate spectra that are distinctly characteristic to histologic type of cancer. Even within a single site of origin such as the thyroid gland, the peptide spectra differ depending on histologic sub-types (FIG. 7). Importantly, the spectra are reproducible. The most common type of thyroid cancer, papillary thyroid carcinoma (PTC), exhibits a recurrent triplet of peptides at approximately at 13.9, 15.1 and 15.9 kDa (bracket, FIG. 7A). Thus, peptides in the 13.9, 15.1 and 15.9 kDa triplet are potential biomarkers for papillary thyroid carcinoma. This triplet has been observed in 21 / 29 (72%) of patients with papillary thyroid cancer before operation who -29- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502have been tested to date and are not observed in plasma from 33 patients with various other cancer types or benign thyroid nodules. This data suggests sensitivity is 78% and because it appears unique to this cancer, specificity could be 100%. One notable patient whose preoperative biopsy was read as PTC proved to have a benign non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). This patient did not have the candidate peptide triplet in her preoperative sample. The potential for this peptide triplet to contain a useful biomarker is illustrated by the finding that after surgical removal of PTC by partial or total thyroidectomy such that the patient should no longer have cancer, the characteristic peptide triplet spectra observed prior to operation (FIG. 8A) disappears after surgery to remove the cancer, as illustrated (FIG. 8B) These peptides are candidates for protein sequencing and potential biomarker development.

[0125] For thyroid cancer, the most widely used blood biomarker is thyroglobulin.2It is of limited utility when native thyroid remains as is commonly the case when the primary operation is a hemithyroidectomy or when patients have anti-thyroglobulin antibodies.25TG is a 660 kDa protein,29and the comparison to peptide databases supports the conclusion that these peaks do not represent thyroglobulin. This observation supports the potential utility of MALDI-TOF assay of exosomal proteins.

[0126] In another case a 30-year-old woman underwent total thyroidectomy and central neck dissection for stage I (Tib, Nla, M0) PTC pre-operative sample. In her initial postoperative plasma sample, the characteristic preoperative peptide triplet persisted (FIGs. 9A and 9B). A metastatic neck lymph node later was detected in the right jugular chain, so she underwent a lateral neck dissection after which the PTC peptide triplet disappeared (FIG. 9C). The patient is currently without evidence of residual thyroid cancer.

[0127] Pancreatic Cyst Fluid Analysis

[0128] In certain embodiments, the biological fluid comprises pancreatic cyst fluid. Pancreatic cysts are increasingly detected incidentally during abdominal imaging, and distinguishing benign cysts from those with malignant potential remains a significant clinical challenge. Current diagnostic approaches often lead to unnecessary surgical interventions for benign cysts or, conversely, missed diagnoses of early pancreatic cancer. The methods disclosed herein address-30- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502this unmet need by providing spectral analysis of exosomal proteins from pancreatic cyst fluid to characterize cyst pathology.

[0129] In one study, pancreatic cyst fluid samples were collected from six patients undergoing diagnostic or therapeutic procedures. Paired plasma samples were also obtained from each patient to enable personalized spectral comparison. Samples were processed to isolate exosomes, and MALDI-TOF mass spectrometry was performed as described herein.

[0130] As shown in FIGs. 10A-10B and 11A-11B, pancreatic cyst fluid exhibited distinct spectral profiles compared to plasma from the same patient. Characteristic peaks were observed in the 4-10 kDa range, including peaks at approximately 4.5, 5.4, 6.3, 7.0, 8.2, 8.7, 9.2, and 9.9 kDa. These spectral features differed between benign and potentially malignant cysts, supporting the utility of the disclosed methods for pancreatic cyst risk stratification.

[0131] The paired analysis of pancreatic cyst fluid and plasma from the same patient exemplifies the personalized cancer spectrum approach disclosed herein. By establishing an individualized baseline spectrum from one biological fluid (e.g., plasma), changes detected in another biological fluid (e.g., pancreatic cyst fluid) or in subsequent samples over time can be identified with greater sensitivity and specificity than comparison to population-based reference spectra alone.

[0132] Accordingly, pancreatic cyst fluid represents a preferred embodiment of the biological fluid for use in the disclosed methods, particularly for applications involving early detection of pancreatic cancer, risk stratification of pancreatic cysts, and personalized monitoring of patients with known pancreatic lesions.

[0133] Equivalents

[0134] 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 technology belongs.

[0135] The present technology illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be

[0136] Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and-31- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.

[0137] Thus, the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology. The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0138] In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0139] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0140] Other aspects are set forth within the following claims.

[0141] Clauses

[0142] Clause 1. A method of treating or managing cancer in a subject, comprising: (a) generating a first mass spectrum using an analytical method from protein cargo of exosomes isolated from a subject’s first biological fluid sample; (b) administering an anti-cancer therapy to the subject; (c) generating a second mass spectrum using the analytical method from protein cargo of exosomes isolated from the subject’s second biological fluid sample after administering the anti-cancer therapy.

[0143] Clause 2. The method of clause 1, further comprising comparing the first mass spectrum and the second mass spectrum to determine whether one or more cancer-associated exosomal protein peaks are reduced or absent in the second mass spectrum relative to the first mass spectrum, optionally wherein the comparison is used to treat or manage the subject.-32- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502

[0144] Clause 3. The method of clause 1 or 2, wherein the biological fluid sample has a volume from about 25 pl to about 200 pl, or from about 25 pl to about 150 pl, or about 25 pl to about lOOpl, or from about 30 pl to about 75 pl, or from about 35 pl to about 50 pl, to about 40pl, or from about 400pl to about 2.5mL, or from about 450 pl to about 2.5mL, or from about 500 pl to about 2.0mL, or from about 150 pl, to about 250pl, or from about ImL to about 2mL, or from about 500 pl to about 2mL, or from about 200 pl, optionally wherein when the biological sample is plasma and the volume is from about 150 pl, to about 250pl, or optionally wherein when the fluid sample is pancreatic cyst fluid and the sample volume is from about ImL to about 2mL, or from about 500 pl to about 2mL.

[0145] Clause 4. The method of any one of clauses 1-3, wherein the cancer is a solid tumor and the anti-cancer therapy comprises tumor resection.

[0146] Clause 5. The method of any one of clauses 1-4, wherein isolating exosomes comprises immunoaffinity capture using one or more exosome markers selected from Flotillin, TSG101, CD9, CD63, and CD81.

[0147] Clause 6. The method of any one of clauses 2-5, further comprising selecting, continuing, discontinuing, or changing the anti-cancer therapy based on the comparing.

[0148] Clause 7. The method of any one of clauses 1-6, wherein the cancer is selected from ileocecal junction carcinoma, thyroid cancer, melanoma, rectal cancer, endometrial cancer, ovarian cancer, pancreatic cancer, breast cancer, lung cancer, prostate cancer, renal cancer, colon cancer, follicular thyroid carcinoma, medullary thyroid cancer, angiosarcoma of the thyroid, papillary thyroid carcinoma (PTC), leukemia, lymphoma, or multiple myeloma.

[0149] Clause 8. The method of clause 7, wherein the cancer is PTC and the one or more cancer-associated exosomal protein peaks comprise peaks at about 13.9 kDa, about 15.1 kDa, and about 15.9 kDa.

[0150] Clause 9. The method of any one of clauses 2-8, wherein isolating exosomes comprises alternating current electrokinesis.

[0151] Clause 10. The method of any one of clauses 1-9, wherein the analytical method comprises MALDLTOF mass spectrometry, and generating the first mass spectrum and generating the second mass spectrum comprises spotting an exosome preparation mixed with a -33- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502sinapinic acid matrix on a MALDI-TOF target plate and acquiring spectra in positive linear mode.

[0152] Clause 11. The method of any one of clauses 1-10, wherein the spectra are acquired over a mass range of about 4 kDa to about 300 kDa, or from about 4kDa to about 200kDa, or from about 4kDa to about lOOkDa, or from about 4kDa to about 50kDa, or from about 4kDa to about 20kDa, or from about 4kDa to about lOkDa.

[0153] Clause 12. The method of clause 10, wherein the exosome preparation is mixed with a solvent comprising acetonitrile and water and further comprising trifluoroacetic acid.

[0154] Clause 13. The method of any one of clauses 1-12, wherein the spectra are acquired using an external calibrant.

[0155] Clause 14. A method of detecting cancer recurrence or minimal residual disease in a subject after anti-cancer therapy, comprising generating a post-treatment mass spectrum using an analytical method from protein cargo of exosomes isolated from a biological fluid sample of the subject, comparing the post-treatment mass spectrum to a reference spectrum, wherein the reference spectrum comprises one or more cancer-associated exosomal protein peaks, and determining that cancer recurrence or minimal residual disease is present when the one or more cancer-associated exosomal protein peaks are present in the post-treatment mass spectrum.

[0156] Clause 15. The method of clause 14, wherein the reference spectrum is a pre-treatment mass spectrum generated using the analytical method from protein cargo of exosomes isolated from the subject’s first biological fluid sample and the post-treatment spectrum is a posttreatment mass spectrum generated using the analytical method from protein cargo of exosomes isolated from the subject’s second biological fluid sample after administering the anti-cancer therapy.

[0157] Clause 16. The method of clause 14 or 15, wherein the biological fluid sample has a volume from about 400pl to about 2.5mL, or from about 450 pl to about 2.5mL, or from about 500 pl to about 2.0mL, or from about 150 pl, to about 250pl, ImL to about 2mL, or about 500 pl to about 2mL, or about 200 pl, or from about 400pl to about 2.5mL, or from about 450 pl to about 2.5mL, or from about 500 pl to about 2.0mL, or from about 150 pl, to about 250pl, or from about ImL to about 2mL, or from about 500 pl to about 2mL, or from about 200 pl,-34- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502optionally wherein when the biological sample is plasma, the fluid sample volume is from about 150 pl to about 250pl, or optionally wherein when the fluid sample is pancreatic cyst fluid, the sample volume is from about ImL to about 2mL, or from about 500 pl to about 2mL.

[0158] Clause 17. The method of any one of clauses 14-16, wherein the cancer is a solid tumor and the anti-cancer therapy comprises tumor resection.

[0159] Clause 18. The method of any one of clauses 14-17, wherein isolating exosomes comprises immunoaffmity capture using one or more exosome markers selected from Flotillin, TSG101, CD9, CD63, and CD81.

[0160] Clause 19. The method of any one of clauses 14-18, further comprising selecting, continuing, discontinuing, or changing the anti-cancer therapy when the one or more cancer-associated exosomal protein peaks are present in the post-treatment mass spectrum.

[0161] Clause 20. The method of any one of clauses 14-19, wherein the cancer is selected from ileocecal junction carcinoma, thyroid cancer, melanoma, endometrial cancer, rectal cancer, ovarian cancer, pancreatic cancer, breast cancer, lung cancer, prostate cancer, renal cancer, colon cancer, follicular thyroid carcinoma, medullary thyroid cancer, angiosarcoma of the thyroid, papillary thyroid carcinoma (PTC), leukemia, lymphoma, or multiple myeloma.

[0162] Clause 21. The method of clause 20, wherein the cancer is PTC and the one or more cancer-associated exosomal protein peaks comprise peaks at about 13.9 kDa, about 15.1 kDa, and about 15.9 kDa.

[0163] Clause 22. The method of any one of clauses 14-21, wherein isolating exosomes comprises alternating current electrokinesis.

[0164] Clause 23. The method of any one of clauses 14-22, wherein the analytical method comprises MALDI-TOF mass spectrometry, and generating the post-treatment mass spectrum and the reference spectrum comprises spotting an exosome preparation mixed with a sinapinic acid matrix on a MALDI-TOF target plate and acquiring spectra in positive linear mode.

[0165] Clause 24. The method of any one of clauses 14-23, wherein the spectra are acquired over a mass range of from about 4 kDa to about 300 kDa, or from about 4 kDa to about 200 kDa, or from about 4 kDa to aboutlOO kDa, or from about 4kDa to about 50kDa, or from about 4kDa to about 20kDa, or from about 4kDa to about lOkDa.-35- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502

[0166] Clause 25. The method of clause 24, wherein the exosome preparation is mixed with a solvent comprising acetonitrile and water and further comprising trifluoroacetic acid.

[0167] Clause 26. The method of any one of clauses 14-25, wherein the spectra are acquired using an external calibrant.

[0168] Clause 27. A method of preparing a personalized cancer spectrum for a subject, comprising: (a) obtaining biological fluid samples from (i) a subject or subjects having the cancer and (ii) control subjects not having the cancer and / or the subject or subjects after anti-cancer therapy;(b) isolating exosomes from the biological fluid samples;(c) generating mass spectra using an analytical method from protein cargo of isolated exosomes;(d) identifying one or more peaks that distinguish the subject having the cancer from the control subjects and / or that decrease or disappear after anti-cancer therapy; and optionally (e) designating the one or more peaks, or a peptide or protein corresponding to the one or more peaks, as a biomarker candidate for the cancer or patient.

[0169] Clause 28. The method of clause 27, further comprising sequencing a peptide or protein corresponding to at least one of the one or more peaks.

[0170] Clause 29. The method of clause 27 or 28, wherein isolating exosomes comprises alternating current electrokinesis.

[0171] Clause 30. The method of any one of clauses 27-29, wherein the analytical method comprises MALDI-TOF mass spectrometry, and generating the mass spectra comprises spotting an exosome preparation mixed with a sinapinic acid matrix on a MALDI target plate and acquiring spectra in positive linear mode over a mass range of about 4 kDa to about 25 kDa.

[0172] Clause 31. The method of any one of clauses 1-13, wherein the biological fluid sample is selected from whole blood, plasma, serum, urine, pancreatic cyst fluid, cerebrospinal fluid, or saliva.

[0173] Clause 32. The method of any one of clauses 1-13, wherein the analytical method is selected from MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESIMS), liquid chromatography-mass spectrometry (LC-MS), tandem mass spectrometry (MS / MS), quadrupole time-of-flight mass spectrometry (Q-TOF), or Orbitrap mass spectrometry.-36- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502

[0174] Clause 33. The method of any one of clauses 1-13, wherein the cancer is a hematologic cancer selected from leukemia, lymphoma, or multiple myeloma.

[0175] Clause 34. The method of any one of clauses 1-13, wherein the biological fluid sample is pancreatic cyst fluid and the cancer is pancreatic cancer.

[0176] Clause 35. The method of any one of clauses 14-26, wherein the biological fluid sample is selected from whole blood, plasma, serum, urine, pancreatic cyst fluid, cerebrospinal fluid, or saliva.

[0177] Clause 36. The method of any one of clauses 14-26, wherein the analytical method is selected from MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESIMS), liquid chromatography-mass spectrometry (LC-MS), tandem mass spectrometry (MS / MS), quadrupole time-of-flight mass spectrometry (Q-TOF), or Orbitrap mass spectrometry.

[0178] Clause 37. The method of any one of clauses 14-26, wherein the cancer is a hematologic cancer selected from leukemia, lymphoma, or multiple myeloma.

[0179] Clause 38. The method of any one of clauses 27-30, wherein the biological fluid sample is selected from whole blood, plasma, serum, urine, pancreatic cyst fluid, cerebrospinal fluid, or saliva.

[0180] Clause 39. The method of any one of clauses 27-30, wherein the analytical method is selected from MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESIMS), liquid chromatography-mass spectrometry (LC-MS), tandem mass spectrometry (MS / MS), quadrupole time-of-flight mass spectrometry (Q-TOF), or Orbitrap mass spectrometry.

[0181] Clause 40. The method of any one of clauses 27-30, wherein the analytical method is MALDI-TOF mass spectrometry and 4 kDa to about 300 kDa, or from about 4kDa to about 200kDa, or from about 4kDa to about lOOkDa and the spectra are acquired over a mass range of about 4 kDa to about 300 kDa, or from about 4kDa to about 200kDa, or from about 4kDa to about lOOkDa.

[0182] Clause 41. The method of any one of clauses 27-30, wherein the biological fluid sample comprises blood and the method further comprises separating plasma from the blood prior to isolating exosomes.-37- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502

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Claims

Atty. Dkt. No.: 059304-0502What is claimed is:

1. A method of treating or managing cancer in a subject, comprising:(a) generating a first mass spectrum using an analytical method from protein cargo of exosomes isolated from a subject’s first biological fluid sample; (b) administering an anti-cancer therapy to the subject; (c) generating a second mass spectrum using the analytical method from protein cargo of exosomes isolated from the subject’s second biological fluid sample after administering the anti-cancer therapy.

2. The method of claim 1, further comprising comparing the first mass spectrum and the second mass spectrum to determine whether one or more cancer-associated exosomal protein peaks are reduced or absent in the second mass spectrum relative to the first mass spectrum, optionally wherein the comparison is used to treat or manage the subject.

3. The method of claim 1 or 2, wherein the biological fluid sample has a volume from about 25 pl to about 200 pl, or from about 25 pl to about 150 pl, or about 25 pl to about lOOpl, or from about 30 pl to about 75 pl, or from about 35 pl to about 50 pl, to about 40pl, or from about 400pl to about 2.5mL, or from about 450 pl to about 2.5mL, or from about 500 pl to about 2.0mL, or from about 150 pl, to about 250pl, or from about ImL to about 2mL, or from about 500 pl to about 2mL, or from about 200 pl, optionally wherein when the biological sample is plasma and the volume is from about 150 pl, to about 250pl, or optionally wherein when the fluid sample is pancreatic cyst fluid and the sample volume is from about ImL to about 2mL, or from about 500 pl to about 2mL.

4. The method of any one of claims 1-3, wherein the cancer is a solid tumor and the anticancer therapy comprises tumor resection.

5. The method of any one of claims 1-4, wherein isolating exosomes comprises immunoaffinity capture using one or more exosome markers selected from Flotillin, TSG101, CD9, CD63, and CD81.

6. The method of any one of claims 2-5, further comprising selecting, continuing, discontinuing, or changing the anti-cancer therapy based on the comparing.

7. The method of any one of claims 1-6, wherein the cancer is selected from ileocecal junction carcinoma, thyroid cancer, melanoma, rectal cancer, endometrial cancer, ovarian cancer,-42- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502pancreatic cancer, breast cancer, lung cancer, prostate cancer, renal cancer, colon cancer, follicular thyroid carcinoma, medullary thyroid cancer, angiosarcoma of the thyroid, papillary thyroid carcinoma (PTC), leukemia, lymphoma, or multiple myeloma.

8. The method of claim 7, wherein the cancer is PTC and the one or more cancer-associated exosomal protein peaks comprise peaks at about 13.9 kDa, about 15.1 kDa, and about 15.9 kDa.

9. The method of any one of claims 2-8, wherein isolating exosomes comprises alternating current electrokinesis.

10. The method of any one of claims 1-9, wherein the analytical method comprises MALDI-TOF mass spectrometry, and generating the first mass spectrum and generating the second mass spectrum comprises spotting an exosome preparation mixed with a sinapinic acid matrix on a MALDI-TOF target plate and acquiring spectra in positive linear mode.

11. The method of any one of claims 1-10, wherein the spectra are acquired over a mass range of about 4 kDa to about 300 kDa, or from about 4kDa to about 200kDa, or from about 4kDa to about lOOkDa, or from about 4kDa to about 50kDa, or from about 4kDa to about 20kDa, or from about 4kDa to about lOkDa.

12. The method of claim 10, wherein the exosome preparation is mixed with a solvent comprising acetonitrile and water and further comprising trifluoroacetic acid.

13. The method of any one of claims 1-12, wherein the spectra are acquired using an external calibrant.

14. A method of detecting cancer recurrence or minimal residual disease in a subject after anti-cancer therapy, comprising generating a post-treatment mass spectrum using an analytical method from protein cargo of exosomes isolated from a biological fluid sample of the subject, comparing the post-treatment mass spectrum to a reference spectrum, wherein the reference spectrum comprises one or more cancer-associated exosomal protein peaks, and determining that cancer recurrence or minimal residual disease is present when the one or more cancer-associated exosomal protein peaks are present in the post-treatment mass spectrum.

15. The method of claim 14, wherein the reference spectrum is a pre-treatment mass spectrum generated using the analytical method from protein cargo of exosomes isolated from the subject’s first biological fluid sample and the post-treatment spectrum is a post-treatment -43- 4932-1015-8235.1Atty. Dkt. No.: 059304-0502mass spectrum generated using the analytical method from protein cargo of exosomes isolated from the subject’s second biological fluid sample after administering the anti-cancer therapy.

16. The method of claim 14 or 15, wherein the biological fluid sample has a volume from about 400pl to about 2.5mL, or from about 450 pl to about 2.5mL, or from about 500 pl to about 2.0mL, or from about 150 pl, to about 250pl, ImL to about 2mL, or about 500 pl to about 2mL, or about 200 pl, or from about 400pl to about 2.5mL, or from about 450 pl to about 2.5mL, or from about 500 pl to about 2.0mL, or from about 150 pl, to about 250pl, or from about ImL to about 2mL, or from about 500 pl to about 2mL, or from about 200 pl, optionally wherein when the biological sample is plasma, the fluid sample volume is from about 150 pl to about 250pl, or optionally wherein when the fluid sample is pancreatic cyst fluid, the sample volume is from about ImL to about 2mL, or from about 500 pl to about 2mL.

17. The method of any one of claims 14-16, wherein the cancer is a solid tumor and the anticancer therapy comprises tumor resection.

18. The method of any one of claims 14-17, wherein isolating exosomes comprises immunoaffinity capture using one or more exosome markers selected from Flotillin, TSG101, CD9, CD63, and CD81.

19. The method of any one of claims 14-18, further comprising selecting, continuing, discontinuing, or changing the anti-cancer therapy when the one or more cancer-associated exosomal protein peaks are present in the post-treatment mass spectrum.

20. The method of any one of claims 14-19, wherein the cancer is selected from ileocecal junction carcinoma, thyroid cancer, melanoma, endometrial cancer, rectal cancer, ovarian cancer, pancreatic cancer, breast cancer, lung cancer, prostate cancer, renal cancer, colon cancer, follicular thyroid carcinoma, medullary thyroid cancer, angiosarcoma of the thyroid, papillary thyroid carcinoma (PTC), leukemia, lymphoma, or multiple myeloma.

21. The method of claim 20, wherein the cancer is PTC and the one or more cancer-associated exosomal protein peaks comprise peaks at about 13.9 kDa, about 15.1 kDa, and about 15.9 kDa.

22. The method of any one of claims 14-21, wherein isolating exosomes comprises alternating current electrokinesis.-44- 4932-1015-8235.1Atty. Dkt. No.: 059304-050223. The method of any one of claims 14-22, wherein the analytical method comprises MALDI-TOF mass spectrometry, and generating the post-treatment mass spectrum and the reference spectrum comprises spotting an exosome preparation mixed with a sinapinic acid matrix on a MALDI-TOF target plate and acquiring spectra in positive linear mode.

24. The method of any one of claims 14-23, wherein the spectra are acquired over a mass range of from about 4 kDa to about 300 kDa, or from about 4 kDa to about 200 kDa, or from about 4 kDa to aboutlOO kDa, or from about 4kDa to about 50kDa, or from about 4kDa to about 20kDa, or from about 4kDa to about lOkDa.

25. The method of claim 24, wherein the exosome preparation is mixed with a solvent comprising acetonitrile and water and further comprising trifluoroacetic acid.

26. The method of any one of claims 14-25, wherein the spectra are acquired using an external calibrant.

27. A method of preparing a personalized cancer spectrum for a subject, comprising: (a) obtaining biological fluid samples from (i) a subject or subjects having the cancer and (ii) control subjects not having the cancer and / or the subject or subjects after anti-cancer therapy;(b) isolating exosomes from the biological fluid samples;(c) generating mass spectra using an analytical method from protein cargo of isolated exosomes;(d) identifying one or more peaks that distinguish the subject having the cancer from the control subjects and / or that decrease or disappear after anti-cancer therapy; and optionally (e) designating the one or more peaks, or a peptide or protein corresponding to the one or more peaks, as a biomarker candidate for the cancer or patient.

28. The method of claim 27, further comprising sequencing a peptide or protein corresponding to at least one of the one or more peaks.

29. The method of claim 27 or 28, wherein isolating exosomes comprises alternating current electrokinesis.

30. The method of any one of claims 27-29, wherein the analytical method comprises MALDI-TOF mass spectrometry, and generating the mass spectra comprises spotting an exosome preparation mixed with a sinapinic acid matrix on a MALDI target plate and acquiring spectra in positive linear mode over a mass range of about 4 kDa to about 25 kDa.-45- 4932-1015-8235.1Atty. Dkt. No.: 059304-050231. The method of any one of claims 1-13, wherein the biological fluid sample is selected from whole blood, plasma, serum, urine, pancreatic cyst fluid, cerebrospinal fluid, or saliva.

32. The method of any one of claims 1-13, wherein the analytical method is selected from MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESI-MS), liquid chromatography-mass spectrometry (LC-MS), tandem mass spectrometry (MS / MS), quadrupole time-of-flight mass spectrometry (Q-TOF), or Orbitrap mass spectrometry.

33. The method of any one of claims 1-13, wherein the cancer is a hematologic cancer selected from leukemia, lymphoma, or multiple myeloma.

34. The method of any one of claims 1-13, wherein the biological fluid sample is pancreatic cyst fluid and the cancer is pancreatic cancer.

35. The method of any one of claims 14-26, wherein the biological fluid sample is selected from whole blood, plasma, serum, urine, pancreatic cyst fluid, cerebrospinal fluid, or saliva.

36. The method of any one of claims 14-26, wherein the analytical method is selected from MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESI-MS), liquid chromatography -mass spectrometry (LC-MS), tandem mass spectrometry (MS / MS), quadrupole time-of-flight mass spectrometry (Q-TOF), or Orbitrap mass spectrometry.

37. The method of any one of claims 14-26, wherein the cancer is a hematologic cancer selected from leukemia, lymphoma, or multiple myeloma.

38. The method of any one of claims 27-30, wherein the biological fluid sample is selected from whole blood, plasma, serum, urine, pancreatic cyst fluid, cerebrospinal fluid, or saliva.

39. The method of any one of claims 27-30, wherein the analytical method is selected from MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESI-MS), liquid chromatography -mass spectrometry (LC-MS), tandem mass spectrometry (MS / MS), quadrupole time-of-flight mass spectrometry (Q-TOF), or Orbitrap mass spectrometry.

40. The method of any one of claims 27-30, wherein the analytical method is MALDI-TOF mass spectrometry and 4 kDa to about 300 kDa, or from about 4kDa to about 200kDa, or from about 4kDa to about lOOkDa and the spectra are acquired over a mass range of about 4 kDa to about 300 kDa, or from about 4kDa to about 200kDa, or from about 4kDa to about lOOkDa.-46- 4932-1015-8235.1Atty. Dkt. No.: 059304-050241. The method of any one of claims 27-30, wherein the biological fluid sample comprises blood and the method further comprises separating plasma from the blood prior to isolating exosomes.4932-1015-8235.1

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