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CN-122003605-A - Methods for detecting the presence of lesions in a subject based on extracellular vesicle analysis

CN122003605ACN 122003605 ACN122003605 ACN 122003605ACN-122003605-A

Abstract

A method for diagnosing a disease, in particular a hematological malignancy or a solid tumor, in a subject, the method comprising the steps of isolating (21, 61) extracellular vesicle pellets (3 a,3 b) obtained from a sample (2) of biological fluid previously taken from the subject, measuring (31, 41, 51-52) parameters representing six biomarkers from the pellets, (I) the size of the extracellular vesicles, (II) the total amount of extracellular vesicles, (III) the amount of extracellular vesicles expressing a first antigen on its own surface and (IV) the amount of extracellular vesicles expressing a first antigen and a second antigen, (V) for the average fluorescence intensity of each of the antigens, and (VI) the nucleic acid content of the extracellular vesicles, assigning a partial score to each parameter or to the six biomarkers based on the first comparison step, calculating a final score, i.e. the subject score, as a sum of partial scores, comparing the final score with at least one of the predetermined diagnoses, i.e. the second threshold value, and estimating the probability of a lesion based on the second threshold value. In the context of personalized medicine, the combination of these six biomarkers overcomes the limitations of prior art methods due to the high degree of clinical heterogeneity between subjects, particularly in terms of sensitivity to a given pathology, i.e. to a certain biomarker selected from these six biomarkers, but not other biomarkers and their related parameters, which behave differently.

Inventors

  • A. Kavanaugh
  • I. Laurenzana
  • D. Lamott
  • L. Deluca
  • S. Trino

Assignees

  • 巴西利卡塔肿瘤参考中心科学研究型诊疗机构

Dates

Publication Date
20260508
Application Date
20241008
Priority Date
20231009

Claims (20)

  1. 1. A method for detecting the presence or absence of a lesion in a subject, the method comprising the steps of: -defining at least a first reference antigen and a second reference antigen; -isolating (21, 61) extracellular vesicle pellets (3 a,3 b) obtained from a sample (2) of a biological fluid, said sample having been previously taken from said subject; -measuring (31, 41, 51-52) from the extracellular vesicle pellet (3 a,3 b) a plurality of parameters representing six biomarkers (I-VI), the biomarkers comprising: -the size (I) of the extracellular vesicles; -the total amount of extracellular vesicles (II); -expressing the first antigen on its own surface, i.e. the amount (III) of the extracellular vesicles positive for the first antigen; -an amount (IV) of the extracellular vesicles expressing both the first antigen and the second antigen; -an average fluorescence intensity (V) for each of the first antigen and the second antigen; Nucleic acid content (VI) of the extracellular vesicles, -Inputting respective predetermined threshold values for the parameter representing the biomarker; -first comparing the parameter representative of the biomarker with the respective threshold value; -assigning a partial score to each of the parameters representing biomarkers or to the six biomarkers based on the first comparing step; -calculating a final score for the subject; -a second comparison of the final score with at least one predetermined diagnostic threshold; -establishing a probabilistic diagnosis of said lesion in said subject based on said second comparison.
  2. 2. The method of claim 1, wherein the biological fluid is selected from the group consisting of blood, urine, saliva, cerebrospinal fluid, bronchoalveolar lavage fluid, amniotic fluid, semen, breast milk, sweat, tears, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, ascites fluid, and vitreous fluid.
  3. 3. The method according to claim 1, wherein the biological fluid is peripheral blood (1).
  4. 4. A method according to claim 3, wherein the extracellular vesicle pellet (3 a,3 b) is extracted from a plasma sample or a serum sample (2) obtained from the peripheral blood sample (1).
  5. 5. The method according to claim 4, wherein the extracellular vesicle pellet (3 a) is extracted from the serum sample (2 a), and the step of isolating (21) extracellular vesicle pellet (3 a) comprises the steps of: -suspending (22) said extracellular vesicle pellet in a saline solution, thereby obtaining a wet extracellular vesicle pellet (3 a).
  6. 6. The method of claim 5, further comprising the step of: -taking (30) a first portion of the wet extracellular vesicle pellet (3 a); And said step of measuring a representative parameter of said size (I) and said total amount (II) of said extracellular vesicles is performed jointly by -Performing nanoparticle tracking analysis (31) of said first portion of said wet extracellular vesicle pellet (3 a), thereby obtaining: -the size distribution curve of said extracellular vesicles, and -A first concentration value of said extracellular vesicles in said wet extracellular vesicle pellet.
  7. 7. The method of claim 5, wherein the step of isolating extracellular vesicle pellet comprises the steps of: -taking (40) a second portion of the wet extracellular vesicle pellet (3 a); and measuring said step of parameters representing: -said total amount (II) of said extracellular vesicles; -said amount (III) of said extracellular vesicles expressing said first antigen; -said amount (IV) of said extracellular vesicles expressing both said first antigen and said second antigen; -said positive fluorescence (V) against said first antigen and against said second antigen The method is carried out by the following steps: -performing flow cytometry (41) on the second fraction of the wet extracellular vesicle pellet, obtaining: -a concentration value (II) of total extracellular vesicles in the second fraction of the wet extracellular vesicle pellet; -a concentration value (III) of extracellular vesicles specific for a first antigen in the second fraction of the wet extracellular vesicle pellet; -a concentration value of extracellular vesicles specific for a second antigen in the second fraction of the wet extracellular vesicle pellet; -a concentration value (IV) of extracellular vesicles specific for both the first antigen and the second antigen in the second fraction of the wet extracellular vesicle pellet; -positive fluorescence (V) for the first antigen and for the second antigen.
  8. 8. The method according to claim 5, wherein said step of measuring a parameter representative of the nucleic acid content (VI) is a step of determining the specific microrna content (VI) and comprises the steps of: -manually extracting (51) nucleic acids from the wet extracellular vesicle pellet; -sequencing (52) the nucleic acid extracted from the wet extracellular vesicle pellet.
  9. 9. The method according to claim 4, wherein said step of measuring a parameter representative of the nucleic acid content (VI) comprises the steps of: -automatically extracting (62) nucleic acids (RNA) from the extracellular vesicle pellet by means of an automatic extractor; -performing fluorescent quantification of the micrornas (63); -reverse transcribing (64, 65) a portion of the RNA into cDNA; -determining (65) said micrornas (VI) using digital PCR techniques, in particular microdroplet digital techniques.
  10. 10. The method according to claims 8 and 9, wherein said step (51) of manually extracting said nucleic acids and said step (62) of automatically extracting nucleic acids (RNA) by means of an automatic extractor are performed on respective first (3 a) and second EV-precipitates (3 b) separated from a first (2 a) and a second (2 b) portion of said serum sample (2), respectively.
  11. 11. The method of claim 1, wherein the final score is calculated as a sum of the partial scores.
  12. 12. The method of claim 1, wherein the step of performing a second comparison of the final score is performed with respect to two diagnostic thresholds, the two diagnostic thresholds comprising: Upper diagnostic threshold, and A lower diagnostic threshold value is chosen, And the diagnosis is: -a high probability of said lesion if said final score is higher than or equal to said upper diagnostic threshold; -a moderate probability of said lesion if said final score is between said upper diagnostic threshold and said lower diagnostic threshold; -a low probability of said lesion if said final score is lower than or equal to said lower diagnostic threshold.
  13. 13. The method of claim 1, wherein in the step of defining a plurality of fractional scores, a fractional score is assigned to each of the six biomarkers (I-VI).
  14. 14. The method of claim 13, wherein for each biomarker (I-VI) of the biomarkers, the partial score is -A first value if the value of at least one of the parameters representing the biomarker: -above a threshold value of the biomarker itself in the threshold values, if the threshold value carries a > sign; -below a threshold value of the biomarker itself in the threshold values, if the threshold value is with a < sign; -a second value lower than said first value, if the value of each of said parameters representing the biomarker: -lower than or equal to a threshold value of the biomarker itself, if the threshold value carries a > sign; -higher than or equal to a threshold value of the biomarker itself, if the threshold value is with a < sign.
  15. 15. The method according to claims 12 and 14, wherein the first and second values are the same value for all the six biomarkers (I-VI), in particular the first and second values are equal to 1 and 0, respectively, and the upper and lower diagnostic threshold values are equal to 4 and 2, respectively.
  16. 16. The method of claim 14, wherein the first value and the second value are assigned independently for each of the six biomarkers (I-VI).
  17. 17. The method of claim 16, wherein the first value -The same first lower value for the following biomarkers: -said size (I) of said extracellular vesicles, and -Said amount (II) of said extracellular vesicles; -the same first intermediate value for the following biomarkers: -said amount (III) of said extracellular vesicles expressing said first antigen, and -The average fluorescence intensity (V) for each of the first antigen and the second antigen, and -The same first higher value for the following biomarkers: -said amount (IV) of said extracellular vesicles expressing both said first antigen and said second antigen, and -The nucleic acid (VI) content of the extracellular vesicles.
  18. 18. The method of claims 12 and 17, wherein the first lower value, the first intermediate value, and the first higher value are equal to 1,2, and 3, respectively, the second value is equal to 0, and the upper diagnostic threshold and the lower diagnostic threshold are equal to 6 and 3, respectively.
  19. 19. The method of claim 13, wherein The step of defining a plurality of fractional scores comprises the step of defining a plurality of fractional sub-scores, wherein each parameter representing the six biomarkers is assigned a fractional sub-score of the fractional sub-scores, and For each of the parameters, the sub-portion score is: -a third value, if the value of the parameter: -above a threshold value of the biomarker itself in the threshold values, if the threshold value carries a > sign; -below a threshold value of the biomarker itself in the threshold values, if the threshold value is with a < sign; -a fourth value lower than said third value, if the value of said parameter: -lower than or equal to a threshold value of the biomarker itself, if the threshold value carries a > sign; Higher than or equal to the threshold value of the biomarker itself, if the threshold value is given by a < sign, And, for each of the biomarkers, the partial score is calculated by summing the sub-partial scores assigned to the parameters representing the biomarker.
  20. 20. Method according to claim 19, wherein said third value and said fourth value are the same value for all said parameters, in particular said third value and said fourth value are equal to 1 and 0, respectively, and said upper diagnostic threshold and said lower diagnostic threshold are equal to 10 and 5, respectively.

Description

Methods for detecting the presence of lesions in a subject based on extracellular vesicle analysis Description Scope of the invention The present invention relates to a method for diagnosing various lesions in a subject. The method is based on analysis of Extracellular Vesicles (EVs) extracted from a sample of biological fluid previously taken from a subject. In particular, the invention relates to diagnosing hematological tumors of lymphoid and myeloid type, e.g. multiple myeloma, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma in general, etc., diagnosing solid tumors, such as nervous system tumors, head or neck tumors, gastrointestinal tumors, pancreatic tumors, liver tumors, testicular tumors, kidney tumors, lung tumors, breast tumors, cervical tumors, bladder tumors, skin tumors, sarcomas, etc., diagnosing autoimmune diseases, such as systemic lupus erythematosus, diabetes, rheumatoid arthritis, diagnosing cardiovascular diseases, such as atherosclerosis, myocardial ischemia, cardiac fibrosis, diagnosing inflammatory diseases, such as diabetes, pancreatitis, fibrosis, and diagnosing degenerative diseases, e.g. alzheimer's disease. Prior Art Diagnosis/prognosis of several diseases, particularly most tumors, has traditionally been performed by analyzing samples from bone marrow biopsies and bone marrow punctures (in the case of hematological tumors), samples from tumor biopsies (in the case of solid tumors), and combining imaging techniques. These methods have significant limitations such as high invasiveness, inapplicability in the case of inaccessible tumors, unrepeatability on the same subject, and inability to assess molecular and spatial heterogeneity of tumors. For this reason, technologies based on finding new tumor biomarkers in the blood are finding increasing use. In particular, so-called Extracellular Vesicles (EVs), as well as circulating free proteins, circulating free DNA/small RNAs and lipids, can be found in the blood along with normal blood cells. EV is a lipid bilayer particle secreted by all cells, released not only in blood, but also in other body fluids (such as urine, semen, cerebrospinal fluid and breast milk). The size of EV ranges from 30nm to 10 μm, and EV can be classified (among others) as exosomes (30 nm to 150 nm), microvesicles (100 nm to 1000 nm), etc. EV can be regarded as a circulating cell biopsy. In fact, their surface contains biomarkers, including proteins and carbohydrates, inherited from the surface of the cell from which they originate. These biomarkers enable classification and targeting of EV that are cell and tissue specific. More specifically, EV-carried inclusions include, but are not limited to, proteins, metabolites, DNA, and RNA. The content depends not only on the originating cell but also on the pathophysiological state of the donor, on the cellular conditions (such as oxidative or metabolic stress) and on the response to the treatment. EV also reflects the molecular/protein changes induced by the disease and/or the treatment received by the patient, including spatial and molecular heterogeneity of the tumor. Thus, EVs can serve as a source of biomarkers for specific conditions of the body or disease. EV-mediated signaling has been shown to play an important role in many physiological and pathological conditions, such as tumors, but also neurodegenerative, cardiovascular and autoimmune diseases, as well as metabolic disorders. Several schemes are available for isolating EVs. Most of these protocols involve the use of differential ultracentrifugation and microfluidic methods, as well as the use of antibody or polymer-based kits. All of these methods isolate specific EV populations such as exosomes or microvesicles. Different characteristics or parameters of EVs, such as their concentration, proteins, lipids, surface antigens and their genomic content (micrornas, mrnas and DNAs), have been analyzed/quantified by different methods. This allows these features or parameters to be defined as potential biomarkers for diagnosing tumors and identifying their molecular profile. In particular, laurenzana i., trino s, lamorte d, the following. De Luca L., caivano A a scheme was developed in Analysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma, International Journal of Nanomedicine 2021:16 3141-3160 wherein the following steps are performed: -obtaining a serum sample from the peripheral blood of a subject; Separating EV pellet from serum by centrifugation, for example in a bench centrifuge; -suspending the EV precipitate in a saline solution; -taking a first portion of the EV precipitate; -characterizing a first part of EV precipitation by Nanoparticle Tracking Analysis (NTA), thereby obtaining: -size distribution curve of EV, and -A first value of EV concentration in a first portion of EV precipitation; -taking a second fraction of EV precipitate; -charact