Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/5752—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the lungs
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/50—Determining the risk of developing a disease
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum

Definitions

• the present disclosure provides insights and technologies for achieving effective lung cancer screening.
• the present disclosure provides insights and technologies that are particularly useful for non-small cell lung cancer screening.
• provided technologies are effective for detection of early stage lung cancers (e.g ., in some embodiments non-small cell lung cancer).
• provided technologies are effective even when applied to populations comprising or consisting of asymptomatic individuals (e.g., due to sufficiently high sensitivity and/or low rates of false positive and/or false negative results).
• provided technologies are effective when applied to populations comprising or consisting of individuals (e.g, asymptomatic individuals) without hereditary risk in developing lung cancer (e.g, in some embodiments non-small cell lung cancer). In some embodiments, provided technologies are effective when applied to populations comprising or consisting of symptomatic individuals (e.g, individuals suffering from one or more symptoms of lung cancer). In some embodiments, provided technologies are effective when applied to populations comprising or consisting of individuals at risk for lung cancer (e.g ., individuals with hereditary and/or life-history associated risk factors for lung cancer).
• provided technologies may be or include one or more compositions (e.g., molecular entities or complexes, systems, cells, collections, combinations, kits, etc.) and/or methods (e.g, of making, using, assessing, etc.), as will be clear to one skilled in the art reading the disclosure provided herein.
• compositions e.g., molecular entities or complexes, systems, cells, collections, combinations, kits, etc.
• methods e.g, of making, using, assessing, etc.
• the present disclosure identifies the source of a problem with certain prior technologies including, for example, certain conventional approaches to detection and diagnosis of lung cancer.
• the present disclosure appreciates that many conventional diagnostic assays, e.g, X-ray imaging, sputum testing, low-dose CT scanning, and/ or molecular tests based on cell-free nucleic acids, serum proteins (e.g, carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA 21-1), neuron-specific enolase (NSE), progastrin-releasing peptide (ProGRP), and/or squamous cell carcinoma antigen (SCCA)), and/or bulk analysis of extracellular vesicles, can be time-consuming, costly, and/or lacking sensitivity and/or specificity sufficient to provide a reliable and comprehensive diagnostic assessment.
• CEA carcinoembryonic antigen
• CYFRA 21-1 cytokeratin 19 fragment
• NSE neuron-specific enolase
• ProGRP prog
• the present disclosure provides technologies (including systems, compositions, and methods) that solve such problems, among other things, by detecting colocalization of a target biomarker signature of lung cancer in individual extracellular vesicles, which comprises at least one extracellular vesicle-associated membrane-bound polypeptide and at least one target biomarker selected from the group consisting of surface protein biomarkers, internal protein biomarkers, and RNA biomarkers.
• the present disclosure provides technologies (including systems, compositions, and methods) that solve such problems, among other things, by detecting such target biomarker signature of lung cancer using a target entity detection approach that was developed by Applicant and described in U.S. Application No.
• Target Entity Detection 16/805,637 (published as US2020/0299780), and International Application PCT/US2020/020529 (published as W02020180741), both filed February 28, 2020 and entitled “Systems, Compositions, and Methods for Target Entity Detection,” which are based on interaction and/or co-localization of at least two or more target entities (e.g, a target biomarker signature) in individual extracellular vesicles.
• target entities e.g, a target biomarker signature
• the present disclosure provides insights that screening of asymptotic individuals, e.g ., regular screening prior to or otherwise in absence of developed symptom(s), can be beneficial, and even important for effective management (e.g, successful treatment) of lung cancer (e.g, in some embodiments non-small cell lung cancer).
• lung cancer screening systems can be implemented to detect lung cancer (e.g, in some embodiments non-small cell lung cancer), including early-stage cancer, in some embodiments in asymptomatic individuals.
• provided technologies are implemented to achieve regular screening of asymptomatic individuals.
• compositions e.g, reagents, kits, components, etc.
• methods of providing and/or using them including strategies that involve regular testing of one or more individuals (e.g, symptomatic or asymptomatic individuals).
• individuals e.g, symptomatic or asymptomatic individuals.
• the present disclosure defines usefulness of such systems, and provides compositions and methods for implementing them.
• provided technologies achieve detection (e.g, early detection, e.g, in asymptomatic individual(s) and/or population(s)) of one or more features (e.g, incidence, progression, responsiveness to therapy, recurrence, etc.) of lung cancer, with sensitivity and/or specificity (e.g, rate of false positive and/or false negative results) appropriate to permit useful application of provided technologies to single-time and/or regular (e.g, periodic) assessment.
• detection e.g, early detection, e.g, in asymptomatic individual(s) and/or population(s)
• features e.g, incidence, progression, responsiveness to therapy, recurrence, etc.
• sensitivity and/or specificity e.g, rate of false positive and/or false negative results
• provided technologies are useful in conjunction with regular medical examinations, such as but not limited to: physicals, general practitioner visits, cholesterol/lipid blood tests, diabetes (type 2) screening, colonoscopies, blood pressure screening, thyroid function tests, prostate cancer screening, mammograms, HPV/Pap smears, and/or vaccinations.
• provided technologies are useful in conjunction with treatment regimen(s); in some embodiments, provided technologies may improve one or more characteristics (e.g, rate of success according to an accepted parameter) of such treatment regimen(s).
• the present disclosure provides methods or assays for classifying a subject (e.g, an asymptomatic subject) as having or being susceptible to lung cancer (e.g, in some embodiments non-small cell lung cancer).
• a provided method or assay comprises (a) detecting, in a blood-derived sample from a subject in need thereof, extracellular vesicles expressing a target biomarker signature of lung cancer, the target biomarker signature comprising: at least one extracellular vesicle-associated membrane- bound polypeptide and at least one target biomarker selected from the group consisting of: surface protein biomarkers, intravesicular protein biomarkers, and intravesicular RNA biomarkers, wherein the surface protein biomarkers are selected from ALCAM, ABCC3, ARSL, B3GNT3, CDCP1, CDH1, CDH3, CD55, CD274 (PD-L1), CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3, CLDN4, CLDN7, CLIC6, DMBT1, DSG2, EGFR, EPCAM, EPHX3, EVA1A, FOLR1, GJB1, GJB2, GPC4, HS6ST2, KDELR3, KRTCAP3, IG1FR, LAMB
• the intravesicular protein biomarkers are selected from AOC1, C12orf45, CRABP2, CST1, ETV4, FAM83A, FOXA2, HMGB3, LGALS3BP, MIF, NAPSA, PPP1R14D, S100A14, SBK1, SCGB3A2, SFTA2, SFTPA1, SFTPA2, SFTPB, SPINK1, TGFA, ZC3H11A, and combinations thereof; and the intravesicular RNA (e.g ., mRNA) biomarkers are selected from ABCC3, AOC1, ARSL, B3GNT3, C12orf45, CDCP1, CDH1, CDH3, CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN
• a subject e.g ., an asymptomatic subject
• lung cancer e.g., in some embodiments non-small cell lung cancer
• the present disclosure provides methods or assays for classifying a subject (e.g, an asymptomatic subject) as having or being susceptible to lung cancer (e.g, in some embodiments non-small cell lung cancer).
• a provided method or assay comprises (a) detecting, in a blood-derived sample from a subject in need thereof, extracellular vesicles expressing a target biomarker signature of lung cancer (e.g, in some embodiments non-small cell lung cancer), the target biomarker signature comprising: at least one extracellular vesicle-associated membrane-bound polypeptide and at least one target biomarker selected from the group consisting of: surface protein biomarkers (as described herein), intravesicular protein biomarkers (as described herein), and intravesicular RNA biomarkers (as described herein); (b) comparing sample information indicative of level of the target biomarker signature-expressing extracellular vesicles in the blood-derived sample to reference information including a reference threshold level; and (c) classifying the subject as having or being susceptible to lung cancer when the blood-derived sample shows an elevated level of target biomarker signature-expressing extracellular vesicles relative to a classification cutoff referencing the reference threshold level.
• At least one such target biomarker is or comprises a surface protein biomarker selected from: ABCA3, ABCC1, ABCC3, ACBD3, ACSL5, AGER, ALCAM, API M2, APHIA, APOO, ATP11A, ATP11B, ATP1B1, ATP6AP2, B4GALT4, BCAP31, BSPRY, CD109, CD55, CD9, CDC42, CDH1, CDH3, CDKALl, CEACAM5, CEACAM6, CELSR1, CIP2A, CISD2, CKAP4, CLCA2, CLDN1, CLIC6, CLPTM1L, CLSTN1, CNTN1, CPD, CYP2S1, CYP4F11, CYP4F3, DPY19L1, DSC2, DSC3, DSG2, DSG3, EGFR, EPCAM, EPHB3, FAT2, FBX045, FERMT1, FOLR1, FZD6, GALNTl, GALNT3, GALNT5, GALNT6, GGCX,
• TTC33 UGT1A6, UPK1B, VAMP8, VMA21, VRK2, VWA1, XPR1, XXYLT1, ADAM28, AXL, BSG, CD274, CD47, CLU, DKK1, ERBB3, FLT4, GM3, HGF, IGF1R, IL6, KDR,
• At least one such target biomarker is or comprises an intravesicular protein biomarkers selected from ABRACL, ACP5, ADH7, AGR2, AIF1, AKRICI, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, AOC1, API M2, APOBEC3B, APOBEC3C, ARNTL2, ASF IB, AURKB, BAIAP2L1, BIRC5, C12orf45, C15orf48, C19orfi3, CIS, C8orf4, CA9, CALML3, CAPNS2, CBLC, CCL19, CCNB2, CDC20, CDC45, CDCA4, CDCA5, CDK1, CDKN2A, CDKN2B, CENPW, CEP55, CES1, CHMP4C, CNN2, CP A3, CRABP2, CST1, CSTA, CTSC, CTSE, CYP2S1, DPYSL3, EFS, E
• At least one such target biomarker is or comprises an intravesicular RNA (e.g ., mRNA) selected from ABCA3, ABCCl, ABCC3, ABRACL, ACP5, ADAM23, ADH7, AGR2, AIF1, AKRICI, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, ANTXRl, AOC1, API M2, APOBEC3B, APOBEC3C, AQP3, AREG, ARNTL2, ARSL, ASF IB, ATP8B1, AURKB, B3GNT3, B3GNT5, BAIAP2L1, BCAM, BIK, BIRC5, C12orf45, C15orf48, C19orf33, CIS, C8orf4, CA12, CA9, CALML3, CAPNS2, CBLC, CCL19, C
• a subject e.g ., an asymptomatic subject
• lung cancer e.g., in some embodiments non-small cell lung cancer such as, e.g., lung adenocarcinoma (LUAD) and/or lung squamous cell carcinoma (LUSC)
• the present disclosure provides methods or assays for classifying a subject (e.g, an asymptomatic subject) as having or being susceptible to lung cancer (e.g, in some embodiments non-small cell lung cancer such as, e.g., LUAD and/or LUSC)).
• a provided method or assay comprises (a) detecting, in a blood- derived sample from a subject in need thereof, extracellular vesicles expressing a target biomarker signature of lung cancer (e.g, in some embodiments non-small cell lung cancer such as, e.g., LUAD and/or LUSC), the target biomarker signature comprising: at least one extracellular vesicle-associated membrane-bound polypeptide and at least one target biomarker selected from the group consisting of: surface protein biomarkers (as described herein), intravesicular protein biomarkers (as described herein), and intravesicular RNA biomarkers (as described herein); (b) comparing sample information indicative of level of the target biomarker signature-expressing extracellular vesicles in the blood-derived sample to reference information including a reference threshold level; and (c) classifying the subject as having or being susceptible to lung cancer (e.g, in some embodiments non-small cell lung cancer such as, e.g
• At least one such target biomarker is or comprises a surface protein biomarker selected from: ABCA3, ACBD3, AGER, ALCAM, API M2, APHIA, APOO, ATP1B1, ATP6AP2, BCAP31, BSPRY, CDC42, CDH1, CDH3, CDKALl, CELSR1, CIP2A, CISD2, CKAP4, CLPTM1L, CLSTN1, CPD, DPY19L1, DSG2, EGFR, EPCAM, FZD6, GALNT1, GALNT3, GALNT5, GALNT6, GGCX, GOLPH3L, GRHL2, HACD3, IER3IP1, IL1RAP, ITGA2, ITGB6, KPNA2, KRTCAP3, LADl, LAMB3, LAMP3, LAMTOR2, LCLAT1, LSR, MAGT1, MARCKSL1, MET, MGAT1, MUC4, NCSTN, NECTIN4, NRAS, NUP210, PEX13, PIGN
• At least one such target biomarker is or comprises an intravesicular protein biomarkers selected from ABRACL, ACP5, AIF1, ALDH1A1, ALGIL, API M2, APOBEC3C, ASF1B, AURKB, BAIAP2L1, BIRC5, C12orf45, C15orf48, C19orf33, CIS, C8orf4, CBLC, CCL19, CCNB2, CDC20, CDCA5, CDK1, CDKN2A, CDKN2B, CEP55, CHMP4C, CNN2, CP A3, CRABP2, CST1, CTSC, DPYSL3, EGLN3, EHF, ELF 3, ELF4, ENAH, ESRP1, ETV4, EVPL, FAM129B, FAM60A, FAM83A, FAM83H, GNA15, GRHL2, HCK, HMGB3, HOXB7, ID1, IMPA2, KIAA1522, KIF2C, KIFC1, KLF
• At least one such target biomarker is or comprises an intravesicular RNA (e.g ., mRNA) selected from ABCA3, ABRACL, ACP5, AIF1, ALDHIAI, ALGIL, ANTXRl, API M2, APOBEC3C, AQP3, AREG, ARSL, ASF1B, ATP8B1, AURKB, BAIAP2L1, BCAM, BIK, BIRC5, C12orf45, C15orf48, C19orf33, CIS, C8orf4, CBLC, CCL19, CCL5, CCNB2, CD24, CD53, CD74, CDC20, CDC42EP1, CDCA5, CDCP1, CDH1, CDH3, CDK1, CDKN2A, CDKN2B, CELSR1, CEP55, CHMP4C, CLDN4, CLDN7, CNN2, CP A3, CRABP2, CST1, CTSC, CX3CL1, CXADR, CXCR4, CY
• TMPRSSl IE TMPRSS4, TNFRSF18, TOP2A, TP53I11, TPD52, TPX2, TREM2, TRIP13, TSPAN1, TSPAN13, TSPAN6, TSPAN7, TUSC3, TYROBP, UBE2C, VAMP8, WLS, YAPl, ZC3H11 A, ZNF217, and combinations thereof.
• a subject e.g ., an asymptomatic subject
• a certain type of lung cancer e.g., in particular embodiments lung adenocarcinoma (LUAD)
• the present disclosure provides methods or assays for classifying a subject (e.g, an asymptomatic subject) as having or being susceptible to a certain type of lung cancer (e.g, in particular embodiments LUAD).
• a provided method or assay comprises (a) detecting, in a blood- derived sample from a subject in need thereof, extracellular vesicles expressing a target biomarker signature of a certain type of lung cancer (e.g, in particular embodiments LUAD), the target biomarker signature comprising: at least one extracellular vesicle-associated membrane- bound polypeptide and at least one target biomarker selected from the group consisting of: surface protein biomarkers (as described herein), intravesicular protein biomarkers (as described herein), and intravesicular RNA biomarkers (as described herein); (b) comparing sample information indicative of level of the target biomarker signature-expressing extracellular vesicles in the blood-derived sample to reference information including a reference threshold level; and (c) classifying the subject as having or being susceptible to a certain type of lung cancer (e.g., in particular embodiments LUAD) when the blood-derived sample shows an elevated level of target biomarker signature-expressing extracellular ves
• At least one such target biomarker that is particularly useful for classifying a subject as having or being susceptible to LUAD is or comprises a surface protein biomarker selected from: ABCC3, ACSL5, ATP11 A, CD55, CEACAM5, CEACAM6, CLIC6, FOLR1, GOLM1, LPCAT1, MSLN, MUC1, NT5E, PLA2G4A, PLCH1, SLC34A2, SMPDL3B, TESC, TMC5, ERBB3, KDR, and combinations thereof.
• At least one such target biomarker that is particularly useful for classifying a subject as having or being susceptible to LUAD is or comprises an intravesicular protein biomarkers selected from AGR2, AOC1, CTSE, FBP1, FOXA2, KRT7, NAPSA, PPP1R14D, S100P, SCGB3A2, SFTA2, SFTPA1, SFTPA2, SFTPB, SPINK1, and combinations thereof.
• At least one such target biomarker that is particularly useful for classifying a subject as having or being susceptible to LUAD is or comprises an intravesicular RNA (e.g, mRNA) selected from ABCC3, AGR2, AOC1, B3GNT3, CEACAM5, CEACAM6, CLDN18, CLDN3, CLIC6, CTSE, FBP1, FOLR1, FOXA2, GJB1, GPRC5A, KRT7, MSLN, MUC1, MUC21, NAPSA, PIGR, PPP1R14D, ROS1, SI OOP, SCGB3A2, SFTA2, SFTPA1, SFTPA2, SFTPB, SLC34A2, SLC44A4, SLC6A14, SMIM22, SMPDL3B, SPINK1, TMC5, TMEM45B, TMPRSS2, TSPAN8, and combinations thereof.
• an intravesicular RNA e.g, mRNA
• mRNA intravesicular RNA
• certain aforementioned target biomarkers are particularly useful in differentiating LUAD from LUSC.
• a subject e.g., an asymptomatic subject
• a certain type of lung cancer e.g, in particular embodiments lung squamous cell carcinoma (LUSC)
• the present disclosure provides methods or assays for classifying a subject (e.g, an asymptomatic subject) as having or being susceptible to a certain type of lung cancer (e.g, in particular embodiments LUSC).
• a provided method or assay comprises (a) detecting, in a blood-derived sample from a subject in need thereof, extracellular vesicles expressing a target biomarker signature of a certain type of lung cancer (e.g, in particular embodiments LUSC), the target biomarker signature comprising: at least one extracellular vesicle-associated membrane-bound polypeptide and at least one target biomarker selected from the group consisting of: surface protein biomarkers (as described herein), intravesicular protein biomarkers (as described herein), and intravesicular RNA biomarkers (as described herein); (b) comparing sample information indicative of level of the target biomarker signature-expressing extracellular vesicles in the blood-derived sample to reference information including a reference threshold level; and (c) classifying the subject as having or being susceptible to a certain type of lung cancer (e.g., in particular embodiments LUSC) when the blood-derived sample shows an elevated level of target biomarker signature-expressing extracellular
• At least one such target biomarker that is particularly useful for classifying a subject as having or being susceptible to LUSC is or comprises a surface protein biomarker selected from: ABCC1, ATP11B, B4GALT4, CD109, CD9, CLCA2, CLDN1, CNTN1, CYP2S1, CYP4F11, CYP4F3, DSC2, DSC3, DSG3, EPHB3, FAT2, FBX045, FERMT1, IGSF3, KLRG2, LAMC2,
• At least one such target biomarker that is particularly useful for classifying a subject as having or being susceptible to LUSC is or comprises an intravesicular protein biomarkers selected from ADH7, AKR1C1, AKR1C2, AKR1C3, ALDH3A1, ALDH3B2, APOBEC3B, ARNTL2, CA9, CALML3, CAPNS2, CDC45, CDCA4, CENPW, CES1, CSTA, CYP2S1, EFS, FAM83D, FERMT1, FOXE1, FOXM1, GBP6, GPX2, GSTA1, IGF2BP2, IRF6, IVL, JUP, KRT13, KRT14, KRT15,
• At least one such target biomarker that is particularly useful for classifying a subject as having or being susceptible to LUSC is or comprises an intravesicular RNA (e.g., mRNA) selected from ABCCl, ADAM23, ADH7, AKR1C1, AKR1C2, AKR1C3, ALDH3A1, ALDH3B2, APOBEC3B, ARNTL2, B3GNT5, CA12, CA9, CALML3, CAPNS2, CD 109, CD9, CDC45, CDCA4, CENPW, CES1, CLCA2, CLDN1, COL17A1, CSTA, CYP2S1, CYP4F11, DAPLl, DSC2, DSC3, DSG3, DSP, EFS, EPHB3, FAM83D, FAT2, FERMT1, FGFR2, FGFR3, FOXE1, FOXM1, FXYD3, GBP6, GJA1, GJB2, GJB3, GJB5, GJB6, GPC1, G
• certain aforementioned target biomarkers are particularly useful in differentiating LUSC from LUAD.
• methods or assays described herein may be performed for at least one more additional target biomarker signature (including, e.g ., at least one, at least two, at least three, or more additional target biomarker signatures).
• a classification cutoff may reference additional reference threshold level(s) corresponding to each additional target biomarker signature.
• an extracellular vesicle-associated membrane-bound polypeptide for use in a target biomarker signature of lung cancer used and/or described herein may be or comprise a tumor-specific biomarker and/or a tissue-specific biomarker (e.g, a lung tissue- specific biomarker).
• a tissue-specific biomarker e.g, a lung tissue- specific biomarker
• such an extracellular vesicle-associated membrane- bound polypeptide biomarker may be or comprise a non-specific marker, e.g, it is present in one or more non-target tumors, and/or in one or more non-target tissues.
• such an extracellular vesicle-associated membrane-bound polypeptide biomarker may be or comprise one or more of surface protein biomarkers described herein.
• such an extracellular vesicle-associated membrane-bound polypeptide biomarker may be or comprise one or more of polypeptide biomarkers selected from: ALCAM, B3GNT3, CDCP1, CDH1, CDH3, CD55, CD274 (PD-L1), CEACAM5, CEACAM6, CLDN3, CLDN4, DSG2, EGFR, EPCAM, FOLR1, GJB1, GJB2, IG1FR, LAMB 3, MET, MSLN, MUC1, PIGT, PODXL2, ROS1, SDC1, SLC34A2, sTn antigen, Tn antigen, T antigen, SMPDL3B, ST14, TACSTD2, TMPRSS4, TNFRSF10B, TSPAN8, and combinations thereof.
• polypeptide biomarkers selected from: ALCAM, B3GNT3, CDCP1, CDH1, CDH3, CD55, CD274 (PD-L1), CEACAM5, CEACAM6, CLDN3, CLDN4, DSG2, EGFR, EPCAM, FOLR
• such an extracellular vesicle-associated membrane-bound polypeptide biomarker may be or comprise SLC34A2, CEACAM5, CEACAM6, and/or EPCAM. In some embodiments, such an extracellular vesicle- associated membrane-bound polypeptide biomarker may be or comprise ALCAM, CD55,
• CDH1, CDH3, CD274 (PD-L1), CEACAM5, CEACAM6, DSG2, EGFR, EPCAM, FOLR1, IG1FR, MET, MSLN, MUC1, SLC34A2, sTn antigen, Tn antigen, T antigen, TACSTD2, TNFRSFIOB, or combinations thereof.
• such an extracellular vesicle- associated membrane-bound polypeptide may be or comprise a SLC34A2 polypeptide.
• such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a CEACAM5 polypeptide.
• such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a CEACAM6 polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise an EPCAM polypeptide.
• a target biomarker signature of lung cancer comprises an extracellular vesicle-associated membrane-bound polypeptide (e.g ., ones described herein) and at least one (including, e.g., 1, 2, 3, or more) additional target surface protein biomarker, which, in some embodiments, may be or comprise ALCAM, ABCC3, ARSL, B3GNT3, CDCP1, CDH1, CDH3, CD55, CD274 (PD-L1), CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3,
• an extracellular vesicle-associated membrane-bound polypeptide for use in a target biomarker signature of lung cancer used and/or described herein may be or comprise a tumor-specific biomarker and/or a tissue-specific biomarker (e.g, a lung tissue- specific biomarker).
• a tissue-specific biomarker e.g, a lung tissue- specific biomarker
• such an extracellular vesicle-associated membrane- bound polypeptide biomarker may be or comprise a non-specific marker, e.g, it is present in one or more non-target tumors, and/or in one or more non-target tissues.
• such an extracellular vesicle-associated membrane-bound polypeptide biomarker may be or comprise one or more of surface protein biomarkers described herein.
• such an extracellular vesicle-associated membrane-bound polypeptide biomarker may be or comprise one or more of polypeptide biomarkers selected from: ABCA3, ABCCl, ABCC3, ACBD3, ACSL5, AGER, ALCAM, API M2, APHIA, APOO, ATP11A, ATP11B, ATP1B1, ATP6AP2, B4GALT4, BCAP31, BSPRY, CD109, CD55, CD9, CDC42, CDH1, CDH3, CDKALl, CEACAM5, CEACAM6, CELSR1, CIP2A, CISD2, CKAP4, CLCA2, CLDN1, CLIC6, CLPTM1L, CLSTN1, CNTN1, CPD, CYP2S1, CYP4F11, CYP4F3, DPY19L1, DSC2, DSC3, DSG2, DSG3, EGFR, EPCAM, EPHB3, FAT2, FBX045, FERMTl, FOLR1, FZD6, GALNT
• MSLN MUC1, MUC4, NCSTN, NECTINl, NECTIN4, NRAS, NT5E, NUP210, PARL,
• TTC33 UGT1A6, UPK1B, VAMP8, VMA21, VRK2, VWA1, XPR1, XXYLT1, ADAM28, AXL, BSG, CD274, CD47, CLU, DKK1, ERBB3, FLT4, GM3, HGF, IGF1R, IL6, KDR,
• a target biomarker signature of lung cancer comprises an extracellular vesicle-associated membrane-bound polypeptide (e.g ., ones described herein) and at least one (including, e.g., 1, 2, 3, or more) additional target surface protein biomarker, which, in some embodiments, may be or comprise ABCA3, ABCCl, ABCC3, ACBD3, ACSL5, AGER, ALCAM, API M2, APHIA, APOO, ATP11A, ATP11B, ATP1B1, ATP6AP2, B4GALT4, BCAP31, BSPRY, CD109, CD55, CD9, CDC42, CDH1, CDH3, CDKALl, CEACAM5, CEACAM6, CEL SRI, CIP2A, CISD2, CKAP4, CLCA2, CLDN1, CLIC6, CLPTM1L,
• additional target surface protein biomarker which, in some embodiments, may be or comprise ABCA3, ABCCl, ABCC3, ACBD3, ACSL5, AGER, ALCAM, API M2,
• MSLN MUC1, MUC4, NCSTN, NECTINl, NECTIN4, NRAS, NT5E, NUP210, PARL,
• PEX13 PIGN, PIGT, PLA2G4A, PLCH1, PLEC, PSMD2, PTDSS1, PTGFRN, PTPRF,
• TTC33 UGT1A6, UPK1B, VAMP8, VMA21, VRK2, VWA1, XPR1, XXYLTl, ADAM28, AXL, BSG, CD274, CD47, CLU, DKK1, ERBB3, FLT4, GM3, HGF, IGF1R, IL6, KDR,
• a target biomarker signature of lung cancer comprises an extracellular vesicle-associated membrane-bound polypeptide (e.g ., ones described herein) and at least one target intravesicular RNA (e.g., mRNA) biomarker, which, in some embodiments, may be or comprise ABCC3, AOC1, ARSL, B3GNT3, C12orf45, CDCP1, CDH1, CDH3, CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3, CLDN4, CLDN7, CLIC6, CRABP2, CST1, DMBT1, DSG2, EPCAM, EPHX3, ETV4, EVA1A, FAM83A, FOLR1, FOXA2, GJB1, GJB2, GPC4, HMGB3, HS6ST2, KDELR3, KRTCAP3, LAMB3, LFNG, LGALS3BP, LSR, MANEAL, MIF, MSLN, MUC1, MUC21, NAPSA, PIGT, PODX
• a target biomarker signature of lung cancer comprises an extracellular vesicle-associated membrane-bound polypeptide (e.g, ones described herein) and at least one target intravesicular RNA (e.g, mRNA) biomarker, which, in some embodiments, may be or comprise ABC A3, ABCCl, ABRACL, ACP5, ADAM23, ADH7, AGR2, AIF1, AKR1C1, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, ANTXR1, API M2, APOBEC3B, APOBEC3C, AQP3, AREG, ARNTL2, ASF1B, ATP8B1, AURKB, B3GNT5, BAIAP2L1, BCAM, BIK, BIRC5, C15orf48, C19orf33, CIS, C8orf4, CA12, CA9, CALML3, CAPNS2, CBLC, CCL
• a target biomarker signature of lung cancer comprises an extracellular vesicle-associated membrane-bound polypeptide (e.g ., ones described herein) and at least one additional target intravesicular protein biomarker, which, in some embodiments, may be or comprise AOC1, C12orf45, CRABP2, CST1, ETV4, FAM83A, FOXA2, HMGB3, LGALS3BP, MIF, NAPSA, PPP1R14D, S100A14, SBK1, SCGB3A2, SFTA2, SFTPA1, SFTPA2, SFTPB, SPINK1, TGFA, ZC3H11A, or combinations thereof.
• a target biomarker signature comprises at least one extracellular vesicle-associated membrane- bound polypeptide, which is or comprises a SCL34A2 polypeptide and/or a CEACAM5 polypeptide; and at least one target biomarker SLC34A2, CEACAM5, CEACAM6 and/or EPCAM.
• a target biomarker signature of lung cancer comprises an extracellular vesicle-associated membrane-bound polypeptide (e.g., ones described herein) and at least one additional target intravesicular protein biomarker, which, in some embodiments, may be or comprise ABRACL, ACP5, ADH7, AGR2, AIF1, AKR1C1, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, API M2, APOBEC3B, APOBEC3C, ARNTL2, ASF IB, AURKB, BAIAP2L1, BIRC5, C15orf48, C19orf33, CIS, C8orf4, CA9, CALML3, CAPNS2, CBLC, CCL19, CCNB2, CDC20, CDC45, CDCA4, CDCA5, CDK1, CDKN2A, CDKN2B, CENPW, CEP55, CES1, CHMP4C, CNN
• a reference threshold level for use in a provided method or assay described herein is determined by levels of target biomarker signature-expressing extracellular vesicles observed in comparable samples from a population of non-lung cancer subjects.
• an extracellular vesicle-associated membrane-bound polypeptide included in a target biomarker signature may be detected using antibody -based agents.
• such an extracellular vesicle-associated membrane-bound polypeptide may be detected using a capture assay comprising an antibody-based agent.
• a capture assay for detecting the presence of an extracellular vesicle-associated membrane-bound polypeptide in an extracellular vesicle may involve contacting a blood-derived sample comprising extracellular vesicles with a capture agent directed to such an extracellular vesicle-associated membrane-bound polypeptide.
• such a capture agent comprises a binding moiety directed to an extracellular vesicle-associated membrane-bound polypeptide (e.g ., ones described herein), which may be optionally conjugated to a solid substrate.
• an exemplary capture agent for an extracellular vesicle-associated membrane-bound polypeptide may be or comprising a solid substrate (e.g ., a magnetic bead) and a binding moiety (e.g., an antibody agent) directed to an extracellular vesicle-associated membrane-bound polypeptide.
• a target biomarker included in a target biomarker signature may be detected using appropriate methods known in the art, which may vary with types of analytes to be detected (e.g, surface proteins, intravesicular proteins, intravesicular RNA (e.g, mRNA)).
• analytes to be detected e.g, surface proteins, intravesicular proteins, intravesicular RNA (e.g, mRNA)
• a surface protein biomarker and/or an intravesicular protein biomarker may be detected using antibody-based agents in some embodiments
• an intravesicular RNA (e.g, mRNA) biomarker may be detected using nucleic acid-based agents, e.g, using quantitative reverse transcription PCR.
• a target biomarker is or comprises a surface protein biomarker and/or an intravesicular protein marker
• a target biomarker may be detected involving a proximity ligation assay, e.g, following a capture assay (e.g, ones as described herein) to capture extracellular vesicles that express an extracellular vesicle-associated membrane-bound polypeptide (e.g, ones as used and/or described herein).
• a proximity ligation assay e.g, following a capture assay (e.g, ones as described herein) to capture extracellular vesicles that express an extracellular vesicle-associated membrane-bound polypeptide (e.g, ones as used and/or described herein).
• such a proximity ligation assay comprises contacting a blood-derived sample comprising extracellular vesicles with a set of detection probes, each directed to a target biomarker, which set comprises at least two distinct detection probes, so that a combination comprising the extracellular vesicles and the set of detection probes is generated, wherein the two detection probes each comprise: (i) a binding moiety directed to a surface protein biomarker and/or an intravesicular protein biomarker; and (ii) an oligonucleotide domain coupled to the binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single- stranded overhang portion extended from one end of the oligonucleotide domain.
• Such single- stranded overhang portions of the detection probes are characterized in that they can hybridize to each other when the detection probes are bound to the same extracellular vesicle.
• Such a combination comprising the extracellular vesicles and the set of detection probes is then maintained under conditions that permit binding of the set of detection probes to their respective targets on the extracellular vesicles such that the detection probes can bind to the same extracellular vesicle to form a double-stranded complex.
• Such a double-stranded complex can be detected by contacting the double-stranded complex with a nucleic acid ligase to generate a ligated template; and detecting the ligated template.
• a ligated template is indicative of presence of extracellular vesicles that are positive for a target biomarker signature of lung cancer. While such a proximity ligation assay may perform better, e.g. , with higher specificity and/or sensitivity, than other existing proximity ligation assays, a person skilled in the art reading the present disclosure will appreciate that other forms of proximity ligation assays that are known in the art may be used instead.
• a target biomarker is or comprises an intravesicular RNA (e.g., mRNA) marker
• a target biomarker may be detected involving a nucleic acid detection assay.
• an exemplary nucleic acid detection assay may be or comprise reverse-transcription PCR.
• a target biomarker is or comprises an intravesicular biomarker (e.g, an intravesicular protein biomarker and/or an intravesicular RNA (e.g, mRNA) biomarker)
• an intravesicular biomarker e.g, an intravesicular protein biomarker and/or an intravesicular RNA (e.g, mRNA) biomarker
• a detection assay e.g, a proximity ligation assay as described herein
• a sample treatment e.g, fixation and/or permeabilization
• the present disclosure recognizes that detection of a single lung cancer-associated serum protein or a plurality of lung cancer-associated biomarkers based on a bulk sample (e.g, a bulk sample of extracellular vesicles), rather than at a resolution of a single extracellular vesicle, typically does not provide sufficient specificity and/or sensitivity in determination of whether a subject from whom the sample is obtained is likely to be suffering from or susceptible to lung cancer.
• a bulk sample e.g, a bulk sample of extracellular vesicles
• the present disclosure provides technologies, including systems, compositions, and/or methods, that solve such problems, including for example by specifically requiring that individual extracellular vesicles for detection be characterized by presence of a target biomarker signature comprising a combination of at least one or more extracellular vesicle-associated membrane-bound polypeptides and at least one or more target biomarkers.
• the present disclosure teaches technologies that require such individual extracellular vesicles be characterized by presence (e.g, by expression) of such a target biomarker signature of lung cancer, while extracellular vesicles that do not comprise the target biomarker signature do not produce a detectable signal (e.g, a level that is above a reference level, e.g, by at least 10% or more, where in some embodiments, a reference level may be a level observed in a negative control sample, such as a sample in which individual extracellular vesicles comprising such a target biomarker signature are absent).
• a detectable signal e.g, a level that is above a reference level, e.g, by at least 10% or more, where in some embodiments, a reference level may be a level observed in a negative control sample, such as a sample in which individual extracellular vesicles comprising such a target biomarker signature are absent.
• a target biomarker signature may be selected for detection of lung cancer.
• a target biomarker signature may be selected for detection of a specific category of lung cancer, including, e.g ., but not limited to lung adenocarcinoma, small cell lung cancer, squamous and transitional cell lung cancer, large cell lung cancer, nonsmall cell carcinoma, other specified carcinomas, sarcomas, and other specified types of lung cancer as known in the art (see, e.g. , SEER Cancer Statistics Review 1975-2017).
• technologies provided herein can be used periodically (e.g, every year) to screen a human subject or across a population of human subjects for early-stage lung cancer or lung cancer recurrence.
• a subject that is amenable to technologies provided herein for detection of incidence or recurrence of lung cancer may be an asymptomatic human subject and/or across an asymptomatic population.
• Such an asymptomatic subject may be a subject who has a family history of lung cancer, who has been previously treated for lung cancer, who is at risk of lung cancer recurrence after cancer treatment, who is in remission after lung cancer treatment, and/or who has been previously or periodically screened for the presence of lung cancer by chest X-ray, sputum analysis, low dose CT, and/or the presence of at least one lung cancer serum biomarker, e.g, but not limited to CEA, CYFRA 21-1, NSE, ProGRP, and/or SCC A serum proteins.
• lung cancer serum biomarker e.g, but not limited to CEA, CYFRA 21-1, NSE, ProGRP, and/or SCC A serum proteins.
• such an asymptomatic subject may be a subject who is determined to have a normal medical diagnosis result from, e.g, chest X-ray, sputum analysis, low dose CT analysis, or serum CEA, CYFRA 21-1, NSE, ProGRP, and/or SCCA levels.
• such an asymptomatic subject may be a subject who is determined to have an abnormal medical diagnosis result from, e.g, chest X-ray, sputum analysis, low dose CT analysis, and/or a serum level of CEA, CYFRA 21-1, NSE, ProGRP, and/or SCCA level when compared to results as typically observed in non-lung cancer subjects and/or normal healthy subjects.
• an asymptomatic subject may be a subject who has not been previously screened for lung cancer, who has not been diagnosed for lung cancer, and/or who has not previously received lung cancer therapy.
• a subject or population of subjects may be selected based on one or more characteristics such as age, race, genetic history, medical history, personal and/or medical history (e.g ., smoking, alcohol, drugs, carcinogenic agents, diet, obesity, diabetes, physical activity, sun exposure, radiation exposure, exposure to infectious agents such as viruses, and/or occupational hazard).
• technologies provided herein can be useful for selecting therapy for a subject who is suffering from or susceptible to lung cancer.
• a lung cancer therapy and/or an adjunct therapy can be selected in light of findings based on technologies provided herein.
• technologies provided herein can be useful for monitoring and/or evaluating efficacy of therapy administered to a subject (e.g., a lung cancer subject).
• the present disclosure provides technologies for managing patient care, e.g, for one or more individual subjects and/or across a population of subjects.
• the present disclosure provides technologies that may be utilized in screening (e.g, temporally or incidentally motivated screening and/or non-temporally or incidentally motivated screening, e.g, periodic screening such as annual, semi-annual, biannual, or with some other frequency).
• provided technologies for use in temporally motivated screening can be useful for screening one or more individual subjects or across a population of subjects (e.g, asymptomatic subjects) who are older than a certain age (e.g, over 50, 55, 60, 65, 70, or older).
• provided technologies for use in temporally motivated screening can be useful for screening one or more individual subjects or across a population of subjects (e.g, asymptomatic subjects) who have a cigarette pack-year history greater than a certain number (e.g, 5 pack years, 10 pack years, 15 pack years, 20 pack years, 25 pack years, 30 pack years, and/or greater than 35 pack years; 1 pack year is equal to 1 pack of cigarettes smoked per day for one year, while 2 packs smoked per day for one year would equal 2 pack years, or 1 ⁇ 2 pack smoked per day for two years would equal 1 pack years, etc.).
• provided technologies for use in incidentally motivated screening can be useful for screening individual subjects who may have experienced an incident or event that motivates screening for lung cancer as described herein.
• an incidental motivation relating to determination of one or more indicators of cancer or susceptibility thereto may be or comprise, e.g, an incident based on their family history (e.g ., a close relative such as blood-related relative was previously diagnosed for lung cancer), identification of one or more risk factors associated with lung cancer (e.g., life history risk factors including but not limited to, e.g, smoking, alcohol, diet, obesity, occupational hazard, etc.) and/or prior incidental findings from genetic tests (e.g, genome sequencing), and/or imaging diagnostic tests (e.g, X-ray, ultrasound, computerized tomography (CT), low dose CT, and/or magnetic resonance imaging (MRI) scans), development of one or more signs or symptoms characteristic of lung cancer (e.g, abnormal imaging results, and/or symptoms potentially indicative of lung cancer, etc.).
• family history e.g a close relative such as blood-related relative was previously diagnosed for lung cancer
• identification of one or more risk factors associated with lung cancer e.g., life history risk factors including but
• provided technologies for managing patient care can inform treatment and/or payment (e.g, reimbursement for treatment) decisions and/or actions.
• provided technologies can provide determination of whether individual subjects have one or more indicators of incidence or recurrence of lung cancer, thereby informing physicians and/or patients when to initiate therapy in light of such findings.
• provided technologies can inform physicians and/or patients of treatment selection, e.g., based on findings of specific responsiveness biomarkers (e.g., lung cancer responsiveness biomarkers).
• provided technologies can provide determination of whether individual subjects are responsive to current treatment, e.g, based on findings of changes in one or more levels of molecular targets associated with lung cancer, thereby informing physicians and/or patients of efficacy of such therapy and/or decisions to maintain or alter therapy in light of such findings.
• provided technologies can inform decision making relating to whether health insurance providers reimburse (or not), e.g, for (1) screening itself (e.g., reimbursement available only for periodic/regular screening or available only for temporally and/or incidentally motivated screening); and/or for (2) initiating, maintaining, and/or altering therapy in light of findings by provided technologies.
• the present disclosure provides methods relating to (a) receiving results of a screening as described herein and also receiving a request for reimbursement of the screening and/or of a particular therapeutic regimen; (b) approving reimbursement of the screening if it was performed on a subject according to an appropriate schedule or response to a relevant incident and/or approving reimbursement of the therapeutic regimen if it represents appropriate treatment in light of the received screening results; and, optionally (c) implementing the reimbursement or providing notification that reimbursement is refused.
• a therapeutic regimen is appropriate in light of received screening results if the received screening results detect a biomarker that represents an approved biomarker for the relevant therapeutic regimen (e.g ., as may be noted in a prescribing information label and/or via an approved companion diagnostic).
• a biomarker that represents an approved biomarker for the relevant therapeutic regimen (e.g ., as may be noted in a prescribing information label and/or via an approved companion diagnostic).
• the present disclosure contemplates reporting systems (e.g., implemented via appropriate electronic device(s) and/or communications system(s)) that permit or facilitate reporting and/or processing of screening results, and/or of reimbursement decisions as described herein.
• a system or kit may comprise detection agents for a tumor biomarker signature of lung cancer (e.g, ones described herein).
• a system or kit may comprise a capture agent for an extracellular vesicle-associated membrane-bound polypeptide present in extracellular vesicles associated with lung cancer (e.g, ones used and/or described herein); and (b) at least one or more detection agents directed to one or more target biomarkers of a target biomarker signature of lung cancer, which may be or comprise additional surface protein biomarker(s) (e.g, ones as used and/or described herein), intravesicular protein biomarker(s) (e.g, ones as used and/or described herein), and/or intravesicular RNA (e.g, mRNA) biomarker(s)(c.£ ⁇ ., ones as used and/or described herein).
• additional surface protein biomarker(s) e.g, ones as used and/or described herein
• intravesicular protein biomarker(s)
• a capture agent included in a system and/or kit may comprise a binding moiety directed to an extracellular vesicle-associated membrane-bound polypeptide (e.g, ones described herein).
• a binding moiety may be conjugated to a solid substrate, which in some embodiments may be or comprise a solid substrate.
• such a solid substrate may be or comprise a magnetic bead.
• an exemplary capture agent included in a provided system and/or kit may be or comprise a solid substrate (e.g., a magnetic bead) and an antibody agent directed to an extracellular vesicle- associated membrane-bound polypeptide conjugated thereto.
• a system and/or kit may include detection agents for performing a proximity ligation assay (e.g., ones as described herein).
• such detection agents for performing a proximity ligation assay may comprise a set of detection probes, each directed to a target biomarker of a target biomarker signature, which set comprises at least two detection probes, wherein the two detection probes each comprise: (i) a polypeptidebinding moiety directed to a target biomarker; and (ii) an oligonucleotide domain coupled to the binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single- stranded overhang portion extended from one end of the oligonucleotide domain, wherein the single-stranded overhang portions of the detection probes are characterized in that they can hybridize to each other when the detection probes are bound to the same extracellular vesicle.
• a provided system and/or kit may comprise a plurality ( e.g ., 2, 3,
• a provided system and/kit may comprise at least one set for detection probes for detection of lung cancer and at least one set of detection probes for detection of a different cancer (e.g, pancreatic cancer).
• two or more detection probes may be directed to different categories of lung cancer, including, e.g, but not limited to lung adenocarcinoma lung cancer, small cell lung cancer, squamous and transitional cell lung cancer, Large cell lung cancer, non-small cell carcinoma lung cancer, other specified carcinoma lung cancer, sarcoma lung cancer, and other specified types of lung cancer as known in the art (see, e.g, SEER Cancer Statistics Review 1975-2017).
• two or more sets may be directed to detection of lung cancer of different categories.
• two or more sets may be directed to detection of lung cancer of the same category.
• two or more sets may be directed to detection of lung cancer of different stages.
• two or more sets may be directed to detection of lung cancer of the same stage.
• detection probes in a provided kit may be provided as a single mixture in a container. In some embodiments, multiple sets of detection probes may be provided as individual mixtures in separate containers. In some embodiments, each detection probe is provided individually in a separate container.
• a system and/or kit may include detection agents for performing a nucleic acid detection assay.
• detection agents for performing a quantitative reverse-transcription PCR for example, which may comprise primers directed to intravesicular RNA (e.g, mRNA) target(s).
• a provided system and/or kit may comprise at least one chemical reagent, e.g. , to process a sample and/or extracellular vesicles therein.
• a provided system and/or kit may comprise at least one chemical reagent to process extracellular vesicles in a sample, including, e.g. , but not limited to a fixation agent, a permeabilization agent, and/or a blocking agent.
• a provided system and/or kit may comprise a nucleic acid ligase and/or a nucleic acid polymerase.
• a provided system and/or kit may comprise one or more primers and/or probes.
• a provided system and/or kit may comprise one or more pairs of primers, for example for PCR, e.g. , quantitative PCR (qPCR) reactions.
• a provided system and/or kit may comprise one or more probes such as, for example, hydrolysis probes which may in some embodiments be designed to increase the specificity of qPCR (e.g, TaqMan probes).
• a provided system and/or kit may comprise one or more multiplexing probes, for example as may be useful when simultaneous or parallel qPCR reactions are employed (e.g, to facilitate or improve readout).
• a provided system and/or kit can be used for screening (e.g, regular screening) and/or other assessment of individuals (e.g, asymptomatic or symptomatic subjects) for detection (e.g, early detection) of lung cancer.
• a provided system and/or kit can be used for screening and/or other assessment of individuals susceptible to lung cancer (e.g, individuals with a known genetic, environmental, or experiential risk, etc.).
• provided system and/or kits can be used for monitoring recurrence of lung cancer in a subject who has been previously treated.
• provided systems and/or kits can be used as a companion diagnostic in combination with a therapy for a subject who is suffering from lung cancer.
• provided systems and/or kits can be used for monitoring or evaluating efficacy of a therapy administered to a subject who is suffering from lung cancer. In some embodiments, provided systems and/or kits can be used for selecting a therapy for a subject who is suffering from lung cancer. In some embodiments, provided systems and/or kits can be used for making a therapy decision and/or selecting a therapy for a subject with one or more symptoms (e.g, non-specific symptoms) associated with lung cancer. [44] Complexes formed by performing methods described herein and/or using systems and/or kits described herein are also within the scope of disclosure.
• a complex comprising: (a) an extracellular vesicle expressing a target biomarker signature, at least two of which include at least one extracellular vesicle-associated membrane-bound polypeptide and at least one target biomarker selected from the group consisting of: surface protein biomarkers, intravesicular protein biomarkers, and intravesicular RNA biomarkers, wherein the surface protein biomarkers are selected from ABCC3, ALCAM, ARSL, B3GNT3, CDCP1, CDH1, CDH3, CD55, CD274 (PD-L1), CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3, CLDN4, CLDN7, CLIC6, DMBT1, DSG2, EGFR, EPCAM, EPHX3, EVA1A,
• intravesicular protein biomarkers are selected from: AOC1, C12orf45, CRABP2, CST1, ETV4, FAM83A, FOXA2, HMGB3, LGALS3BP, MIF, NAPSA, PPP1R14D, S100A14, SBK
• Such a complex further comprises at least two detection probes directed to at least one target biomarker of the target biomarker signature present in the extracellular vesicle, wherein each detection probe is bound to such a target biomarker and each comprises: (i) a binding directed to the target biomarker; and (ii) an oligonucleotide domain coupled to the binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single-stranded overhang portion extended from one end of the oligonucleotide domain, wherein the single-stranded overhang portions of the detection probes are hybridized to each other.
• an extracellular vesicle-associated membrane-bound polypeptide biomarker present in an extracellular vesicle that forms a complex comprises one or more of surface protein biomarkers described herein.
• such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise one or more of polypeptides selected from: ALCAM, B3GNT3, CDCP1, CDHA1, CDH3, CD55, CD274 (PD- Ll), CEACAM5, CEACAM6, CLDN3, CLDN4, DSG2, EGFR, EPCAM, FOLR1, GJB1, GJB2, IG1FR, LAMB 3, MET, MSLN, MUC1, PIGT, PODXL2, ROS1, SDC1, SLC34A2, SMPDL3B, ST14, sTn antigen, Tn antigen, T antigen, TACSTD2, TMPRSS4, TSPAN8, TNFRSF10B, and combinations thereof.
• such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a SLC34A2 polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a CEACAM5 polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a CEACAM6 polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise an EPCAM polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise an ALCAM polypeptide.
• such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a CD55 polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a CDH1 polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a CDH3 polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a CD274 (PD-L1) polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a DSG2 polypeptide.
• PD-L1 CD274
• such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a EGFR polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a FOLR1 polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a IG1FR polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a MET polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a MSLN polypeptide.
• such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a MUC1 polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a sTn antigen polypeptide glycosylation. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a Tn antigen polypeptide glycosylation. In some embodiments, such an extracellular vesicle- associated membrane-bound polypeptide may be or comprise a T antigen polypeptide glycosylation. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a TACSTD2 polypeptide. In some embodiments, such an extracellular vesicle-associated membrane-bound polypeptide may be or comprise a TNFRSF10B polypeptide.
• FIG. 1 is a schematic diagram illustrating an exemplary workflow of profiling individual extracellular vesicles (EVs).
• the figure shows purification of EVs from plasma using size exclusion chromatography (SEC) and immunoaffmity capture of EVs displaying a specific membrane-bound protein marker (Panel A); detection of co-localized target markers (e.g, intravesicular proteins or surface proteins) on captured EVs using a target entity detection assay according to some embodiments described herein (Panel B).
• SEC size exclusion chromatography
• Panel A specific membrane-bound protein marker
• target entity detection assay according to some embodiments described herein
• FIG. 2 is a schematic diagram illustrating a target entity detection assay according to some embodiments described herein.
• a target entity detection assay uses a combination of detection probes, which combination is specific for detection of cancer.
• a duplex system includes a first detection probe for a target protein 1 (e.g, cancer marker 1) and a second detection probe for a target protein 2 (e.g, cancer marker 2) are added to a sample comprising a biological entity (e.g, extracellular vesicle).
• a target protein 1 e.g, cancer marker 1
• a target protein 2 e.g, cancer marker 2
• a biological entity e.g, extracellular vesicle
• detection probes each comprise a target binding moiety (e.g, an antibody agent against a target protein) coupled to an oligonucleotide domain, which comprises a double- stranded portion and a single-stranded overhang extended from one end of the oligonucleotide domain.
• a detection signal is generated when distinct target binding moieties (e.g ., antibody agents against target protein 1 and target protein 2, respectively) of the first and second detection probes are localized to the same biological entity (e.g., an extracellular vesicle) in close proximity such that the corresponding single-stranded overhangs hybridize to each other, thus allowing ligation of their oligonucleotide domains to occur.
• a control entity e.g, a biological entity from a healthy subject sample
• target protein 1 e.g, cancer marker 1
• target protein 2 e.g, cancer marker 2
• a biological entity from a cancer sample e.g, lung cancer
• target protein 1 and target protein 2 e.g, cancer marker 2
• a detection signal is generated.
• Figure 3 shows a graphical representation of the prevalence of different lung and bronchus cancer histological subtypes. Data obtained from the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) Program Cancer Statistics Review 1975- 2017 the entire contents of which are incorporated herein by reference. Category “Other” includes large cell carcinoma, non-small cell carcinoma, other specified carcinomas, carcinoma NOS, sarcoma, and other specified types. Determination and grouping of histological classifications are noted within the review.
• Figure 4 is a graphical representation of the population demographics of a pilot patient cohort. A total of 39 patient plasma samples obtained from this cohort were analyzed, an overview of age, sex, and cohort size for the patient lung cancer diagnostic stage are shown. Cohort samples were subsequently evaluated by exemplary assays described herein.
• Figure 5 is a graphical representation of an exemplary lung adenocarcinoma (LUAD) diagnostic assay as described herein. Normalized signals of healthy controls and LUAD patient cohorts using SLC34A2 antibody based EV capture with CEACAM6 + CEACAM6 detection probes. The horizontal cutoff line represents a 100% specificity threshold. Sensitivities of 16.7% for stage I LUAD, 60% for stage II LUAD, 50% for stage III LUAD, and 100% for stage IV LUAD were achieved.
• Figure 6 is a graphical representation of an exemplary lung adenocarcinoma diagnostic assay as described herein.
• Figure 7 is a graphical representation of an exemplary lung adenocarcinoma diagnostic assay as described herein. Normalized signals of healthy controls and LUAD patient cohorts using CEACAM5 antibody based EV capture with CEACAM6 + SLC34A2 detection probes. The horizontal cutoff line represents a 100% specificity threshold. Sensitivities of 16.7% for stage I LUAD, 20% for stage II LUAD, 50% for stage III LUAD, and 75% for stage IV LUAD were achieved.
• Figure 8 is a graphical representation of the correlation between exemplary lung adenocarcinoma diagnostic assays as described herein. Signal from SLC34A2 antibody based capture with CEACAM6 + CEACAM6 detection probes is depicted along the x-axis, while signal from SLC34A2 antibody based capture with CEACAM6 + EPCAM detection probes is depicted along the y-axis. Correlations were determined using the Pearson product-moment correlation coefficient. Strong correlations were observed, especially for stage III and stage IV LUAD samples.
• Figure 9 is a graphical representation of the correlation between exemplary Lung Adenocarcinoma diagnostic assays as described herein. Signal from SLC34A2 antibody based capture with CEACAM6 + CEACAM6 detection probes is depicted along the x-axis, while signal from CEACAM5 antibody based capture with CEACAM6 + SLC34A2 detection probes is depicted along the y-axis. Correlations were determined using the Pearson product-moment correlation coefficient. Strong correlations were observed, especially for stage III and stage IV LUAD samples.
• Figure 10 is a graphical representation of the correlation between exemplary Lung Adenocarcinoma diagnostic assays as described herein. Signal from SLC34A2 antibody based capture with CEACAM6 + EPCAM detection probes is depicted along the x-axis, while signal from CAECAM5 antibody based capture with CEACAM6 + SLC34A2 detection probes is depicted along the y-axis. Correlations were determined using the Pearson product-moment correlation coefficient. Strong correlations were observed, especially for stage III and stage IV LUAD samples.
• Figure 11 is a graphical representation of the population demographics of an expanded patient cohort. A total of 138 patient plasma samples obtained from this cohort were analyzed, an overview of age, sex, and cohort size for the patient lung cancer diagnostic stage are shown. Cohort samples were subsequently evaluated by exemplary assays described herein.
• Figure 12 is a graphical representation of an exemplary lung adenocarcinoma diagnostic assay as described herein. Normalized signals of healthy controls and LUAD patient cohorts using SLC34A2 antibody based EV capture with CEACAM6 + CEACAM6 detection probes.
• the horizontal cutoff line represents a 99.9% specificity threshold. Sensitivities of 50% for stage II LUAD, 55.5% for stage III LUAD, and 71.4% for stage IV LUAD were achieved.
• FIG. 13 is a schematic diagram illustrating a target entity detection assay according to some embodiments described herein.
• the figure shows an exemplary triplex target entity detection system, in which in some embodiments, three or more detection probes, each for a target protein, can be added to a sample comprising a biological entity (e.g ., extracellular vesicle).
• detection probes each comprise a target binding moiety (e.g., an antibody agent against a target protein) coupled to an oligonucleotide domain, which comprises a double-stranded portion and a single-stranded overhang extended from one end of the oligonucleotide domain.
• a target binding moiety e.g., an antibody agent against a target protein
• a detection signal is generated when the corresponding single-stranded overhangs of all three or more detection probes hybridize to each other to form a linear double- stranded complex, and ligation of at least one strand of the double-stranded complex occurs, thus allowing a resulting ligated product to be detected.
• Figure 14 is a non-limiting example of a double-stranded complex comprising four detection probes connected to each other in a linear arrangement through hybridization of their respective single-stranded overhangs.
• FIG. 15 is a schematic diagram illustrating a target entity detection assay of an exemplary embodiment described herein.
• a plurality of detection probes are added to a sample comprising a biological entity (e.g, extracellular vesicle).
• detection probes each comprise a target binding moiety (e.g, an antibody agent) coupled to an oligonucleotide domain, which comprises a double-stranded portion and a single-stranded overhang extended from one end of the oligonucleotide domain.
• a target binding moiety e.g, an antibody agent
• a detection signal is generated when all detection probes are localized to the same biological entity (e.g ., an extracellular vesicle or analyte) in close proximity such that the corresponding single- stranded overhangs hybridize to form a linear double-stranded complex, and ligation of at least one strand of the resulting linear double-stranded complex occurs, thereby allowing a ligated product to be detected.
• the same biological entity e.g ., an extracellular vesicle or analyte
• Figure 16A is a graphical representation of the discriminatory power of a biomarker combination for LUAD detection by simulating “healthy patients” and “cancer patients.”
• plasma samples from 5000 simulated “healthy patients” and 5000 simulated “cancer patients” were randomly selected from normal and cancer tissue databases, respectively.
• Simulated “cancer patients” were modeled to have tumors of varying size, ranging from lg to lOOOg. Based on the two pools of “healthy patients” and “cancer patients,” sensitivity of a biomarker combination to detect LUAD at 99% specificity was calculated.
• Figure 16B is an exemplary heatmap illustrating the ability of each possible combination of biomarkers from the list in Table 3 to detect LUAD based on simulated sensitivities for a lOOg tumor as described herein.
• Each row represents one biomarker combination and each column represents one LUAD cancer patient.
• Light grey indicates that a LUAD patient’s cancer was not detected using a given biomarker combination, and dark grey indicates that a LUAD patient’s cancer was detected using a given biomarker combination.
• the heatmap shows the sensitivity thresholds based on a lOOg tumor.
• Figure 16C is a histogram illustrating the AUC values for each possible LUAD biomarker combination based on the list in Table 3 against other cancer types.
• EV scores for biomarker combinations (wherein an EV score is a multiplication of all TPM expression values in a given biomarker combination) were calculated for lung adenocarcinomas and all other tumor types in a cancer molecular database (e.g., the Cancer Genome Atlas), except for lung squamous carcinoma, and then the AUC was calculated.
• the histogram shows the distribution of AUC values against all other cancers.
• biomarker combination described herein are shown to be specific to LUAD over other cancer types.
• Figure 17A is a graphical representation of certain biomarker combinations relative to a Healthy Smoker Sample Pool for detection of Early Stage lung adenocarcinoma (LUAD). Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Early Stage LUAD Sample Pool from the Healthy Smoker Sample Pool.
• the x- axis represents the difference in Ct value obtained from Healthy Smoker Pooled samples and Early Stage LEI AD Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probe).
• Figure 17B is a graphical representation of certain biomarker combinations relative to a Healthy Smoker Sample Pool for detection of Late Stage LUAD, Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Late Stage LUAD Sample Pool from the Healthy Smoker Sample Pool.
• the x-axis represents the difference in Ct value obtained from Healthy Smoker Pooled samples and Late Stage LUAD Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probe).
• Figure 18A is a graphical representation of certain biomarker combinations relative to a Healthy Smoker Sample Pool for detection of Early Stage lung squamous cell cancer (LUSC). Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Early Stage LUSC Sample Pool from the Healthy Smoker Sample Pool.
• the x- axis represents the difference in Ct value obtained from Healthy Smoker Pooled samples and Early Stage LUSC Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probe).
• Figure 18B is a graphical representation of certain biomarker combinations relative to a Healthy Smoker Sample Pool for detection of Late Stage LUSC. Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Late Stage LUSC Sample Pool from the Healthy Smoker Sample Pool.
• the x-axis represents the difference in Ct value obtained from Healthy Smoker Pooled samples and Late Stage LUSC Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probe).
• Figure 19A is a graphical representation of certain biomarker combinations relative to a Healthy Non-Smoker Sample Pool for detection of Early Stage LUAD. Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Early Stage LUAD Sample Pool from the Healthy Non-Smoker Sample Pool.
• the x-axis represents the difference in Ct value obtained from Healthy Non-Smoker Pooled samples and Early Stage LUAD Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probe).
• Figure 19B is a graphical representation of certain biomarker combinations biomarker combinations relative to a Healthy Non-Smoker Sample Pool for detection of Late Stage LUAD. Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Late Stage LUAD Sample Pool from the Healthy Non-Smoker Sample Pool.
• the x-axis represents the difference in Ct value obtained from Healthy Non-Smoker Pooled samples and Late Stage LUAD Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probe).
• Figure 20A is a graphical representation of certain biomarker combinations relative to a Healthy Non-Smoker Sample Pool for detection of Early Stage LUSC. Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Early Stage LUSC Sample Pool from the Healthy Non-Smoker Sample Pool.
• the x-axis represents the difference in Ct value obtained from Healthy Non-Smoker Pooled samples and Early Stage LUSC Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probe).
• Figure 20B is a graphical representation of certain biomarker combinations relative to a Healthy Non-Smoker Sample Pool for detection of Late Stage LUSC. Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Late Stage LUSC Sample Pool from the Healthy Non-Smoker Sample Pool.
• the x-axis represents the difference in Ct value obtained from Healthy Non-Smoker Pooled samples and Late Stage LUSC Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probe).
• Figure 21A is a graphical representation of certain biomarker combinations relative to a Healthy Smoker Sample Pool for detection of Early Stage LUAD. Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Early Stage LUAD Sample Pool from the Healthy Smoker Sample Pool. The x-axis represents the difference in Ct value obtained from Healthy Smoker Pooled samples and Early Stage LUAD Pooled samples. The y-axis represents certain biomarker combinations (target of capture probe, targets of detection probes).
• Figure 21B is a graphical representation of certain biomarker combinations relative to a Healthy Smoker Sample Pool for detection of Late Stage LUAD.
• Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Late Stage LUAD Sample Pool from the Healthy Smoker Sample Pool.
• the x-axis represents the difference in Ct value obtained from Healthy Smoker Pooled samples and Late Stage LUAD Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probes).
• Figure 22A is a graphical representation of certain biomarker combinations relative to a Healthy Smoker Sample Pool for detection of Early Stage LUSC. Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Early Stage LUSC Sample Pool from the Healthy Smoker Sample Pool.
• the x-axis represents the difference in Ct value obtained from Healthy Smoker Pooled samples and Early Stage LUSC Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probes).
• Figure 22B is a graphical representation of certain biomarker combinations relative to a Healthy Smoker Sample Pool for detection of Late Stage LUSC. Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Late Stage LUSC Sample Pool from the Healthy Smoker Sample Pool.
• the x-axis represents the difference in Ct value obtained from Healthy Smoker Pooled samples and Late Stage LUSC Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probes).
• Figure 23A is a graphical representation of certain biomarker combinations relative to a Healthy Non-Smoker Sample Pool for detection of Early Stage LUAD. Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Early Stage LUAD Sample Pool from the Healthy Non-Smoker Sample Pool.
• the x-axis represents the difference in Ct value obtained from Healthy Non-Smoker Pooled samples and Early Stage LUAD Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probes).
• Figure 23B is a graphical representation of certain biomarker combinations relative to a Healthy Non-Smoker Sample Pool for detection of Late Stage LUAD. Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Late Stage LUAD Sample Pool from the Healthy Non-Smoker Sample Pool.
• the x-axis represents the difference in Ct value obtained from Healthy Non-Smoker Pooled samples and Late Stage LUAD Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probe).
• Figure 24A is a graphical representation of certain biomarker combinations relative to a Healthy Non-Smoker Sample Pool for detection of Early Stage LUSC. Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Early Stage LUSC Sample Pool from the Healthy Non-Smoker Sample Pool.
• the x-axis represents the difference in Ct value obtained from Healthy Non-Smoker Pooled samples and Early Stage LUSC Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probes).
• Figure 24B is a graphical representation of certain biomarker combinations relative to a Healthy Non-Smoker Sample Pool for detection of Late Stage LUSC. Biomarker combinations were ranked (highest rank at the top of the chart) by their ability to distinguish the Late Stage LUSC Sample Pool from the Healthy Non-Smoker Sample Pool.
• the x-axis represents the difference in Ct value obtained from Healthy Non-Smoker Pooled samples and Late Stage LUSC Pooled samples.
• the y-axis represents certain biomarker combinations (target of capture probe, targets of detection probe).
• administering typically refers to the administration of a composition to a subject to achieve delivery of an agent that is, or is included in, a composition to a target site or a site to be treated.
• routes may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
• administration may be parenteral.
• administration may be oral.
• administration may involve only a single dose.
• administration may involve application of a fixed number of doses.
• administration may involve dosing that is intermittent (e.g ., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing ( e.g ., perfusion) for at least a selected period of time.
• Amplification refers to a template-dependent process that results in an increase in the amount and/or levels of a nucleic acid molecule relative to its initial amount and/or level.
• a template-dependent process is generally a process that involves template-dependent extension of a primer molecule, wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, J. D. et al., In: Molecular Biology of the Gene, 4th Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1987); which is incorporated herein by reference for the purpose described herein).
• antibody agent refers to an agent that specifically binds to a particular antigen.
• the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding.
• Exemplary antibody agents include, but are not limited to monoclonal antibodies or polyclonal antibodies.
• an antibody agent may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies.
• an antibody agent may include one or more sequence elements are humanized, primatized, chimeric, etc., as is known in the art.
• an antibody agent utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g, Zybodies®, etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated complementary determining regions (CDRs) or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g, shark single domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g, Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM ); single chain or Tandem diabodies (TandAb®); VHHs
• SMIPsTM Small Modular ImmunoPharmaceuticals
• an antibody may lack a covalent modification (e.g ., attachment of a glycan) that it would have if produced naturally.
• an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g, a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or other pendant group [e.g, poly-ethylene glycol, etc.]).
• an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g, at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR.
• CDR complementarity determining region
• an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR.
• an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR.
• an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain.
• an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.
• Antibody agents can be made by the skilled person using methods and commercially available services and kits known in the art. For example, methods of preparation of monoclonal antibodies are well known in the art and include hybridoma technology and phage display technology. Further antibodies suitable for use in the present disclosure are described, for example, in the following publications: Antibodies A Laboratory Manual, Second edition. Edward A. Greenfield. Cold Spring Harbor Laboratory Press (September 30, 2013); Making and Using Antibodies: A Practical Handbook , Second Edition. Eds. Gary C. Howard and Matthew R. Kaser. CRC Press (July 29, 2013); Antibody Engineering: Methods and Protocols , Second Edition (Methods in Molecular Biology). Patrick Chames.
• Antibodies may be produced by standard techniques, for example by immunization with the appropriate polypeptide or portion(s) thereof, or by using a phage display library. If polyclonal antibodies are desired, a selected host animal (e.g. , mouse, rabbit, goat, horse, chicken, etc.) is immunized with an immunogenic polypeptide bearing a desired epitope(s), optionally haptenized to another polypeptide. Depending on the host species, various adjuvants may be used to increase immunological response.
• Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
• Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to the desired epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffmity chromatography or any other method known in the art. Techniques for producing and processing polyclonal antisera are well known in the art.
• aptamer typically refers to a nucleic acid molecule or a peptide molecule that binds to a specific target molecule (e.g ., an epitope).
• a nucleic acid aptamer may be described by a nucleotide sequence and is typically about 15-60 nucleotides in length.
• a nucleic acid aptamer may be or comprise a single stranded and/or double-stranded structure.
• a nucleic acid aptamer may be or comprise DNA.
• a nucleic acid aptamer may be or comprise RNA.
• a peptide aptamer may be described to have one or more peptide loops of variable sequence displayed by a protein scaffold.
• Peptide aptamers can be isolated from combinatorial libraries and often subsequently improved by directed mutation or rounds of variable region mutagenesis and selection.
• aptamers may be obtained for a wide array of molecular targets, including proteins and small molecules.
• aptamers typically have very high affinities for their targets (e.g., affinities in the picomolar to low nanomolar range for proteins or polypeptides). Because aptamers are typically synthetic molecules, aptamers are amenable to a variety of modifications, which can optimize their function for particular applications.
• Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other.
• a particular biological phenomenon e.g, expression of a specific biomarker
• lung cancer e.g, a specific type of lung cancer and/or stage of lung cancer
• its presence correlates with incidence of and/or susceptibility of lung cancer (e.g, across a relevant population).
• biological entity may be utilized to refer to an entity or component that is present in a biological sample, e.g ., in some embodiments derived or obtained from a subject, which, in some embodiments, may be or comprise a cell or an organism, such as an animal or human, or, in some embodiments, may be or comprise a biological tissue or fluid.
• a biological entity is or comprises a cell or microorganism, or a fraction, extract, or component thereof (including, e.g. , intracellular components and/or molecules secreted by a cell or microorganism).
• a biological entity is or comprises a cell.
• a biological entity is or comprises an extracellular vesicle.
• a biological entity is or comprises a biological analyte (e.g, a metabolite, carbohydrate, protein or polypeptide, enzyme, lipid, organelle, cytokine, receptor, ligand, and any combinations thereof).
• a biological entity present in a sample is in a native state (e.g, proteins or polypeptides remain in a naturally occurring conformational structure).
• a biological entity is processed, e.g, by isolating from a sample or deriving from a naturally occurring biological entity.
• a biological entity can be processed with one or more chemical agents such that it is more desirable for detection utilizing technologies provided herein.
• a biological entity may be a cell or extracellular vesicle that is contacted with a fixative agent (e.g, but not limited to methanol and/or formaldehyde) to cause proteins and/or peptides present in the cell or extracellular vesicle to form crosslinks.
• a biological entity is in an isolated or pure form (e.g, isolated from a bodily fluid sample such as, e.g, a blood, serum, plasma sample, etc.).
• a biological entity may be present in a complex matrix (e.g, a bodily fluid sample such as, e.g, a blood, serum, or plasma sample, etc.).
• Biomarker typically refers to an entity, event, or characteristic whose presence, level, degree, type, and/or form, correlates with a particular biological event or state of interest, so that it is considered to be a “marker” of that event or state.
• a biomarker may be or comprise a marker for a particular disease state, or for likelihood that a particular disease, disorder or condition may develop, occur, or reoccur.
• a biomarker may be or comprise a marker for a particular disease or therapeutic outcome, or likelihood thereof.
• a biomarker may be or comprise a marker for a particular tissue (e.g, but not limited to brain, breast, colon, ovary and/or other tissues associated with a female reproductive system, pancreas, prostate and/or other tissues associated with a male reproductive system, liver, lung, and skin).
• a marker for a particular tissue in some embodiments, may be specific for a healthy tissue, specific for a diseased tissue, or in some embodiments may be present in a normal healthy tissue and diseased tissue (e.g ., a tumor); those skilled in the art, reading the present disclosure, will appreciate appropriate contexts for each such type of biomarker.
• a biomarker may be or comprise a cancer-specific marker (e.g., a marker that is specific to a particular cancer). In some embodiments, a biomarker may be or comprise a non-specific cancer marker (e.g, a marker that is present in at least two or more cancers).
• a cancer-specific marker e.g., a marker that is specific to a particular cancer.
• a biomarker may be or comprise a non-specific cancer marker (e.g, a marker that is present in at least two or more cancers).
• a non-specific cancer marker may be or comprise, in some embodiments, a generic marker for cancers (e.g, a marker that is typically present in cancers, regardless of tissue types), or in some embodiments, a marker for cancers of a specific tissue (e.g, but not limited to brain, breast, colon, ovary and/or other tissues associated with a female reproductive system, pancreas, prostate and/or other tissues associated with a male reproductive system, liver, lung, and skin).
• a biomarker is predictive; in some embodiments, a biomarker is prognostic; in some embodiments, a biomarker is diagnostic, of the relevant biological event or state of interest.
• a biomarker may be or comprise an entity of any chemical class, and may be or comprise a combination of entities.
• a biomarker may be or comprise a nucleic acid, a polypeptide, a lipid, a carbohydrate, a small molecule, an inorganic agent (e.g, a metal or ion), or a combination thereof.
• a biomarker is or comprises a portion of a particular molecule, complex, or structure; e.g, in some embodiments, a biomarker may be or comprise an epitope.
• a biomarker is a surface marker (e.g, a surface protein marker) of an extracellular vesicle associated with lung cancer.
• a biomarker is intravesicular (e.g, a protein or RNA marker that is present within an extracellular vesicle).
• a biomarker may be or comprise a genetic or epigenetic signature.
• a biomarker may be or comprise a gene expression signature.
• a “biomarker” appropriate for use in accordance with the present disclosure may refer to presence, level, and/or form of a molecular entity (e.g, epitope) present in a target marker.
• Blood-derived sample refers to a sample derived from a blood sample (i.e., a whole blood sample) of a subject in need thereof.
• blood-derived samples include, but are not limited to, blood plasma (including, e.g ., fresh frozen plasma), blood serum, blood fractions, plasma fractions, serum fractions, blood fractions comprising red blood cells (RBC), platelets, leukocytes, etc., and cell lysates including fractions thereof (for example, cells, such as red blood cells, white blood cells, etc., may be harvested and lysed to obtain a cell lysate).
• a blood-derived sample that is used with methods, systems, and/or kits described herein is a plasma sample.
• cancer is used herein to generally refer to a disease or condition in which cells of a tissue of interest exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation.
• cancer may comprise cells that are precancerous (e.g, benign), malignant, pre-metastatic, metastatic, and/or non-metastatic.
• precancerous e.g, benign
• malignant e.g., pre-metastatic, metastatic, and/or non-metastatic.
• Classification cutoff refers to a level, value, or score, or a set of values, or an indicator that is used to predict a subject’s risk for a disease or condition (e.g, lung cancer), for example, by defining one or more dividing lines among two or more subsets of a population (e.g, normal healthy subjects and subjects with inflammatory conditions vs. lung cancer subjects).
• a classification cutoff may be determined referencing at least one reference threshold level (e.g, reference cutoff) for a target biomarker signature described herein, optionally in combination with other appropriate variables, e.g, age, life-history-associated risk factors, hereditary factors, physical and/or medical conditions of a subject.
• a classification cutoff may be the same as a reference threshold (e.g, cutoff) pre-determined for the single target biomarker signature.
• a classification cutoff may reference two or more reference thresholds (e.g, cutoffs) each individually pre-determined for the corresponding target biomarker signatures, and optionally incorporate one or more appropriate variables, e.g, age, life-history-associated risk factors, hereditary factors, physical and/or medical conditions of a subject.
• a classification cutoff may be determined via a computer algorithm-mediated analysis that references at least one reference threshold level (e.g ., reference cutoff) for a target biomarker signature described herein, optionally in combination with other appropriate variables, e.g., age, life-history-associated risk factors, hereditary factors, physical and/or medical conditions of a subject.
• reference threshold level e.g., reference cutoff
• close proximity refers to a distance between two detection probes (e.g. two detection probes in a pair) that is sufficiently close enough such that an interaction between the detection probes (e.g, through respective oligonucleotide domains) is expected to likely occur.
• probability of two detection probes interacting with each other e.g, through respective oligonucleotide domains
• probability of two detection probes interacting with each other is at least 50% or more, including, e.g, at least 60%, at least 70%, at least 80%, at least 90% or more.
• a distance between two detection probes when they are in sufficiently close proximity to each other may range between approximately 0.1- 1000 nm, or 0.5-500 nm, or 1-250 nm.
• a distance between two detection probes when they are in sufficiently close proximity to each other may range between approximately 0.1-10 nm or between approximately 0.5-5 nm. In some embodiments, a distance between two detection probes when they are in sufficiently close proximity to each other may be less than 100 nm or shorter, including, e.g, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, less than 10 nm, less than 5 nm, less than 1 nm, or shorter. In some embodiments, a distance between two detection probes when they are in sufficiently close proximity to each other may range between approximately 40-1000 nm or 40 nm-500 nm.
• Comparable refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
• comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
• Complementary is used in reference to oligonucleotide hybridization related by base-pairing rules.
• sequence “C-A-G- T” is complementary to the sequence “G-T-C-A ”
• Complementarity can be partial or total.
• any degree of partial complementarity is intended to be included within the scope of the term “complementary” provided that the partial complementarity permits oligonucleotide hybridization.
• Partial complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules.
• Total or complete complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules.
• detecting is used broadly herein to include appropriate means of determining the presence or absence of an extracellular vesicle expressing a target biomarker signature of lung cancer or any form of measurement indicative of such an extracellular vesicle.
• detecting may include determining, measuring, assessing, or assaying the presence or absence, level, amount, and/or location of an entity of interest (e.g ., a surface protein biomarker, an intravesicular protein biomarker, or an intravesicular RNA biomarker) that corresponds to part of a target biomarker signature in any way.
• an entity of interest e.g ., a surface protein biomarker, an intravesicular protein biomarker, or an intravesicular RNA biomarker
• “detecting” may include determining, measuring, assessing, or quantifying a form of measurement indicative of an entity of interest (e.g., a ligated template indicative of a surface protein biomarker and/or an intravesicular protein biomarker, or a PCR amplification product indicative of an intravesicular mRNA). Quantitative and qualitative determinations, measurements or assessments are included, including semi-quantitative. Such determinations, measurements or assessments may be relative, for example when an entity of interest (e.g, a surface protein biomarker, an intravesicular protein biomarker, or an intravesicular RNA biomarker) or a form of measurement indicative thereof is being detected relative to a control reference, or absolute.
• an entity of interest e.g, a surface protein biomarker, an intravesicular protein biomarker, or an intravesicular RNA biomarker
• the term “quantifying” when used in the context of quantifying an entity of interest can refer to absolute or to relative quantification.
• Absolute quantification may be accomplished by correlating a detected level of an entity of interest (e.g ., a surface protein biomarker, an intravesicular protein biomarker, or an intravesicular RNA biomarker) or a form of measurement indicative thereof to known control standards (e.g., through generation of a standard curve).
• relative quantification can be accomplished by comparison of detected levels or amounts between two or more different entities of interest (e.g, different surface protein biomarkers, intravesicular protein biomarkers, or intravesicular RNA biomarkers) to provide a relative quantification of each of the two or more different entities of interest, i.e., relative to each other.
• entities of interest e.g, different surface protein biomarkers, intravesicular protein biomarkers, or intravesicular RNA biomarkers
• Detection label refers to any element, molecule, functional group, compound, fragment or moiety that is detectable. In some embodiments, a detection label is provided or utilized alone. In some embodiments, a detection label is provided and/or utilized in association with (e.g, joined to) another agent.
• detection labels include, but are not limited to: various ligands, radionuclides (e.g, 3 H, 14 C, 18 F, 19 F, 32 P, 35 S, 135 I, 125 I, 123 I, 64 CU, 187 Re, U1 ln, 90 Y, 99m Tc, 177 Lu, 89 Zr, etc.), fluorescent dyes, chemiluminescent agents (such as, for example, acridinium esters, stabilized dioxetanes, and the like), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots), metal nanoparticles (e.g, gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (such as, for example, dyes, colloidal gold, and the like), biotin, digoxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available
• Detection probe typically refers to a probe directed to detection and/or quantification of a specific target.
• a detection probe is a quantification probe, which provides an indicator representing level of a specific target.
• a detection probe refers to a composition comprising a target binding entity, directly or indirectly, coupled to an oligonucleotide domain, wherein the target binding entity specifically binds to a respective target (e.g, molecular target), and wherein at least a portion of the oligonucleotide domain is designed to permit hybridization with a portion of an oligonucleotide domain of another detection probe for a distinct target.
• an oligonucleotide domain appropriate for use in the accordance with the present disclosure comprises a double-stranded portion and at least one single-stranded overhang.
• an oligonucleotide domain may comprise a double-stranded portion and a single-stranded overhang at each end of the double-stranded portion.
• Double-stranded in the context of oligonucleotide domain is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen-bonded, helical arrangement typically associated with, for example, nucleic acid such as DNA.
• double-stranded as used herein is also meant to refer to those forms which include mismatches (e.g ., partial complementarity) and/or structural features as bulges, loops, or hairpins.
• Double-stranded complex typically refers to a complex comprising at least two or more (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) detection probes (e.g, as provided and/or utilized herein), each directed to a target (which can be the same target or a distinct target), connected or coupled to one another in a linear arrangement through hybridization of complementary single- stranded overhangs of the detection probes.
• a double-stranded complex may comprise an extracellular vesicle, wherein respective target binding moieties of the detection probes are simultaneously bound to the extracellular vesicle.
• epitope includes any moiety that is specifically recognized by an immunoglobulin (e.g, antibody or receptor) binding component or an aptamer.
• an epitope is comprised of a plurality of chemical atoms or groups on an antigen.
• such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation.
• such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation.
• at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized).
• Extracellular vesicle typically refers to a vesicle outside of a cell, e.g, secreted by a cell.
• secreted vesicles include, but are not limited to exosomes, microvesicles, microparticles, ectosomes, oncosomes, and apoptotic bodies.
• exosomes are nanometer-sized vesicles (e.g, between 40 nm and 120 nm) of endocytic origin that may form by inward budding of the limiting membrane of multi vesicular endosomes (MVEs), while microvesicles typically bud from the cell surface and their size may vary between 50 nm and 1000 nm.
• MVEs multi vesicular endosomes
• an extracellular vesicle is or comprises an exosome and/or a microvesicle.
• a sample comprising an extracellular vesicle is substantially free of apoptotic bodies.
• a sample comprising extracellular vesicles may comprise extracellular vesicles shed or derived from one or more tissues (e.g ., cancerous tissues and/or non-cancerous or healthy tissues).
• an extracellular vesicle in a sample may be shed or derived from a lung cancer tumor; in some embodiments, an extracellular vesicle is shed or derived from a tumor of a nondung cancer. In some embodiments, an extracellular vesicle is shed or derived from a healthy tissue. In some embodiments, an extracellular vesicle is shed or derived from a benign lung tumor. In some embodiments, an extracellular vesicle is shed or derived from a tissue of a subject with symptoms (e.g., non-specific symptoms) associated with lung cancer.
• Extracellular vesicle-associated membrane-bound polypeptide refers to a polypeptide that is present in the membrane of an extracellular vesicle.
• such a polypeptide may be tumor-specific.
• such a polypeptide may be tissue-specific (e.g, lung tissue-specific).
• such a polypeptide may be non-specific, e.g, it is present in one or more non-target tumors, and/or in one or more non-target tissues.
• Hybridization As used herein, the term “hybridizing”, “hybridize”, “hybridization”, “annealing”, or “anneal” are used interchangeably in reference to pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. Hybridization and the strength of hybridization (e.g, strength of the association between the nucleic acids) is impacted by various factors including, e.g, the degree of complementarity between the nucleic acids, stringency of the conditions involved, the melting temperature (T) of the formed hybridization complex, and the G:C ratio within the nucleic acids.
• T melting temperature
• Intravesicular protein biomarker refers to a marker indicative of the state (e.g, presence, level, and/or activity) of a polypeptide that is present within a biological entity (e.g, a cell or an extracellular vesicle). In many embodiments, an intravesicular protein biomarker is associated with or present within an extracellular vesicle.
• Intravesicular RNA biomarker refers to a marker indicative of the state (e.g ., presence and/or level) of a RNA (e.g, mRNA) that is present within a biological entity (e.g, a cell or an extracellular vesicle). In many embodiments, an intravesicular RNA biomarker is associated with or present within an extracellular vesicle.
• a RNA e.g., mRNA
• a biological entity e.g, a cell or an extracellular vesicle.
• an intravesicular RNA biomarker is associated with or present within an extracellular vesicle.
• Ligase As used herein, the term “ligase” or “nucleic acid ligase” refers to an enzyme for use in ligating nucleic acids. In some embodiments, a ligase is enzyme for use in ligating a 3'- end of a polynucleotide to a 5 '-end of a polynucleotide. In some embodiments, a ligase is an enzyme for use to perform a sticky-end ligation. In some embodiments, a ligase is an enzyme for use to perform a blunt-end ligation. In some embodiments, a ligase is or comprises a DNA ligase.
• Life-history-associated risk factors refers to individuals’ actions, experiences, medical history, and/or exposures in their lives which may directly or indirectly increase such individuals’ risk for a condition, e.g, lung cancer, relative to individuals who do not have such actions, experiences, medical history, and/or exposures in their lives.
• non-limiting examples of life-history-associated risk factors include smoking, alcohol, drugs, carcinogenic agents, diet, obesity, diabetes, chronic obstructive pulmonary disease (COPD), physical activity, sun exposure, radiation exposure, bituminous smoke exposure, exposure to infectious agents such as viruses and bacteria, and/or occupational hazard (see e.g., Jyoti Malhotra et ah, “Risk Factors for Lung Cancer Worldwide ” European Respiratory journal (2016) 48: 889-902; which is incorporated herein by reference for the purpose described herein).
• COPD chronic obstructive pulmonary disease
• Ligation refers to a method or composition known in the art for joining two oligonucleotides or polynucleotides.
• a ligation may be or comprise a sticky-end ligation or a blunt-end ligation.
• ligation involved in provided technologies is or comprises a sticky-end ligation.
• ligation refers to joining a 3' end of a polynucleotide to a 5' end of a polynucleotide.
• ligation is facilitated by use of a nucleic acid ligase.
• Non-cancer subjects generally refers to subjects who do not have non-benign lung cancer.
• a noncancer subject is a healthy subject.
• a non-cancer subject is a healthy subject below age 55.
• a non-cancer subject is a healthy subject with age 55 or above.
• a non-cancer subject is a subject with non-lung related health diseases, disorders, or conditions.
• a non-cancer subject is a subject having a benign lung tumor (e.g ., a benign mass observed in the thoracic or pulmonary cavity).
• nucleic acid/ Oligonucleotide refers to a polymer of at least 10 nucleotides or more.
• a nucleic acid is or comprises DNA.
• a nucleic acid is or comprises RNA.
• a nucleic acid is or comprises peptide nucleic acid (PNA).
• PNA peptide nucleic acid
• a nucleic acid is or comprises a single stranded nucleic acid.
• a nucleic acid is or comprises a double-stranded nucleic acid.
• a nucleic acid comprises both single and double-stranded portions.
• a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and/or one or more peptide bonds, e.g. , as in a “peptide nucleic acid”.
• a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues.
• natural residues e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil.
• a non-natural residue comprises a nucleoside analog (e.g, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 - methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 - propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof).
• a nucleoside analog e
• a non-natural residue comprises one or more modified sugars (e.g, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues.
• a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide.
• a nucleic acid has a nucleotide sequence that comprises one or more introns.
• a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g, by polymerization based on a complementary template, e.g., in vivo or in vitro , reproduction in a recombinant cell or system, or chemical synthesis.
• a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
• nucleotide refers to its art-recognized meaning. When a number of nucleotides is used as an indication of size, e.g ., of an oligonucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g. , of an oligonucleotide.
• a patient refers to any organism who is suffering or at risk of a disease or disorder or condition. Typical patients include animals (e.g, mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disease or disorder or condition. In some embodiments, a patient has been diagnosed with one or more diseases or disorders or conditions. In some embodiments, a disease or disorder or condition that is amenable to provided technologies is or includes cancer, or presence of one or more tumors. In some embodiments, a patient is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition.
• animals e.g, mammals such as mice, rats, rabbits, non-human primates, and/or humans.
• a patient is a human.
• a patient is suffering from or susceptible to one or more diseases or disorders
• Polypeptide typically has its art-recognized meaning of a polymer of at least three amino acids or more. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional, biologically active, or characteristic fragments, portions or domains ( e.g ., fragments, portions, or domains retaining at least one activity) of such complete polypeptides.
• polypeptides may contain L-amino acids, D-amino acids, or both and/or may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc.
• polypeptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof (e.g, may be or comprise peptidomimetics).
• Prevent or prevention when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.
• Primer refers to an oligonucleotide capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced (e.g, in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
• a primer is preferably single stranded for maximum efficiency in amplification.
• a primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of a primer can depend on many factors, e.g, temperature.
• Reference describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. In some embodiments, a reference or control in the context of a reference level of a target refers to a level of a target in a normal healthy subject or a population of normal healthy subjects.
• a reference or control in the context of a reference level of a target refers to a level of a target in a subject prior to a treatment.
• a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment.
• risk of a disease, disorder, and/or condition refers to a likelihood that a particular individual will develop the disease, disorder, and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
• sample typically refers to an aliquot of material obtained or derived from a source of interest.
• a sample is obtained or derived from a biological source (e.g ., a tissue or organism or cell culture) of interest.
• a source of interest may be or comprise a cell or an organism, such as an animal or human.
• a source of interest is or comprises biological tissue or fluid.
• a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humour, vomit, and/or combinations or component(s) thereof.
• a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravesicular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid.
• a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g, oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g, bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage).
• a biological sample is or comprises a liquid biopsy.
• a biological sample is or comprises cells obtained from an individual.
• a sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
• the term “sample” refers to a preparation that is obtained by processing ( e.g ., by removing one or more components of and/or by adding one or more agents to) a primary sample.
• a sample is a preparation that is processed by using a semi-permeable membrane or an affinity -based method such antibody -based method to separate a biological entity of interest from other non-target entities.
• Such a “processed sample” may comprise, for example, in some embodiments extracellular vesicles, while, in some embodiments, nucleic acids and/or proteins, etc., extracted from a sample.
• a processed sample can be obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc.
• Selective or specific when used herein with reference to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities, states, or cells. For example, in some embodiments, an agent is said to bind “specifically” to its target if it binds preferentially with that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute.
• specificity may be evaluated relative to that of a target-binding moiety for one or more other potential target entities (e.g, competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding moiety. In some embodiments, specificity is evaluated relative to that of a reference non-specific binding moiety. In some embodiments, a target-binding moiety does not detectably bind to the competing alternative target under conditions of binding to its target entity. In some embodiments, a target-binding moiety binds with higher on-rate, lower off-rate, increased affinity, decreased dissociation, and/or increased stability to its target entity as compared with the competing alternative target(s).
• Small molecule means a low molecular weight organic and/or inorganic compound.
• a “small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size.
• a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD.
• the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D.
• a small molecule is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not a protein or polypeptide ( e.g ., is not an oligopeptide or peptide). In some embodiments, a small molecule is not a polynucleotide (e.g., is not an oligonucleotide).
• a small molecule is not a polysaccharide. In some embodiments, a small molecule does not comprise a polysaccharide (e.g, is not a glycoprotein, proteoglycan, gly colipid, etc.). In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is biologically active. In some embodiments, suitable small molecules may be identified by methods such as screening large libraries of compounds (Beck- Sickinger & Weber (2001) Combinational Strategies in Biology and Chemistry (John Wiley & Sons, Chichester, London); by structure-activity relationship by nuclear magnetic resonance (Shuker etal.
• a small molecule may have a dissociation constant for a target in the nanomolar range.
• Specific binding refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur.
• a target-binding moiety that interacts with one particular target when other potential targets are present is said to "bind specifically" to the target with which it interacts.
• specific binding is assessed by detecting or determining degree of association between a targetbinding moiety and its partner; in some embodiments, specific binding is assessed by detecting or determining degree of dissociation of a target-binding moiety-partner complex; in some embodiments, specific binding is assessed by detecting or determining ability of a target-binding moiety to compete an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations.
• Stage of cancer refers to a qualitative or quantitative assessment of the level of advancement of a cancer (e.g ., lung cancer).
• criteria used to determine the stage of a cancer may include, but are not limited to, one or more of where the cancer is located in a body, tumor size, whether the cancer has spread to lymph nodes, whether the cancer has spread to one or more different parts of the body, etc.
• cancer may be staged using the AJCC staging system.
• the AJCC staging system is a classification system, developed by the American Joint Committee on Cancer for describing the extent of disease progress in cancer patients, which utilizes in part the TNM scoring system: Tumor size, Lymph Nodes affected, Metastases.
• cancer may be staged using a classification system that in part involves the TNM scoring system, according to which T refers to the size and extent of the main tumor, usually called the primary tumor; N refers to the number of nearby lymph nodes that have cancer; and M refers to whether the cancer has metastasized.
• a cancer may be referred to as Stage 0 (abnormal cells are present but have not spread to nearby tissue, also called carcinoma in situ , or CIS; CIS is not cancer, but it may become cancer), Stage I-III (cancer is present; the higher the number, the larger the tumor and the more it has spread into nearby tissues), or Stage IV (the cancer has spread to distant parts of the body).
• a cancer may be assigned to a stage selected from the group consisting of: in situ (abnormal cells are present but have not spread to nearby tissue); localized (cancer is limited to the place where it started, with no sign that it has spread); regional (cancer has spread to nearby lymph nodes, tissues, or organs): distant (cancer has spread to distant parts of the body); and unknown (there is not enough information to figure out the stage).
• Subject refers to an organism from which a sample is obtained, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g, mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans.
• a subject is a human subject, e.g, a human male or female subject.
• a subject is suffering from lung cancer.
• a subject is susceptible to lung cancer.
• a subject displays one or more symptoms or characteristics of lung cancer.
• a subject displays one or more non-specific symptoms of lung cancer.
• a subject does not display any symptom or characteristic of lung cancer.
• a subject is someone with one or more features characteristic of susceptibility to or risk of lung cancer.
• a subject is a patient.
• a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
• a subject is a subject (e.g ., male or female subject) determined to have a thoracic or pulmonary mass(es).
• a subject is an asymptotic subject.
• Such an symptomatic subject may be a subject (e.g., male or female subject) at average population risk, with life-history associated risk, or with hereditary risk.
• an asymptomatic subject may be a subject who has a family history of cancer, who has been previously treated for cancer, who is at risk of cancer recurrence after cancer treatment, who is in remission after cancer treatment, and/or who has been previously or periodically screened for the presence of at least one cancer biomarker.
• an asymptomatic subject may be a subject who has not been previously screened for cancer, who has not been diagnosed for cancer, and/or who has not previously received cancer therapy.
• a subject amenable to provided technologies is an individual selected based on one or more characteristics such as age, race, genetic history, medical history, personal history (e.g, smoking, alcohol, drugs, carcinogenic agents, diet, obesity, physical activity, sun exposure, radiation exposure, exposure to infectious agents such as viruses, and/or occupational hazard).
• characteristics such as age, race, genetic history, medical history, personal history (e.g, smoking, alcohol, drugs, carcinogenic agents, diet, obesity, physical activity, sun exposure, radiation exposure, exposure to infectious agents such as viruses, and/or occupational hazard).
• surface polypeptide or surface protein refers to a polypeptide or protein with one or more domains or regions present in and/or on the surface of the membrane of a biological entity (e.g, a cell, an extracellular vesicle, etc.).
• a surface protein may comprise one or more domains or regions spanning and/or associated with the plasma membrane of a biological entity (e.g, a cell, an extracellular vesicle, etc.).
• a surface protein may comprise one or more domains or regions spanning and/or associated with the plasma membrane of a biological entity (e.g ., a cell, an extracellular vesicle, etc.) and also protruding into the intracellular and/or intravesicular space.
• a biological entity e.g ., a cell, an extracellular vesicle, etc.
• a surface protein may comprise one or more domains or regions associated with the plasma membrane of a biological entity (e.g., a cell, an extracellular vesicle, etc.), for example, via one or more non-peptidic linkages.
• a surface protein may comprise one or more domains or regions that is/are anchored into either side of plasma membrane of a biological entity (e.g, a cell, an extracellular vesicle, etc.).
• a surface protein is associated with or present within an extracellular vesicle.
• a surface polypeptide or membrane-bound polypeptide may be associated with or present within a lung cancer-associated extracellular vesicle (e.g, an extracellular vesicle obtained or derived from a blood or blood-derived sample of a subject suffering from or susceptible to lung cancer).
• a lung cancer-associated extracellular vesicle e.g, an extracellular vesicle obtained or derived from a blood or blood-derived sample of a subject suffering from or susceptible to lung cancer.
• detection of the presence of at least a portion of a surface polypeptide or surface protein on/within extracellular vesicles can facilitate separation and/or isolation of lung cancer-associated extracellular vesicles from a biological sample (e.g, a blood or blood-derived sample) from a subject.
• detection of the presence of a surface polypeptide or surface protein may be or comprise detection of an intravesicular portion (e.g, an intravesicular epitope) of such a surface polypeptide or surface protein. In some embodiments, detection of the presence of a surface polypeptide or surface protein may be or comprise detection of a membrane-spanning portion of such a surface polypeptide or surface protein. In some embodiments, detection of the presence of a surface polypeptide or surface protein may be or comprise detection of an extravesicular portion of such a surface polypeptide or surface protein.
• an intravesicular portion e.g, an intravesicular epitope
• detection of the presence of a surface polypeptide or surface protein may be or comprise detection of a membrane-spanning portion of such a surface polypeptide or surface protein. In some embodiments, detection of the presence of a surface polypeptide or surface protein may be or comprise detection of an extravesicular portion of such a surface polypeptide or surface protein.
• surface protein biomarker refers to a marker indicative of the state (e.g, presence, level, and/or activity) of a surface protein (e.g, as described herein) of a biological entity (e.g, a cell or an extracellular vesicle).
• a surface protein refers to a polypeptide or protein with one or more domains or regions located in or on the surface of the membrane of a biological entity (e.g, a cell or an extracellular vesicle).
• a surface protein biomarker may be or comprise an epitope that is present on the interior side (intravesicular) or the exterior side (extravesicular) of the membrane. In some embodiments, a surface protein biomarker is associated with or present in an extracellular vesicle.
• Susceptible to An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not have been diagnosed with the disease, disorder, and/or condition.
• an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
• Target-binding moiety In general, the terms “target-binding moiety” and “binding moiety” are used interchangeably herein to refer to any entity or moiety that binds to a target of interest (e.g ., molecular target of interest such as a biomarker or an epitope).
• a target-binding moiety of interest is one that binds specifically with its target (e.g., a target biomarker) in that it discriminates its target from other potential binding partners in a particular interaction context.
• a target-binding moiety may be or comprise an entity or moiety of any chemical class (e.g, polymer, non-polymer, small molecule, polypeptide, carbohydrate, lipid, nucleic acid, etc.).
• a target-binding moiety is a single chemical entity.
• a target-binding moiety is a complex of two or more discrete chemical entities associated with one another under relevant conditions by non-covalent interactions.
• a target-binding moiety may comprise a “generic” binding moiety (e.g, one of biotin/avidin/streptavidin and/or a class-specific antibody) and a “specific” binding moiety (e.g, an antibody or aptamers with a particular molecular target) that is linked to the partner of the generic biding moiety.
• a “generic” binding moiety e.g, one of biotin/avidin/streptavidin and/or a class-specific antibody
• a “specific” binding moiety e.g, an antibody or aptamers with a particular molecular target
• Target biomarker signature refers to a combination of (e.g, at least 2 or more, including, e.g, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, or more) biomarkers, which combination correlates with a particular biological event or state of interest, so that one skilled in the art will appreciate that it may appropriately be considered to be a “signature” of that event or state.
• a target biomarker signature may correlate with a particular disease or disease state, and/or with likelihood that a particular disease, disorder or condition may develop, occur, or reoccur.
• a target biomarker signature may correlate with a particular disease or therapeutic outcome, or likelihood thereof.
• a target biomarker signature may correlate with a specific cancer and/or stage thereof.
• a target biomarker signature may correlate with lung cancer and/or a stage and/or a subtype thereof.
• a target biomarker signature comprises a combination of (e.g ., at least 2 or more, including, e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, or more) biomarkers that together are specific for an lung cancer or a subtype and/or a disease stage thereof), though one or more biomarkers in such a combination may be directed to a target (e.g, a surface protein biomarker, an intravesicular protein biomarker, and/or an intravesicular RNA) that is not specific to the lung cancer.
• a target e.g., a surface protein biomarker, an intravesicular protein biomarker, and/or an intravesicular RNA
• a target biomarker signature may comprise at least one biomarker specific to an lung cancer or a stage and/or subtype thereof (i.e., an lung cancer- specific target), and may further comprise a biomarker that is not necessarily or completely specific for the lung cancer (e.g, that may also be found on some or all biological entities such as, e.g, cells, extracellular vesicles, etc., that are not cancerous, are not of the relevant cancer, and/or are not of the particular stage and/or subtype of interest).
• a combination of biomarkers utilized in a target biomarker signature is or comprises a plurality of biomarkers that together are specific for the relevant target biological entities of interest (e.g., lung cancer cells of interest or extracellular vesicles secreted by lung cancer cells) (i.e., sufficiently distinguish the relevant target biological entities (e.g, lung cancer cells of interest or extracellular vesicles secreted by lung cancer cells) for detection from other biological entities not of interest for detection), such a combination of biomarkers is a useful target biomarker signature in accordance with certain embodiments of the present disclosure.
• the relevant target biological entities of interest e.g., lung cancer cells of interest or extracellular vesicles secreted by lung cancer cells
• a combination of biomarkers is a useful target biomarker signature in accordance with certain embodiments of the present disclosure.
• therapeutic agent refers to an agent or intervention that, when administered to a subject or a patient, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
• a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
• a therapeutic agent or therapy is a medical intervention (e.g ., surgery, radiation, phototherapy) that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
• a medical intervention e.g ., surgery, radiation, phototherapy
• Threshold level refers to a level that are used as a reference to attain information on and/or classify the results of a measurement, for example, the results of a measurement attained in an assay.
• a threshold level e.g., a cutoff
• a threshold level means a value measured in an assay that defines the dividing line between two subsets of a population (e.g, normal and/or non-lung cancer vs. lung cancer).
• a value that is equal to or higher than the threshold level defines one subset of the population, and a value that is lower than the threshold level defines the other subset of the population.
• a threshold level can be determined based on one or more control samples or across a population of control samples.
• a threshold level can be determined prior to, concurrently with, or after the measurement of interest is taken.
• a threshold level can be a range of values.
• Treat refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
• Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition.
• treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
• treatment may be administered to a subject at a later-stage of disease, disorder, and/or condition.
• Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g ., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook el al, Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for the purpose described herein.
• Lung cancer was responsible for an estimated 148,869 deaths in 2016 in the United States (U.S. Cancer statistics working group, 2019; which is incorporated herein by reference for the purpose described herein). The majority of these deaths are attributable to late diagnosis; the 5- year total survival rate for lung cancer in the United States from 2001 to 2007 was 15.6%. Patients with localized disease at diagnosis had a 5-year survival rate of 52%; however, the majority of patients received initial diagnosis when distant metastasis had already formed and those patients have a dismal 5-year survival rate of approximately 3.6% (Cruz et al, 2011; which is incorporated herein by reference for the purpose described herein).
• the present disclosure identifies the source of a problem with certain prior technologies including, for example, certain conventional approaches to detection and diagnosis of lung cancer.
• certain conventional diagnostic assays e.g, X-ray imaging, sputum testing, low-dose CT scanning, and / or molecular tests based on cell-free nucleic acids, serum proteins (e.g, CEA, CYFRA 21-1, NSE, ProGRP, and/or SCCA) and/or bulk analysis of extracellular vesicles, can be time- consuming, costly, and/or lacking sensitivity and/or specificity sufficient to provide a reliable and comprehensive diagnostic assessment.
• serum proteins e.g, CEA, CYFRA 21-1, NSE, ProGRP, and/or SCCA
• the present disclosure provides technologies (including systems, compositions, and methods) that solve such problems, among other things, by identification of biomarker combinations that are predicted to exhibit high sensitivity and specificity for lung cancer based on bioinformatics analysis.
• the present disclosure provides technologies (including systems, compositions, and methods) that solve such problems, by detecting co-localization of a target biomarker signature of lung cancer (e.g, identified by bioinformatics analysis) in individual extracellular vesicles, which comprises at least one extracellular vesicle-associated membrane-bound polypeptide and at least one target biomarker selected from the group consisting of surface protein biomarkers, internal protein biomarkers, and RNA biomarkers present in extracellular vesicles associated with lung cancer.
• a target biomarker signature of lung cancer e.g, identified by bioinformatics analysis
• the present disclosure provides technologies (including systems, compositions, and methods) that solve such problems, among other things, by detecting such target biomarker signature of lung cancer using a target entity detection approach that was developed by Applicant and described in U.S. Application No. 16/805,637, and International Application PCT/US2020/020529, both filed February 28, 2020 and entitled “Systems, Compositions, and Methods for Target Entity Detection,” which are based on interaction and/or co-localization of a target biomarker signature in individual extracellular vesicles.
• the contents of each of the aforementioned disclosures is incorporated herein by reference in their entirety.
• the present disclosure provides insights and technologies for achieving effective lung cancer screening, e.g, for early detection of lung cancer.
• the present disclosure provides technologies for early detection of lung cancer in subjects who may be experiencing one more symptoms associated with lung cancer.
• the present disclosure provides technologies for early detection of lung cancer in subjects who are at hereditary risks for lung cancer.
• the present disclosure provides technologies for early detection of lung cancer in subjects who may be at hereditary risk and/or experiencing one or more symptoms associated with lung cancer.
• the present disclosure provides technologies for early detection of lung cancer in subjects who may have life-history risk factors (e.g ., but not limited to smoking).
• the present disclosure provides technologies for screening individuals, e.g., individuals with certain risks (e.g, hereditary risk, life-history associated risk, or average risk), for early stage non-small cell lung cancers such as, e.g, lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC).
• non-small cell lung cancers are the most common subtype of lung cancer, in which 54% of cases are detected at an advanced stage (SEER Cancer Statistics Review 1975-2017).
• provided technologies are effective for detection of early stage lung cancers.
• provided technologies are effective when applied to populations comprising or consisting of individuals having one or more symptoms that may be associated with lung cancer.
• provided technologies are effective even when applied to populations comprising or consisting of asymptomatic or symptomatic individuals (e.g, due to sufficiently high sensitivity and/or low rates of false positive and/or false negative results). In some embodiments, provided technologies are effective when applied to populations comprising or consisting of individuals (e.g, asymptomatic or symptomatic individuals) without hereditary risk, and/or life-history related risk of developing lung cancer. In some embodiments, provided technologies are effective when applied to populations comprising or consisting of individuals (e.g, asymptomatic or symptomatic individuals) with hereditary risk, and/or life-history related risk of developing lung cancer.
• provided technologies are effective when applied to populations comprising or consisting of individuals susceptible to lung cancer (e.g, individuals with a known genetic, environmental, or experiential risk, etc.).
• provided technologies may be or include one or more compositions (e.g, molecular complexes, systems, collections, combinations, kits, etc.) and/or methods (e.g, of making, using, assessing, etc.), as will be clear to one skilled in the art reading the disclosure provided herein.
• provided technologies achieve detection (e.g, early detection, e.g, in asymptomatic individual(s) and/or population(s)) of one or more features (e.g, incidence, progression, responsiveness to therapy, recurrence, etc.) of lung cancer, with sensitivity and/or specificity (e.g, rate of false positive and/or false negative results) appropriate to permit useful application of provided technologies to single-time and/or regular (e.g, periodic) assessment.
• detection e.g, early detection, e.g, in asymptomatic individual(s) and/or population(s)
• features e.g, incidence, progression, responsiveness to therapy, recurrence, etc.
• sensitivity and/or specificity e.g, rate of false positive and/or false negative results
• provided technologies are useful in conjunction with an individual’s regular medical examinations, such as but not limited to: physicals, general practitioner visits, cholesterol/lipid blood tests, diabetes (type 2) screening, colonoscopies, blood pressure screening, thyroid function tests, prostate cancer screening, mammograms, HPV/Pap smears, and/or vaccinations.
• provided technologies are useful in conjunction with treatment regimen(s); in some embodiments, provided technologies may improve one or more characteristics (e.g ., rate of success according to an accepted parameter) of such treatment regimen(s).
• the present disclosure provides insights that screening of asymptotic individuals, e.g., regular screening prior to or otherwise in absence of developed symptom(s), can be beneficial, and even important for effective management (e.g, successful treatment) of lung cancer.
• the present disclosure provides lung cancer screening systems that can be implemented to detect lung cancer, including early-stage cancer, in some embodiments in asymptomatic individuals (e.g, without hereditary, and/or life- history associated risks in lung cancer).
• provided technologies are implemented to achieve regular screening of asymptomatic individuals (e.g, with or without hereditary and/or life-history associated risk(s) in lung cancer).
• provided technologies are implemented to achieve regular screening of symptomatic individuals (e.g, with or without hereditary and/or life-history associated risk(s) in lung cancer).
• the present disclosure provides, for example, compositions (e.g, reagents, kits, components, etc.), and methods of providing and/or using them, including strategies that involve regular testing of one or more individuals (e.g, asymptomatic individuals).
• compositions e.g, reagents, kits, components, etc.
• methods of providing and/or using them including strategies that involve regular testing of one or more individuals (e.g, asymptomatic individuals).
• the present disclosure defines usefulness of such systems, and provides compositions and methods for implementing them.
• SEER Surveillance, Epidemiology and End Results
• Lung cancer arises from the cells of the respiratory epithelium and can be divided into two broad categories.
• SCLC Small cell lung cancer
• SCLC is highly malignant and derived from cells exhibiting neuroendocrine characteristics, SCLC makes up -12 % of all lung cancer cases ( Figure 3).
• NSCLC Non-small cell lung cancer
• NSCLC accounts for the remaining 85% of cases, and is divided into three pathological subtypes: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.
• Adenocarcinoma itself accounts for -50% of all new lung cancer cases in the United States ( Figure 3), with squamous cell carcinoma accounting for -23%, and large cell carcinoma and other lung and bronchial cancers accounting for the remaining -15% ( Figure 3).
• the 5 year survival rates for all patients with invasive non-small cell cancer of the lung and bronchus is -25%. Patients with localized lung cancer at initial diagnosis have -63% 5 year survival rate, patients with regional lung cancer metastasis at initial diagnosis have -35% 5 year survival rates, while those with distant lung cancer metastasis have a poor -7% 5 year survival rates (SEER 1975-2017 review, Table 15.12). These data indicate that diagnoses for early-stage lung cancer is important as it can increase survival rates of lung cancer patients.
• Certain risk factors for lung cancer include age and a history of smoking.
• the International Agency for Research on Cancer has identified at least 50 known carcinogens in tobacco smoke.
• carcinogens include, but are not limited to tobacco-specific A-nitrosamines (TSNAs) formed by nitrosation of nicotine during tobacco processing and during smoking.
• TSNAs tobacco-specific A-nitrosamines
• NNK 4-(methylnitrosamino)-l(3-pyridyl)-l-butanone
• NNK is known to induce adenocarcinoma of the lung in experimental animals.
• NNK is known to bind to DNA and create DNA adducts, leading to DNA damage. Failure to repair this damage can lead to permanent mutations.
• NNK is associated with DNA mutations resulting in the activation of K-ras oncogenes, which is detected in 24% of human lung adenocarcinomas.
• High risk individuals and/or populations as defined by the CDC are 55-77 years of age, have a >30 cigarette pack-year history, are current smokers, or quit smoking within the last 15 years.
• low-dose CT scanning is currently the recommended lung cancer screening tool.
• low-dose CT in high-risk (e.g ., patients as defined by the CDC guidelines) populations can be considered relatively expensive, of limited access, and to have unreasonably high levels of false positives (e.g., the proportion of all positive tests that were falsely positive may be as great as 97.5%; Raghu et al, 2020 and Kinsinger et al, 2017 which are both incorporated herein by reference for the purpose described herein).
• the present disclosure provides a cost-effective screening assay with sufficiently high specificity and/or sensitivity.
• the present disclosure provides an insight that there is a need for development of a lung cancer liquid biopsy assay for screening subjects with a hereditary and/or life-history associated risk for lung cancer and/or subjects who may be experiencing one or more symptoms associated with lung cancer.
• the present disclosure provides an insight that there is a need for development of a lung cancer liquid biopsy assay for screening symptomatic or asymptomatic subjects e.g., prior to other screening methods, e.g, imaging methods for lung cancer detection such as, e.g. , MRI, CT scan, etc.
• the present disclosure provides technologies for effective screening of lung cancer in individuals at hereditary risk, or in individuals with life-history associated-risks. In some embodiments, the present disclosure provides technologies for effective screening of lung cancer in average-risk individuals. In some embodiments, the present disclosure provides technologies for effective screening of lung cancer in individuals with one or more symptoms associated with lung cancer. In some embodiments, the present disclosure provides technologies for effective screening of lung cancer in asymptomatic individuals.
• asymptomatic and/or average-risk individuals e.g, individuals under the age of 55 years, or individuals over the age of 55 years who have no history of smoking or have quit smoking for more than 15 years.
• asymptomatic and/or average-risk individuals e.g, individuals under the age of 55 years, or individuals over the age of 55 years who have no history of smoking or have quit smoking for more than 15 years.
• This is due, in part, to the cost, limited available, potential side effects, and/or poor performance (e.g, high false positive rate, or ineffectualness) of existing lung cancer screening technologies.
• inadequate test specificities ⁇ 99.5%
• the present disclosure provides an insight that a particularly useful lung cancer screening test would be characterized by: (1) ultrahigh specificity (>99.5%) to minimize the number of false positives, and (2) high sensitivity (>40%) for stage I and II lung cancer (i.e., when prognosis is most favorable).
• a particularly useful lung cancer screening test may be characterized by a specificity of >98% and a sensitivity of >50%, for example, for stage I and II lung cancer.
• a particularly useful lung cancer screening test may be characterized by a specificity of >98% and a sensitivity of >60%, for example, for stage I and II lung cancer.
• a particularly useful lung cancer screening test may be characterized by a specificity of >98% and a sensitivity of >70%, for example, for stage I and II lung cancer.
• a particularly useful lung cancer screening test may be characterized by a specificity of >99.5% and a sensitivity of >65%, for example, for stage I and II lung cancer. In some embodiments, a particularly useful lung cancer screening test may be characterized by a specificity of >99.5% and a sensitivity of >60%, for example, for stage I and II lung cancer. In some embodiments, a particularly useful lung cancer screening test may be characterized by a specificity of 99% or higher and a sensitivity of >10% or higher (including, e.g ., >15%, >20%, >25%. In some embodiments, a particularly useful lung cancer screening test may be characterized by a specificity of 99% or higher and a sensitivity of 50% or higher.
• the present disclosure provides an insight that a lung cancer screening test involving more than one set of biomarker combinations (e.g, at least two orthogonal biomarker combinations as described herein) can increase sensitivity of such an assay, as compared to that is achieved by one set of biomarker combination.
• a lung cancer screening test involving at least two orthogonal biomarker combinations can achieve a specificity of at least 98% and a sensitivity of at least 50%.
• a lung cancer screening test involving at least two orthogonal biomarker combinations can achieve a specificity of at least 98% and a sensitivity of at least 60%.
• a lung cancer screening test involving at least two orthogonal biomarker combinations can achieve a specificity of 99% and a sensitivity of 50% or higher.
• the present disclosure provides an insight that a particularly useful lung cancer screening test may be characterized by an acceptable positive predictive value (PPV) at an economically justifiable cost.
• PPV positive predictive value
• PPV is the likelihood a patient has the disease following a positive test, and is influenced by sensitivity, specificity, and/or disease prevalence.
• One clinician consensus for the minimum PPV needed to screen for lung cancer is 10%. With a 10% PPV, there would be nine false positives for every one true positive (Lung Cancer Screening: Recommendation Statement Am Fam Physician. 2005 Mar 15;71(6): 1165-1168). These false positives place a significant burden on both the healthcare system and subjects being screened as they lead to additional tests, unnecessary surgeries, and emotional and physical distress.
• assays described herein are particularly useful for early lung cancer detection that achieves a PPV of greater than 10% or higher, including, e.g., greater than 15%, greater than 20%, or greater than 25% or higher, with a specificity cutoff of at least 98% for subjects at hereditary risk for lung cancer, or with a specificity cutoff of at least 99.5% for subjects experiencing one or more symptoms associated with lung cancer.
• assays described herein can be useful for early lung cancer detection that achieves a PPV of greater than 2% or higher, including, e.g, greater than 3%, greater than 4%, greater than 5%, greater than 6% greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 15%, greater than 20%, or greater than 25% or higher.
• assays described herein can achieve a specificity cutoff of at least 95% or higher (e.g, a specificity cutoff of at least 98% for subjects at risk for lung cancer, or with a specificity cutoff of at least 99.5% for subjects experiencing one or more symptoms associated with lung cancer).
• ctDNA circulating tumor DNA
• CTCs circulating tumor cells
• EVs extracellular vesicles
• EVs are particularly promising due to their abundance and stability in the bloodstream relative to ctDNA and CTCs, suggesting improved sensitivity for early stage cancers.
• EVs contain cargo (i.e., proteins, RNA, metabolites) that originated from the same cell, providing superior specificity over bulk protein measurements. While the diagnostic utility EVs has been studied, much of this work has pertained to bulk EV measurements or low-throughput single-EV analyses.
• target biomarkers or combinations thereof for lung cancer.
• target biomarker signatures that are predicted to exhibit high sensitivity and specificity for lung cancer were discovered by a multi-pronged bioinformatics analysis and biological approach, which for example, in some embodiments involve computational analysis of a diverse set of data, e.g, in some embodiments comprising one or more of sequencing data, expression data, mass spectrometry, histology, post-translational modification data, and/or in vitro and/or in vivo experimental data through machine learning and/or computational modeling.
• a target biomarker signature of lung cancer comprises at least one or more (e.g ., 1, 2, 3, 4, 5, 6, 7, 8, or more) extracellular vesicle-associated membrane-bound polypeptide (e.g., surface polypeptide present in extracellular vesicles associated with lung cancer) and at least one or more (e.g, 1, 2, 3, 4, 5, 6, 7, 8, or more) target biomarkers selected from the group consisting of surface protein biomarker(s), intravesicular protein biomarker(s), and intravesicular RNA biomarker(s), such that the combination of such extracellular vesicle- associated membrane-bound polypeptide(s) and such target biomarker(s) present a target biomarker signature of lung cancer that provides (a) high specificity (e.g, greater than 98% or higher such as greater than 99% or greater than 99.5%) to minimize the number of false positives, and (b) high sensitivity (e.g, greater than 40%, greater than 50%, greater than 60%
• sensitivity and specificity rates for subjects with different lung cancer risk levels may vary depending upon the risk tolerance of the attending physician and/or the guidelines set forth by interested medical consortia.
• subjects at risk of lung cancer may be best served with a 99.5% specificity rate with 70% sensitivity or a 98% specificity rate with 80% sensitivity.
• at risk subjects with life-history-associated risk factors may be best served with a 99.5% specificity rate with 70% sensitivity or a 98% specificity rate with 80% sensitivity.
• an assay described herein for detection of lung cancer in at-risk subjects may have a set sensitivity rate that is lower than 80% sensitivity, including e.g, less than 70%, less than 60%, less than 50% or lower sensitivity rate.
• non-symptomatic subjects may be best served with a 99.5% specificity rate with 70% sensitivity or a 98% specificity rate with 80% sensitivity.
• an assay described herein for detection of lung cancer in non- symptomatic subjects may have a set sensitivity rate that is lower than 80% sensitivity, including e.g, less than 70%, less than 60%, less than 50% or lower sensitivity rate.
• technologies and/or assays described herein for detection of lung cancer in a symptomatic subject may have a lower sensitivity and/or specificity requirement than those for detection of lung cancer in an asymptomatic subject.
• an assay described herein for detection of lung cancer in a symptomatic subject may have a set specificity rate that is lower than 99.5% specificity, including e.g, less than 99% sensitivity, less than 95%, less than 90%, or less than 85% specificity rate.
• an assay described herein for detection of lung cancer in a symptomatic subject may have a set sensitivity rate that is lower than 80% sensitivity, including e.g. , less than 70%, or less than 60% sensitivity rate.
• the present disclosure observes that the gold standard for screening high-risk smokers is a chest CT, which had a reported positive predictive value of 3.8% in such a high-risk population in a National Lung Screening Trial study (National Lung Screening Trial Research Team (2013) “Results of initial low-dose computed tomographic screening for lung cancer. New England Journal of Medicine,” 368(21): 1980-1991).
• the present disclosure appreciates that a biomarker signature of lung cancer that provides a positive predictive value (PPV) of 3.8% or higher is particularly useful for screening individuals at risk for lung cancer.
• a target biomarker signature of lung cancer comprises at least one extracellular vesicle-associated membrane-bound polypeptide (e.g, surface polypeptide present in extracellular vesicles associated with lung cancer) and at least one target biomarker selected from the group consisting of surface protein biomarker(s), intravesicular protein biomarker(s), and intravesicular RNA biomarker(s), such that the combination of such extracellular vesicle-associated membrane-bound polypeptide(s) and such target biomarker(s) present a target biomarker signature of lung cancer that provides a positive predictive value (PPV) of at least 3.8% or higher, including, e.g, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10% or higher, at least 15% or higher, at least 20% or higher, at least 25% or higher, and/or at least 30% or higher, in high-risk population.
• PSV positive predictive value
• a target biomarker signature of lung cancer comprises at least one extracellular vesicle-associated membrane-bound polypeptide (e.g, surface polypeptide present in extracellular vesicles associated with lung cancer) and at least one target biomarker selected from the group consisting of surface protein biomarker(s), intravesicular protein biomarker(s), and intravesicular RNA biomarker(s), such that the combination of such extracellular vesicle- associated membrane-bound polypeptide(s) and such target biomarker(s) is specific for lung cancer.
• a target biomarker signature of lung cancer is or comprises:
• CD 166 antigen (ALCAM) polypeptide N-acetyllactosaminide beta- 1,3 -N- acetylglucosaminyltransferase 3 polypeptide encoded by the UDP-GlcNAc:betaGal beta-1, 3-N- acetylglucosaminyltransferase 3 (B3GNT3) gene, CUB domain containing protein 1 polypeptide encoded by the CUB domain-containing protein 1 (CDCP1) gene, Cadherin-1 (CDH1) polypeptide, cadherin 3 polypeptide encoded by the cadherin 3 (CDH3) gene, Complement decay-accelerating factor (CD55) polypeptide, Programmed cell death 1 ligand 1 (CD274; also known as PD-L1) polypeptide, carcinoembryonic antigen cell adhesion molecule 5 polypeptide encoded by the carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5) gene, carcinoembry
• a target biomarker signature of lung cancer is or comprises a surface protein biomarker selected from the group consisting of: Phospholipid-transporting ATPase ABCA3 (ABCA3) polypeptide, Multidrug resistance-associated protein 1 (ABCC1) polypeptide, ATP -binding cassette sub-family C member 3 (ABCC3) polypeptide, Golgi resident protein GCP60 (ACBD3) polypeptide, Long-chain-fatty-acid— CoA ligase 5 (ACSL5) polypeptide, Advanced glycosylation end product-specific receptor (AGER) polypeptide, CD 166 antigen (ALCAM) polypeptide, AP-1 complex subunit mu-2 (AP1M2) polypeptide, Gamma- secretase subunit APH-IA (APHIA) polypeptide, MICOS complex subunit MIC26 (APOO) polypeptide, Phospholipid-transporting ATPase IH (ATP11 A) polypeptide, Phospholipid-
• Calcineurin B homologous protein 3 (TESC) polypeptide, Transferrin receptor protein 1 (TFRC) polypeptide, Transmembrane channel-like protein 5 (TMC5) polypeptide, Calcium load- activated calcium channel (TMCOl) polypeptide, Transmembrane emp24 domain-containing protein 2 (TMED2) polypeptide, Transmembrane emp24 domain-containing protein 3 (TMED3) polypeptide, Transmembrane protein 132A (TMEM132A) polypeptide, Transmembrane protein 33 (TMEM33) polypeptide, Transmembrane protease serine 4 (TMPRSS4) polypeptide, Protein O-mannosyl-transferase TMTC3 (TMTC3) polypeptide, Mitochondrial import receptor subunit TOM22 homolog (TOMM22) polypeptide, Torsin-lA-interacting protein 2, isoform IFRG15 (TORI AIP2) polypeptide, Translocating chain-associated membrane protein 1 (TRAM1) polypeptide, Trans
• extracellular vesicle-associated membrane-bound polypeptide(s) included in a target biomarker signature of lung cancer is or comprises: SLC34A2 polypeptide, CEACAM5 polypeptide, CEACAM6 polypeptide, EpCAM polypeptide, and/or combinations thereof.
• SLC34A2 polypeptide is a multi-pass membrane transporter than has been studied as a therapeutic target for non-small cell lung cancer (Lin et al ., 2015; which is incorporated herein by reference for the purpose described herein).
• CEACAM5 polypeptide a member of the carcinoembryonic antigen (CEA) family of cell adhesion molecules (CAM), is a cell surface glycoprotein that has been implicated in gastrointestinal cancers and is thought to be involved in cellular differentiation, apoptosis, and polarity.
• CEACAM6 polypeptide is a member of the same protein family as CEACAM5, and has been implicated in Crohn’s disease and pancreatic adenocarcinoma, and is thought to be involved in the innate immune system and cell surface interactions.
• EpCAM polypeptide is implicated in gastrointestinal carcinomas and is thought to function as a homotypic calcium-independent cell adhesion molecule.
• SLC34A2 polypeptide, CEACAM5 polypeptide, CEACAM6 polypeptide, and/or EpCAM polypeptide are detected as intact EV associated trans-membrane proteins. In some embodiments of the present disclosure, SLC34A2 polypeptide, CEACAM5 polypeptide, CEACAM6 polypeptide, and/or EPCAM polypeptide are detected as EV associated trans-membrane polypeptides.
• a target biomarker included in a target biomarker signature of lung cancer is or comprises a surface protein biomarker selected from the group consisting of: CD166 antigen (ALCAM) polypeptide, canalicular multispecific organic anion transporter 2 polypeptide encoded by the ATP binding cassette subfamily C member 3 (ABCC3) gene, arylsulfatase L polypeptide encoded by the arylsulfatase L (ARSL) gene, N-acetyllactosaminide beta- 1,3 -N- acetylglucosaminyltransferase 3 polypeptide encoded by the UDP-GlcNAc:betaGal beta-1, 3-N- acetylglucosaminyltransferase 3 (B3GNT3) gene, CUB domain containing protein 1 polypeptide encoded by the CUB domain-containing protein 1 (CDCP1) gene, cadherin 1 polypeptide encoded by the cadher
• ACAM CD166 anti
• a target biomarker included in a target biomarker signature of lung cancer is or comprises a surface protein biomarker selected from the group consisting of: SLC34A2 polypeptide, CEACAM5 polypeptide, CEACAM6 polypeptide, EpCAM polypeptide, and combinations thereof.
• a target biomarker included in a target biomarker signature of lung cancer is or comprises a surface protein biomarker selected from the group consisting of: ALCAM polypeptide, CD55 polypeptide, CDH1 polypeptide, CDH3 polypeptide, CD274 (PD- Ll) polypeptide, CEACAM5 polypeptide, CEACAM6 polypeptide, DSG2 polypeptide, EGFR polypeptide, EPCAM polypeptide, FOLR1 polypeptide, IG1FR polypeptide, MET polypeptide, MSLN polypeptide, MUC1 polypeptide, SLC34A2 polypeptide, sTn antigen polypeptide glycosylation, Tn antigen polypeptide glycosylation, T antigen polypeptide glycosylation, TACSTD2 polypeptide, TNFRSF10B polypeptide, and combinations thereof.
• a target biomarker signature comprises one or more extracellular vesicle-associated membrane-bound polypeptide biomarkers, selected from a list consisting of a ABC A3 polypeptide, a ABCC1 polypeptide, a ABCC3 polypeptide, a ACBD3 polypeptide, a ACSL5 polypeptide, a AGER polypeptide, a ALCAM polypeptide, a AP1M2 polypeptide, a APHl A polypeptide, a APOO polypeptide, a ATP11 A polypeptide, a ATP1 IB polypeptide, a ATP1B1 polypeptide, a ATP6AP2 polypeptide, a B4GALT4 polypeptide, aBCAP31 polypeptide, a BSPRY polypeptide, a CD 109 polypeptide, a CD55 polypeptide, a CD9 polypeptide, a CDC42 polypeptide, a CDH1 polypeptide, a CD
• a target biomarker signature comprises one or more extracellular vesicle-associated membrane-bound polypeptide biomarkers, selected from a list consisting of a HS6ST2 polypeptide, a CYP2S1 polypeptide, a HAS3 polypeptide, a LAMC2 polypeptide, a ADAM23 polypeptide, a ABCA13 polypeptide, a TMPRSS4 polypeptide, a UGT1 A6 polypeptide, a ILDRl polypeptide, a CYP4F11 polypeptide, a PIGT polypeptide, a LAMB3 polypeptide, a PRSS21 polypeptide, a DSG3 polypeptide, a SDK2 polypeptide, and combinations thereof.
• a target biomarker signature comprises one or more extracellular vesicle-associated membrane-bound polypeptide biomarkers, selected from a list consisting of a HS6ST2 polypeptide, a CYP2S1 polypeptide, a HAS3 polypeptide, a LAMC2 polypeptide, a ADAM23 polypeptide, a ABCA13 polypeptide, a TMPRSS4 polypeptide, a UGT1 A6 polypeptide, a ILDRl polypeptide, a CYP4F11 polypeptide, a PIGT polypeptide, a LAMB3 polypeptide, a PRSS21 polypeptide, a DSG3 polypeptide, a SDK2 polypeptide, a FERMT1 polypeptide, a EPCAM polypeptide, a SDC1 polypeptide, a PANX2 polypeptide, a ULBP2 polypeptide, a ECE2 polypeptide, a KRTCAP
• a target biomarker included in a target biomarker signature of lung cancer is or comprises an intravesicular protein biomarker selected from the group consisting of: amiloride-sensitive amine oxidase [copper-containing] polypeptide encoded by the amine oxidase copper containing 1 (AOC1) gene, uncharacterized protein C12orf45 polypeptide encoded by the chromosome 12 open reading frame 45 (C12orf45) gene, cellular retinoic acid binding protein 2 polypeptide encoded by the cellular retinoic acid binding protein 2 (CRABP2) gene, cystatin SN polypeptide encoded by the cystatin SN (CST1) gene, ETS translocation variant 4 polypeptide encoded by the ETS variant transcription factor 4 (ETV4) gene, protein FAM83 A polypeptide encoded by the family with sequence similarity 83 member A (FAM83 A) gene, hepatocyte nuclear factor 3 -beta polypeptide encoded by the forkhead box
• AOC1 amiloride
• a target biomarker in a target biomarker signature of lung cancer is or comprises an intravesicular protein biomarker selected from the group consisting of: a ABRACL polypeptide, a ACP5 polypeptide, a ADH7 polypeptide, a AGR2 polypeptide, a AIF1 polypeptide, a AKR1C1 polypeptide, a AKR1C2 polypeptide, a AKR1C3 polypeptide, a ALDHl A1 polypeptide, a ALDH3A1 polypeptide, a ALDH3B2 polypeptide, a ALGIL polypeptide, a AP1M2 polypeptide, a APOBEC3B polypeptide, a APOBEC3C polypeptide, a ARNTL2 polypeptide, a ASF IB polypeptide, a AURKB polypeptide, a BAIAP2L1 polypeptide, a BIRC5 polypeptide,
• a target biomarker included in a target biomarker signature of lung cancer is or comprises an intravesicular RNA (e.g, mRNA) biomarker selected from the group consisting of: ABCC3 RNA, AOC1 RNA, ARSL RNA, B3GNT3 RNA, C12orf45 RNA, CDCP1 RNA, CDH1 RNA, CDH3 RNA, CEACAM5 RNA, CEACAM6 RNA, CELSR1 RNA, CLDN18 RNA, CLDN3 RNA, CLDN4 RNA, CLDN7 RNA, CLIC6 RNA, CRABP2 RNA, CST1 RNA, DMBT1 RNA, DSG2 RNA, EPCAM RNA, EPHX3 RNA, ETV4 RNA, EVA1A RNA, FAM83A RNA, FOLR1 RNA, FOXA2 RNA, GJB1 RNA, GJB2 RNA, GPC4 RNA, HMGB3 RNA
• a target biomarker signature for lung cancer comprises at least one or more (e.g ., 1, 2, 3, 4, 5, 6, 7, 8, or more) extracellular vesicle-associated membrane-bound polypeptides ( e.g ., ones described herein) and at least one or more ( e.g ., 1, 2, 3, 4, 5, 6, 7, 8, or more) surface protein biomarkers (e.g., ones described herein).
• at least one extracellular vesicle-associated membrane-bound polypeptide and at least one surface protein biomarker are the same.
• At least one extracellular vesicle- associated membrane-bound polypeptide and at least one surface protein biomarker(s) of a target biomarker signature for lung cancer are distinct.
• a target biomarker signature for lung cancer comprises at least one extracellular vesicle-associated membrane-bound polypeptide, which is or comprises a SLC34A2 polypeptide, and at least one surface protein biomarker, which is or comprises a CEACAM6 polypeptide, and/or an EpCAM polypeptide.
• a target biomarker signature for lung cancer comprises at least one extracellular vesicle-associated membrane-bound polypeptide, which is or comprises a CEACAM5 polypeptide, and at least one surface protein biomarker, which is or comprises a CEACAM6 polypeptide, and/or a SLC34A2 polypeptide.
• a target biomarker signature for lung cancer comprises at least one or more (e.g, 1, 2, 3, 4, 5, 6, 7, 8, or more) extracellular vesicle-associated membrane-bound polypeptides (e.g, ones described herein) and at least one or more (e.g, 1, 2, 3, 4, 5, 6, 7, 8, or more) intravesicular protein biomarkers (e.g, ones described herein).
• the extracellular vesicle-associated membrane-bound polypeptide(s) and the intravesicular protein biomarker(s) can be encoded by the same gene, while the former is expressed in the membrane of the extracellular vesicle and the latter is expressed within the extracellular vesicle.
• the extracellular vesicle-associated membrane- bound polypeptide(s) and the intravesicular protein biomarker(s) can be encoded by different genes.
• a target biomarker signature for lung cancer comprises at least one or more (e.g, 1, 2, 3, 4, 5, 6, 7, 8, or more) extracellular vesicle-associated membrane-bound polypeptides (e.g, ones described herein) and at least one or more (e.g, 1, 2, 3, 4, 5, 6, 7, 8, or more) intravesicular RNA (e.g, mRNA) biomarkers (e.g, ones described herein).
• the extracellular vesicle-associated membrane-bound polypeptide(s) and the intravesicular RNA (e.g, mRNA) biomarker(s) can be encoded by the same gene.
• a target biomarker signature for lung cancer comprises a combination of biomarkers as depicted in Table 4.
• a biomarker in such a combination is utilized as a capture probe polypeptide target (as an extracellular vesicle- associated membrane-bound polypeptide), for example, as depicted in Table 4.
• a biomarker in such a combination is utilized as a detection probe polypeptide target (as a target surface protein biomarker); for example, as depicted in Table 4.
• a target biomarker signature for lung cancer comprises at least a SLC34A2 polypeptide (as an extracellular vesicle-associated membrane-bound polypeptide) and a CEACAM6 polypeptide (as a target surface protein biomarker).
• a target biomarker signature for lung cancer comprises at least a SLC34A2 polypeptide (as an extracellular vesicle-associated membrane-bound polypeptide) and an EpCAM polypeptide (as a target surface protein biomarker).
• a target biomarker signature for lung cancer comprises at least a CEACAM5 polypeptide (as an extracellular vesicle-associated membrane-bound polypeptide) and a CEACAM6 polypeptide (as a target surface protein biomarker).
• a target biomarker signature for lung cancer comprises at least a CEACAM5 polypeptide (as an extracellular vesicle-associated membrane-bound polypeptide) and a SLC34A2 polypeptide (as a target surface protein biomarker).
• a target biomarker signature for lung cancer comprises at least a SLC34A2 polypeptide (as an extracellular vesicle-associated membrane-bound polypeptide) and at least two target surface protein biomarkers, which may be or comprise a CEACAM6 polypeptide and an EpCAM polypeptide.
• a target biomarker signature for lung cancer comprises at least a CEACAM5 polypeptide (as an extracellular vesicle-associated membrane-bound polypeptide) and at least two target surface protein biomarkers, which may be or comprise a CEACAM6 polypeptide and a SLC34A2 polypeptide.
• any one of provided biomarkers can be detected and/or measured by protein and/or RNA (e.g, mRNA) expression levels in wild-type form.
• protein and/or RNA e.g, mRNA
• any one of provided biomarkers can be detected and/or measured by protein and/or RNA (e.g, mRNA) expression levels in mutant form.
• protein and/or RNA e.g, mRNA
• mutant-specific detection of provided biomarkers e.g ., proteins and/or RNA such as, e.g., mRNAs
• provided biomarkers e.g ., proteins and/or RNA such as, e.g., mRNAs
• a biomarker is or comprises a particular form of one or more polypeptides or proteins (e.g, a pro- form, a truncated form, a modified form such as a glycosylated, phosphorylated, phosphatidylated, lipidated form, etc.).
• detection of such form detects a plurality (and, in some embodiments, substantially all) polypeptides present in that form (e.g, containing a particular modification such as, for example, a particular glycosylation, e.g, sialyl-Tn (sTn) glycosylation, e.g, a truncated O-glycan containing a sialic acid a-2,6 linked to GalNAc a-0-Ser/Thr.
• a particular modification such as, for example, a particular glycosylation, e.g, sialyl-Tn (sTn) glycosylation, e.g, a truncated O-glycan containing a sialic acid a-2,6 linked to GalNAc a-0-Ser/Thr.
• a surface protein biomarker can be or comprise a glycosylation moiety (e.g, an sTn antigen moiety, a Tn antigen moiety, or a T antigen moiety).
• Thompsen-nouvelle (Tn) antigen is an O-linked glycan that is thought to be associated with a broad array of tumors.
• Tn is a single alpha-linked GalNAc added to Ser or Thr as the first step of a major O-linked glycosylation pathway.
• T antigen typically refers to an O-linked glycan with the structure Gaipi-
• a surface protein biomarker can be or comprise a tumor-associated post-translational modification.
• a post-translational modification can be or comprise tumor-specific glycosylation patterns such as mucins with glycans aberrantly truncated at the initial GalNAc (e.g, Tn), or combinations thereof.
• a target biomarker signature comprises targets of a combination as depicted in Table 4, wherein a target may be used in a capture probe and/or detection probe.
• a target biomarker signature comprises a target of capture probe as depicted in Table 4 and at least one or more (including, e.g, at least two or more) targets of detection probes (e.g, detection probe 1 and/or detection probe 2).
• certain biomarker combinations as depicted in Table 4 that may be particularly useful (e.g, with higher sensitivity, specificity and/or PPV) for lung cancer detection can undergo an initial round of screening using an advanced stage (e.g, late stage, e.g, stage III and/or IV) lung cancer sample pool and the healthy control sample pool as a reference.
• select combinations can be further tested using early-stage lung cancer sample pools (e.g, stage I and/or II, optionally differentiated as appropriate), benign lung tumor plasma sample pools ( e.g ., as described herein), non-lung cancer sample pools ( e.g ., as described herein), and/or any combination thereof.
• biomarker combination performance can be determined by calculating the difference in assay signal (e.g., on a Ct basis) between the healthy sample pools and lung cancer sample pools.
• certain biomarker combinations for lung cancer detection can be selected with a delta Ct greater than inter-assay variability.
• biomarker combinations with a delta Ct greater than 2.0 (corresponding to a fourfold difference) or 1.0 (corresponding to a twofold difference) are considered to provide particularly effective diagnostic utility (e.g, providing a signal greater than inter-assay variability). See, e.g, Example 10, which provides an exemplary analysis of certain combinations described herein.
• a target biomarker signature for lung cancer can be or comprise targets of a combination as described in Table 4.
• a target biomarker signature for lung cancer comprises a set of markers that differentiates late stage lung cancer samples from a control sample (e.g, compared to healthy smoker samples, and/or compared to healthy nonsmoker samples; see e.g, Table 4).
• a target biomarker signature for lung cancer comprises a set of markers that differentiates early stage lung cancer samples from a control sample (e.g, compared to healthy smoker samples, and/or compared to healthy nonsmoker samples; see e.g, Table 4).
• an assay directed to detection of a target biomarker signature for lung cancer can comprise a combination of capture and detection probes as described in Table 4.
• a target biomarker signature for lung cancer can be or comprise targets of a combination as described in Example 10, Table 4 that differentiates subjects suffering from early stage lung cancer (e.g, early stage non-small cell lung cancer such as LUAD and/or LUSC) from subjects who do not have lung cancer (e.g, healthy subjects or subjects with a condition that is not lung cancer or is not associated with a lung condition).
• early stage lung cancer e.g, early stage non-small cell lung cancer such as LUAD and/or LUSC
• subjects who do not have lung cancer e.g, healthy subjects or subjects with a condition that is not lung cancer or is not associated with a lung condition.
• a target biomarker signature for lung cancer can be or comprise targets of a combination as described in Example 10, Table 4 that differentiates subjects suffering from late stage lung cancer (e.g, late stage non-small cell lung cancer such as LUAD and/or LUSC) from subjects who do not have lung cancer (e.g, healthy subjects or subjects with a condition that is not lung cancer or is not associated with a lung condition).
• late stage lung cancer e.g, late stage non-small cell lung cancer such as LUAD and/or LUSC
• subjects who do not have lung cancer e.g, healthy subjects or subjects with a condition that is not lung cancer or is not associated with a lung condition.
• a target biomarker signature for lung cancer can be or comprise targets of a combination as described in Example 10, Table 4 that differentiates subjects suffering from early stage lung cancer (e.g., early stage non-small cell lung cancer such as LUAD and/or LUSC) from subjects who are suffering from late stage lung cancer (e.g, late stage non-small cell lung cancer such as LUAD and/or LUSC).
• early stage lung cancer e.g., early stage non-small cell lung cancer such as LUAD and/or LUSC
• late stage non-small cell lung cancer e.g, late stage non-small cell lung cancer such as LUAD and/or LUSC
• a target biomarker signature for lung cancer can be or comprise targets of a combination that differentiate lung cancer from healthy samples in at least 8 out of 8 conditions tested as described in Example 10, Table 4. In some embodiments, a target biomarker signature for lung cancer can be or comprise targets of a combination that differentiate lung cancer from healthy samples in at least 7 out of 8 conditions tested as described in Example 10, Table 4. In some embodiments, a target biomarker signature for lung cancer can be or comprise targets of a combination that differentiate lung cancer from healthy samples in at least 6 out of 8 conditions tested as described in Example 10, Table 4. In some embodiments, a target biomarker signature for lung cancer can be or comprise targets of a combination that differentiate lung cancer from healthy samples in at least 5 out of 8 conditions tested as described in Example 10, Table 4.
• a target biomarker signature for lung cancer can be or comprise targets of a combination that differentiate lung cancer from healthy samples in at least 4 out of 8 conditions tested as described in Example 10, Table 4. In some embodiments, a target biomarker signature for lung cancer can be or comprise targets of a combination that differentiate lung cancer from healthy samples in at least 3 out of 8 conditions tested as described in Example 10, Table 4. In some embodiments, a target biomarker signature for lung cancer can be or comprise targets of a combination that differentiate lung cancer from healthy samples in at least 2 out of 8 conditions tested as described in Example 10, Table 4. In some embodiments, a target biomarker signature for lung cancer can be or comprise targets of a combination that differentiate lung cancer from healthy samples in at least 1 out of 8 conditions tested as described in Example 10, Table 4.
• a target biomarker signature for lung cancer detection comprises: a TNFRSF10B biomarker and a PD-L1 biomarker; or a TNFRSF10B biomarker and a CEACAM6 biomarker; or a TNFRSF10B biomarker and a EGFR biomarker; or a TNFRSF10B biomarker and a IGF1R biomarker; or a ALCAM biomarker and a EPCAM biomarker; or a CEACAM6 biomarker and a MUC1 biomarker; or a EGFR biomarker and a T antigen biomarker; or a EPCAM biomarker and a T biomarker; or a FOLR1 biomarker and a T antigen biomarker; or a Tn antigen biomarker and a TACSTD2 biomarker; or a TNFRSF10B biomarker and a FOLR1 biomarker; or a TNFRSF10B biomarker and a FO
• a target biomarker signature for lung cancer detection comprises a TNFRSF10B biomarker and a PD-L1 biomarker.
• a target biomarker signature for lung cancer detection comprises a TNFRSF10B biomarker and a CEACAM6 biomarker.
• a target biomarker signature for lung cancer detection comprises a TNFRSF10B biomarker and an EGFR biomarker.
• a target biomarker signature for lung cancer detection comprises a TNFRSF10B biomarker and an IGF1R biomarker.
• a target biomarker signature for lung cancer detection comprises an ALCAM biomarker and an EPCAM biomarker.
• a target biomarker signature for lung cancer detection comprises a CEACAM6 biomarker and a MUC1 biomarker.
• a target biomarker signature for lung cancer detection comprises an EGFR biomarker and a T antigen biomarker.
• a target biomarker signature for lung cancer detection comprises an EPCAM biomarker and a T antigen biomarker.
• a target biomarker signature for lung cancer detection comprises a FOLR1 biomarker and a T antigen biomarker.
• a target biomarker signature for lung cancer detection comprises a Tn antigen biomarker and a TACSTD2 biomarker.
• a target biomarker signature for lung cancer detection comprises a TNFRSF10B biomarker and a FOLR1 biomarker.
• a target biomarker signature for lung cancer detection comprises a TNFRSF10B biomarker and a sTn antigen biomarker.
• a target biomarker signature for lung cancer detection comprises a ALCAM biomarker and a PD-L1 biomarker.
• a target biomarker signature for lung cancer detection comprises a EPCAM biomarker and a MUC1 biomarker.
• a target biomarker signature for lung cancer detection comprises a TNFRSF10B biomarker and a CD55 biomarker.
• a target biomarker signature for lung cancer detection comprises a TNFRSF10B biomarker and a MUC1 biomarker.
• a target biomarker signature for lung cancer detection comprises a FOLR1 biomarker and a TACSTD2 biomarker.
• a target biomarker signature for lung cancer detection comprises a MET biomarker and a MUC1 biomarker.
• a target biomarker signature for lung cancer detection comprises a MET biomarker and a sTn antigen biomarker.
• a target biomarker signature for lung cancer detection comprises a MUC1 biomarker and a TACSTD2 biomarker.
• a target biomarker signature for lung cancer detection comprises a PD-L1 biomarker and a MUC1 biomarker. In certain embodiments, a target biomarker signature for lung cancer detection comprises a PD-L1 biomarker and a Tn antigen biomarker. In certain embodiments, a target biomarker signature for lung cancer detection comprises a SLC34A2 biomarker and a MET biomarker. In certain embodiments, a target biomarker signature for lung cancer detection comprises a SLC34A2 biomarker and a T antigen biomarker. In certain embodiments, a target biomarker signature for lung cancer detection comprises a TNFRSF10B biomarker and a CEACAM5 biomarker.
• a target biomarker signature for lung cancer detection comprises a TNFRSF10B biomarker and a MSLN biomarker. In certain embodiments, a target biomarker signature for lung cancer detection comprises a TNFRSF10B biomarker and a Tn antigen biomarker.
• a target biomarker signature comprises a combination of at least two target biomarkers, which combination can be selected from the following: a CYP2S1 polypeptide and a HS6ST2 polypeptide; or a ADAM23 polypeptide and a CYP2S1 polypeptide; or a ADAM23 polypeptide and a CYP4F11 polypeptide; or a ADAM23 polypeptide and a UGT1A6 polypeptide; or a ADAM23 polypeptide and a TMPRSS4 polypeptide; or a ADAM23 polypeptide and a ILDR1 polypeptide; or a DSG3 polypeptide and a UPK1B polypeptide; or a ABCA13 polypeptide and a CYP2S1 polypeptide; or a ABCA13 polypeptide and a ADAM23 polypeptide; or a ADAM23 polypeptide and a LAMC2 polypeptide; or a ADAM23 polypeptide
• a target biomarker signature comprises at least two target biomarkers, which is or comprises a CYP2S1 polypeptide and a HS6ST2 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a ADAM23 polypeptide and a CYP2S1 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a ADAM23 polypeptide and a TMPRSS4 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a ADAM23 polypeptide and a ILDRl polypeptide.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises a ABCA13 polypeptide and a CYP2S1 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a ABCA13 polypeptide and a ADAM23 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a HS6ST2 polypeptide and a LAMC2 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a CYP4F11 polypeptide and a HS6ST2 polypeptide.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises a ABCA13 polypeptide and a HS6ST2 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a ADAM23 polypeptide and a CYP4F11 polypeptide.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises a ADAM23 polypeptide and a UGT1 A6 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a ADAM23 polypeptide and a LAMC2 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a ADAM23 polypeptide and a HAS3 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a ADAM23 polypeptide and a LAMB3 polypeptide.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises a ADAM23 polypeptide and a ULBP2 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a ADAM23 polypeptide and a DSG3 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a CYP4F11 polypeptide and a LAMC2 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a CYP4F11 polypeptide and a SLC7A11 polypeptide.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises a ADAM23 polypeptide and a FERMT1 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a ADAM23 polypeptide and a CYP4F3 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a RACGAP1 polypeptide and a TFRC polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a CYP4F11 polypeptide and a HAS3 polypeptide.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises a CD9 polypeptide and a DSG3 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a DSG3 polypeptide and a UPK1B polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a DSG3 polypeptide and a RACGAP1 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a EPCAM polypeptide and a LAMP3 polypeptide.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises a DSG3 polypeptide and a ITGA2 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a CDH1 polypeptide and a DSG3 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a CDH3 polypeptide and a DSG3 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a KPNA2 polypeptide and a ULBP2 polypeptide.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises a TFRC polypeptide and a ULBP2 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a DSG3 polypeptide and a VTCN1 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a ULBP2 polypeptide and a UPK1B polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a DSG3 polypeptide and a NECTINl polypeptide.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises a DSG3 polypeptide and a PTPRZ1 polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a KPNA2 polypeptide and a UPK1B polypeptide. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises a RACGAPl polypeptide and a ULBP2 polypeptide. In some embodiments, a target biomarker in the foregoing combinations may be used as a target of a capture probe and/or a target of a detection probe of assays described herein.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and PD-L1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and CEACAM6 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and EGFR biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and IGF 1R biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises ALCAM and EPCAM biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CEACAM6 and MUC1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises EGFR and T antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises EPCAM and T biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises FOLR1 and T biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises Tn antigen and TACSTD2 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and FOLR1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and sTn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises ALCAM and PD-L1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises EPCAM and MUC1 biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and CD55 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and MUC1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises FOLR1 and TACSTD2 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises MET and MUC1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises MET and sTn antigen biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises MUC1 and TACSTD2 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises PD-L1 and MUC1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises PD-L1 and Tn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises SLC34A2 and MET biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises SLC34A2 and T antigen biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and CEACAM5 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and MSLN biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and Tn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises ALCAM and T antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises ALCAM and TACSTD2 biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises CD55 and PD-L1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CDH3 and CDH1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CEACAM5 and MUC1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CEACAM6 and EGFR biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CEACAM6 and EPCAM biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises CEACAM6 and FOLR1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CEACAM6 and MSLN biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CEACAM6 and sTn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CEACAM6 and TACSTD2 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises DSG2 and CEACAM6 biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises EGFR and MUC1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises FOLR1 and MUC1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises MUC1 and sTn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises SLC34A2 and MSLN biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises T and CDH1 biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises Tn antigen and IGF1R biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and ALCAM biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CD55 and EPCAM biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CEACAM6 and PD-L1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CEACAM6 and SLC34A2 biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises DSG2 and CEACAM5 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises DSG2 and EPCAM biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises DSG2 and MET biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises DSG2 and T biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises EGFR and sTn antigen biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises EGFR and TACSTD2 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises EPCAM and TACSTD2 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises IGF1R and T biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises MET and Tn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises MSLN and T antigen biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises PD-L1 and sTn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises PD- L1 and T antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises SLC34A2 and MUC1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises SLC34A2 and PD-L1 biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises SLC34A2 and sTn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises SLC34A2 and Tn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TACSTD2 and sTn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and CDH3 biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and EPCAM biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSF10B and MET biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises ALCAM and CDH3 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises ALCAM and MET biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises ALCAM and MUC1 biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises ALCAM and SLC34A2 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises ALCAM and sTn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CD55 and CDH1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CD55 and MET biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CDH3 and T biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises CEACAM5 and MSLN biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CEACAM5 and TACSTD2 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises CEACAM6 and Tn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises DSG2 and CDH1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises DSG2 and CDH3 biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises DSG2 and MUC1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises DSG2 and SLC34A2 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises DSG2 and sTn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises EGFR and MSLN biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises FOLR1 and sTn antigen biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises FOLR1 and Tn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises MET and T antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises MSLN and sTn antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises MUC1 and T antigen biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises MUC1 and Tn antigen biomarkers.
• a target biomarker signature comprises at least two target biomarkers, which is or comprises SLC34A2 and EGFR biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSFIOB and CDH1 biomarkers. In some embodiments, a target biomarker signature comprises at least two target biomarkers, which is or comprises TNFRSFIOB and SLC34A2 biomarkers. In some embodiments, a target biomarker in the foregoing combinations may be used as a target of a capture probe and/or a target of a detection probe of assays described herein.
• a target biomarker signature comprises a combination of at least three target biomarkers, which combination can be selected from the following: a ADAM23 polypeptide, a CYP2S1 polypeptide, and a LAMC2 polypeptide; or a CYP2S1 polypeptide, a HS6ST2 polypeptide, and a LAMC2 polypeptide; or a CYP2S1 polypeptide, a HS6ST2 polypeptide, and a PIGT polypeptide; or a ADAM23 polypeptide, a ILDR1 polypeptide, and a LAMC2 polypeptide; or a ABCA13 polypeptide, a CYP2S1 polypeptide, and aDSG2 polypeptide; or a CYP2S1 polypeptide, a HS6ST2 polypeptide, and a KPNA2 polypeptide; or a ABCA13 polypeptide, a ADAM23 polypeptide, and a
• a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a CYP2S1 polypeptide, and a LAMC2 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a CYP2S1 polypeptide, a HS6ST2 polypeptide, and a LAMC2 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a CYP2S1 polypeptide, a HS6ST2 polypeptide, and a PIGT polypeptide.
• a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a ILDR1 polypeptide, and a LAMC2 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a ABCA13 polypeptide, a CYP2S1 polypeptide, and a DSG2 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a CYP2S1 polypeptide, a HS6ST2 polypeptide, and a KPNA2 polypeptide.
• a target biomarker signature comprises at least three target biomarkers, which is or comprises a ABCA13 polypeptide, a ADAM23 polypeptide, and a UGT1 A6 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a CYP2S1 polypeptide, a HS6ST2 polypeptide, and a ULBP2 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a CYP2S1 polypeptide, and aNECTINl polypeptide.
• a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a UGT1 A6 polypeptide, and a ULBP2 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a LAMC2 polypeptide, and a UGT1 A6 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a CDH3 polypeptide, and a EPCAM polypeptide.
• a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a LAMP3 polypeptide, and a UGT1 A6 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a NRCAM polypeptide, and a UGT1 A6 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a PIGT polypeptide, and a UGT1 A6 polypeptide.
• a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a ECE2 polypeptide, and a UGT1 A6 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a LAMC2 polypeptide, and a PRSS21 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a LAMB3 polypeptide, and a LAMC2 polypeptide.
• a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a FXYD3 polypeptide, and a ULBP2 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a ADAM23 polypeptide, a RAP2B polypeptide, and a UGT1 A6 polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a DSG3 polypeptide, a EPCAM polypeptide, and a UPK1B polypeptide.
• a target biomarker signature comprises at least three target biomarkers, which is or comprises a DSG3 polypeptide, a KPNA2 polypeptide, and a UPK1B polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a DSG3 polypeptide, a LAMP3 polypeptide, and a UPK1B polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a DSG3 polypeptide, a ULBP2 polypeptide, and a UPK1B polypeptide.
• a target biomarker signature comprises at least three target biomarkers, which is or comprises a DSG3 polypeptide, a RAP2B polypeptide, and a UPK1B polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a DSG3 polypeptide, a LMNB2 polypeptide, and a UPK1B polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a ABCCl polypeptide, a DSG3 polypeptide, and a UPK1B polypeptide.
• a target biomarker signature comprises at least three target biomarkers, which is or comprises a DSG3 polypeptide, a TFRC polypeptide, and a UPK1B polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a CD9 polypeptide, a DSG3 polypeptide, and a UPK1B polypeptide. In some embodiments, a target biomarker signature comprises at least three target biomarkers, which is or comprises a DSG3 polypeptide, a LAMB3 polypeptide, and a UPK1B polypeptide. In some embodiments, at least one target biomarker in the foregoing combinations may be used as a target of a capture probe, and at least two target biomarkers may be used as targets of detection probes.
• a target biomarker signature for lung cancer detection comprises a combination of at least three target biomarkers, which combination can be selected from the following: a CEACAM6 polypeptide, a MUC1 polypeptide, and a sTn antigen polypeptide; or a TNFRSF10B polypeptide, a MUC1 polypeptide, and a CEACAM6 polypeptide; or a SLC34A2 polypeptide, a MUC1 polypeptide, and a CEACAM6 polypeptide; or a CEACAM6 polypeptide, a MUC1 polypeptide, and a MSLN polypeptide; or a FOLR1 polypeptide, a T antigen polypeptide, and a EGFR polypeptide; or a DSG2 polypeptide, a MFJC1 polypeptide, and a CEACAM6 polypeptide; or combinations thereof.
• at least one target biomarker in the foregoing combinations may be used as
• a target biomarker signature for lung cancer detection comprises a CEACAM6 polypeptide, a MUC1 and a sTn antigen polypeptide.
• a target polypeptide signature for lung cancer detection comprises a TNFRSF10B polypeptide, a MUC1 polypeptide, and a CEACAM6 polypeptide.
• a target polypeptide signature for lung cancer detection comprises a SLC34A2 polypeptide, a MUC1 polypeptide, and a CEACAM6 polypeptide.
• a target polypeptide signature for lung cancer detection comprises a CEACAM6 polypeptide, a MUC1 polypeptide, and a MSLN polypeptide.
• a target polypeptide signature for lung cancer detection comprises a FOLR1 polypeptide, a T antigen polypeptide, and a EGFR polypeptide.
• a target polypeptide signature for lung cancer detection comprises a DSG2 polypeptide, a MUC1 polypeptide, and a CEACAM6 polypeptide.
• at least one target biomarker in the foregoing combinations may be used as a target of a capture probe, and at least two target biomarkers may be used as targets of detection probes.
• the present disclosure provides technologies according to which a target biomarker signature is analyzed and/or assessed in a blood-derived sample comprising extracellular vesicles from a subject in need thereof; in some embodiments, a diagnosis or therapeutic decision is made based on such analysis and/or assessment.
• methods of detecting a target biomarker signature include methods for detecting one or more provided markers of a target biomarker signature as proteins.
• Exemplary protein-based methods of detecting one or more provided markers include, but are not limited to, proximity ligation assay, mass spectrometry (MS) and immunoassays, such as immunoprecipitation; Western blot; ELISA; immunohistochemistry; immunocytochemistry; flow cytometry; and immuno-PCR.
• an immunoassay can be a chemiluminescent immunoassay.
• an immunoassay can be a high- throughput and/or automated immunoassay platform.
• methods of detecting one or more provided markers as proteins in a sample comprise contacting a sample with one or more antibody agents directed to the provided markers of interest. In some embodiments, such methods also comprise contacting the sample with one or more detection labels. In some embodiments, antibody agents are labeled with one or more detection labels.
• detecting binding between a biomarker of interest and an antibody agent for the biomarker of interest includes determining absorbance values or emission values for one or more detection agents.
• the absorbance values or emission values are indicative of amount and/or concentration of biomarker of interest expressed by extracellular vesicles ( e.g ., higher absorbance is indicative of higher level of biomarker of interest expressed by extracellular vesicles).
• absorbance values or emission values for detection agents are above a threshold value.
• absorbance values or emission values for detection agents is at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.5, at least 3.0, at least 3.5 fold or greater than a threshold value.
• the threshold value is determined across a population of a control or reference group (e.g., non-cancer subjects).
• methods of detecting one or more provided markers include methods for detecting one or more provided markers as nucleic acids.
• Exemplary nucleic acid- based methods of detecting one or more provided markers include, but are not limited to, performing nucleic acid amplification methods, such as polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA).
• a nucleic acid-based method of detecting one or more provided markers includes detecting hybridization between one or more nucleic acid probes and one or more nucleotides that encode a biomarker of interest.
• the nucleic acid probes are each complementary to at least a portion of one of the one or more nucleotides that encode the biomarker of interest.
• the nucleotides that encode the biomarker of interest include DNA (e.g ., cDNA).
• the nucleotides that encode the biomarker of interest include RNA (e.g., mRNA).
• methods of detecting one or more provided markers involve proximity-ligation-immuno quantitative polymerase chain reaction (pliq-PCR).
• Pliq-PCR can have certain advantages over other technologies to profile EVs.
• pliq-PCR can have a sensitivity three orders of magnitude greater than other standard immunoassays, such as
• a pliq-PCR reaction can be designed to have an ultra- low LOD, which enables to detect trace levels of tumor-derived EVs, for example, down to a thousand EVs per mL.
• methods for detecting one or more provided markers may involve other technologies for detecting EVs, including, e.g, Nanoplasmic Exosome (nPLEX) Sensor (Im et al., 2014; which is incorporated herein by reference for the purpose described herein) and the Integrated Magnetic-Electrochemical Exosome (iMEX) Sensor (Jeong etal., 2016; which is incorporated herein by reference for the purpose described herein), which have reported LODs of ⁇ 10 3 and ⁇ 10 4 EVs, respectively (Shao etal., 2018; which is incorporated herein by reference for the purpose described herein).
• nPLEX Nanoplasmic Exosome
• iMEX Integrated Magnetic-Electrochemical Exosome
• methods for detecting one or more provided biomarkers in extracellular vesicles can be based on bulk EV sample analysis.
• methods for detecting one or more provided biomarkers in extracellular vesicles can be based on profiling individual EVs (e.g, single-EV profiling assays), which is further discussed in the section entitled “Exemplary Methods for Profiling Individual Extracellular Vesicles (EVs)” below.
• profiling individual EVs e.g, single-EV profiling assays
• extracellular vesicles in a sample may be captured or immobilized on a solid substrate prior to detecting one or more provided biomarkers in accordance with the present disclosure.
• extracellular vesicles may be captured on a solid substrate surface by non-specific interaction, including, e.g, adsorption.
• extracellular vesicles may be selectively captured on a solid substrate surface.
• a solid substrate surface may be coated with an agent that specifically binds to extracellular vesicles (e.g, an antibody agent specifically targeting extracellular vesicles, e.g, associated with lung cancer).
• a solid substrate surface may be coated with a member of an affinity binding pair and an entity of interest (e.g ., extracellular vesicles) to be captured may be conjugated to a complementary member of the affinity binding pair.
• an exemplary affinity binding pair includes, e.g., but is not limited to biotin and avidin-like molecules such as streptavidin.
• other appropriate affinity binding pairs can also be used to facilitate capture of an entity of interest to a solid substrate surface.
• an entity of interest may be captured on a solid substrate surface by application of a current, e.g, as described in Ibsen etal.
• a solid substrate may be provided in a form that is suitable for capturing extracellular vesicles and does not interfere with downstream handling, processing, and/or detection.
• a solid substrate may be or comprise a bead (e.g, a magnetic bead).
• a solid substrate may be or comprise a surface.
• such a surface may be a capture surface of an assay chamber (including, e.g, a tube, a well, a microwell, a plate, a filter, a membrane, a matrix, etc.).
• a method described herein comprises, prior to detecting provided biomarkers in a sample, capturing or immobilizing extracellular vesicles on a solid substrate.
• a sample may be processed, e.g, to remove undesirable entities such as cell debris or cells, prior to capturing extracellular vesicles on a solid substrate surface.
• a sample may be subjected to centrifugation, e.g, to remove cell debris, cells, and/or other particulates.
• centrifugation e.g., to remove cell debris, cells, and/or other particulates.
• size-exclusion-based purification or filtration Various size-exclusion-based purification or filtration are known in the art and those skilled in the art will appreciate that in some cases, a sample may be subjected to a spin column purification based on specific molecular weight or particle size cutoff.
• appropriate molecular weight or particle size cutoff for purification purposes can be selected, e.g, based on the size of extracellular vesicles.
• size-exclusion separation methods may be applied to samples comprising extracellular vesicles to isolate a fraction of extracellular vesicles that are of a certain size (e.g, greater than 30 nm and no more than 1000 nm, or greater than 70 nm and no more than 200 nm).
• extracellular vesicles may range from 30 nm to several micrometers in diameter.
• nanoparticles having a size range of about 30 nm to 1000 nm may be isolated, for example, in some embodiments by one or more size-exclusion separation methods, for detection assay.
• specific EV subtype(s) may be isolated, for example, in some embodiments by one or more size-exclusion separation methods, for detection assay.
• extracellular vesicles in a sample may be processed prior to detecting one or more provided biomarkers of a target biomarker signature for lung cancer.
• Different sample processing and/or preparation can be performed, e.g. , to stabilize targets (e.g, target biomarkers) in extracellular vesicles to be detected, and/or to facilitate exposure of targets (e.g, intravesicular proteins and/or RNA such as mRNA) to a detection assay (e.g, as described herein), and/or to reduce non-specific binding.
• targets e.g, intravesicular proteins and/or RNA such as mRNA
• sample processing and/or preparation examples include, but are not limited to, crosslinking molecular targets (e.g, fixation), permeabilization of biological entities (e.g, cells or extracellular vesicles), and/or blocking non-specific binding sites.
• crosslinking molecular targets e.g, fixation
• permeabilization of biological entities e.g, cells or extracellular vesicles
• blocking non-specific binding sites e.g., cell or extracellular vesicles
• the present disclosure provides a method for detecting whether a target biomarker signature of lung cancer is present or absent in a biological sample from a subject in need thereof, which may be in some embodiments a blood-derived sample comprising extracellular vesicles.
• such a method comprises (a) detecting, in a biological sample such as a blood-derived sample (e.g, a plasma sample) from a subject, biological entities of interest (including, e.g, extracellular vesicles) expressing a target biomarker signature of lung cancer; and (b) comparing sample information indicative of the level of the target biomarker signature-expressing biological entities of interest (e.g, extracellular vesicles) in the biological sample (e.g, blood-derived sample) to reference information including a reference threshold level.
• a biological sample such as a blood-derived sample (e.g, a plasma sample) from a subject
• biological entities of interest including, e.g, extracellular vesicles
• a target biomarker signature of lung cancer e.g, extracellular vesicles
• a reference threshold level corresponds to a level of biological entities of interest (e.g, extracellular vesicles) that express such a target biomarker signature in comparable samples from a population of reference subjects, e.g, noncancer subjects.
• biological entities of interest e.g, extracellular vesicles
• exemplary non-cancer subjects include healthy subjects (e.g, healthy subjects of specified age ranges, such as e.g, below age 55 or above age 55), subjects with non-lung related health diseases, disorders, or conditions (including, e.g, subjects having non-lung cancer such as ovarian cancer, colorectal cancer, etc., or subjects having symptoms of chronic obstructive pulmonary disease or chronic lung infections), subjects having benign lung tumors (e.g, a benign mass observed in the lung through imaging such as chest X- ray or low-dose CT scan), and combinations thereof.
• healthy subjects e.g, healthy subjects of specified age ranges, such as e.g, below age 55 or above age 55
• subjects with non-lung related health diseases, disorders, or conditions including, e.g, subjects having non-lung cancer such as ovarian cancer, colorectal cancer, etc., or subjects having symptoms of chronic obstructive pulmonary disease or chronic lung infections
• benign lung tumors e.g, a benign mass observed in the lung through imaging
• a sample is pre-screened for certain characteristics prior to utilization in an assay as described herein.
• a sample meeting certain prescreening criteria is more suitable for diagnostic applications than a sample failing pre-screening criteria.
• samples are visually inspected for appearance using known standards, e.g., is the sample normal, hemolyzed (red), icteric (yellow), and/or lipemic (turbid).
• samples can then be rated on a known standard scale (e.g, 1, 2,
• samples are visually inspected for hemolysis (e.g, heme) and rated on a scale from 1-5, where the visual inspection correlates with a known concentration, e.g, where 1 denotes approximately 0 mg/dL, 2 denotes approximately 50 mg/dL, 3 denotes approximately 150 mg/dL, 4 denotes approximately 250 mg/dL, and 5 denotes approximately 525 mg/dL.
• hemolysis e.g, heme
• concentration e.g, where 1 denotes approximately 0 mg/dL, 2 denotes approximately 50 mg/dL, 3 denotes approximately 150 mg/dL, 4 denotes approximately 250 mg/dL, and 5 denotes approximately 525 mg/dL.
• samples are visually inspected icteric levels (e.g, bilirubin) and rated on a scale from 1-5, where the visual inspection correlates with a known concentration, e.g., where 1 denotes approximately 0 mg/dL, 2 denotes approximately 1.7 mg/dL, 3 denotes approximately 6.6 mg/dL, 4 denotes approximately 16 mg/dL, and 5 denotes approximately 30 mg/dL.
• samples are visually inspected for turbidity (e.g.
• lipids and rated on a scale from 1-5, where the visual inspection correlates with a known concentration, e.g, where 1 denotes approximately 0 mg/dL, 2 denotes approximately 125 mg/dL, 3 denotes approximately 250 mg/dL, 4 denotes approximately 500 mg/dL, and 5 denotes approximately 1000 mg/dL.
• samples scoring lower than a certain level on one or more metrics may be utilized in an assay as described herein.
• samples scoring lower than a certain level on one or more metrics e.g, equal to or lower than a score of 3 may be utilized in an assay as described herein.
• samples scoring lower than a certain level on one or more metrics e.g, equal to or lower than a score of 2 may be utilized in an assay as described herein.
• samples scoring lower than a certain level on all three metrics e.g, hemolyzed, icteric, and lipemic
• a certain level on all three metrics e.g, hemolyzed, icteric, and lipemic
• low visual inspection scores on pre-screening criteria such as hemolysis, bilirubin, and/or lipemia (e.g, equal to or lower than a score of 2) may have no appreciable effect (e.g, not be correlated with) on diagnostic properties (e.g, Ct values) produced in an assay as described herein.
• a sample is determined to have extracellular vesicles expressing a target biomarker signature (e.g, ones described herein) when it shows an elevated level of target biomarker signature-expressing extracellular vesicles relative to a reference threshold level (e.g, ones described herein).
• a sample is determined to be positive for target biomarker signature-expressing extracellular vesicles if its level is at least 30% or higher, including, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or higher, as compared to a reference threshold level.
• a sample is determined to be positive for target biomarker signature-expressing extracellular vesicles if its level is at least 2-fold or higher, including, e.g, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1000-fold, at least 2500-fold, at least 5000-fold, or higher, as compared to a reference threshold level.
• a binary classification system may be used to determine whether a sample is positive for target biomarker signature-expressing extracellular vesicles. For example, in some embodiments, a sample is determined to be positive for target biomarker signatureexpressing extracellular vesicles if its level is at or above a reference threshold level, e.g, a cutoff value. In some embodiments, such a reference threshold level (e.g, a cutoff value) may be determined by selecting a certain number of standard deviations away from an average value obtained from control subjects such that a desired sensitivity and/or specificity of a lung cancer detection assay (e.g, ones described herein) can be achieved.
• a reference threshold level e.g, a cutoff value
• such a reference threshold level (e.g, a cutoff value) may be determined by selecting a certain number of standard deviations away from a maximum assay signal obtained from control subjects such that a desired sensitivity and/or specificity of a lung cancer detection assay (e.g, ones described herein) can be achieved.
• such a reference threshold level (e.g ., a cutoff value) may be determined by selecting the less restrictive of either (i) a certain number of standard deviations away from an average value obtained from control subjects, or (ii) a certain number of standard deviations away from a maximum assay signal obtained from control subjects, such that a desired sensitivity and/or specificity of a lung cancer detection assay (e.g., ones described herein) can be achieved.
• control subjects for determination of a reference threshold level (e.g, a cutoff value) may include, but are not limited to healthy subjects, subjects with inflammatory conditions (e.g, chronic obstructive pulmonary disease (COPD)), subjects with benign lung tumors, and combinations thereof.
• COPD chronic obstructive pulmonary disease
• a reference threshold level e.g, a cutoff value
• subjects with benign lung tumors are not included in determination of a reference threshold level (e.g, a cutoff value).
• a reference threshold level e.g, a cutoff value
• a desired specificity e.g, at least 95% or higher specificity [including, e.g, at least 96%, at least 97%, at least 98%, at least 99%, or higher specificity] such as in some embodiments at least 99.8% specificity
• a lung cancer detection assay e.g, ones described herein
• a reference threshold level (e.g, a cutoff value) may be determined by selecting at least 2.9 SDs (e.g, at least 2.93 SDs) away from (i) an average value obtained from control subjects, or (ii) a maximum assay signal obtained from control subjects, such that a desired specificity (e.g, at least 99%, or higher specificity) of a lung cancer detection assay (e.g, ones described herein) can be achieved.
• a desired specificity e.g, at least 99%, or higher specificity
• a reference threshold level (e.g, a cutoff value) may be determined by selecting at least 2.9 SDs (e.g, at least 2.93 SDs) away from the less restrictive of (i) an average value obtained from control subjects, or (ii) a maximum assay signal obtained from control subjects, such that a desired specificity (e.g, at least 99%, or higher specificity) of a lung cancer detection assay (e.g, ones described herein) can be achieved.
• a reference threshold level (e.g., a cutoff value) may be determined based on expression level (e.g, transcript level) of a target biomarker in normal healthy tissues vs.
• a reference threshold level (e.g, a cutoff value) may vary dependent on, for example, lung cancer stages and/or subtypes and/or patient characteristics, for example, patient age, risks factors for lung cancer (e.g, hereditary risk vs. average risk, life-history-associated risk factors), symptomatic/asymptomatic status, and combinations thereof.
• such a reference threshold level may be determined based on a log-normal distribution around healthy subjects (e.g, of specified age ranges), and optionally subjects with inflammatory conditions (e.g, COPD) and selection of the number of standard deviations (SDs) (e.g, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6 or higher SDs) necessary to achieve the specificity of interest (e.g, at least 95% or higher specificity [including, e.g, at least 96%, at least 97%, at least 98%, at least 99%, or higher specificity] such as in some embodiments at least
• the present disclosure also provides technologies for determining whether a subject as having or being susceptible to lung cancer. For example, in some embodiments, when a blood-derived sample from a subject in need thereof shows a level of target biomarker signature-expressing extracellular vesicles that is at or above a reference threshold level, e.g, cutoff value (e.g, as determined in accordance with the present disclosure), then the subject is classified as having or being susceptible to lung cancer.
• a reference threshold level e.g, cutoff value
• a reference threshold level (e.g, cutoff value) may be determined based on a lognormal distribution around healthy subjects (e.g, of specified age ranges), and optionally subjects with inflammatory conditions (e.g, COPD) and selection of the number of standard deviations (SDs) (e.g, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6 or higher SDs) necessary to achieve the specificity of interest (e.g, at least 95% or higher specificity [including, e.g, at least 96%, at least 97%, at least 98%, at least 99%, or higher specificity] such as in some embodiments at least 99.8% specificity),
• SDs standard
• a reference threshold level (e.g, a cutoff value) may be determined based on expression level (e.g, transcript level) of individual target biomarker(s) of a target biomarker signature in normal healthy tissues vs. in lung cancer samples such that the specificity and/or sensitivity of interest (e.g, as described herein) can be achieved.
• a reference threshold level (e.g, a cutoff value) may vary dependent on, for example, lung cancer stages and/or subtypes and/or patient characteristics, for example, patient age, risks factors for lung cancer (e.g, hereditary risk vs. average risk, life- history-associated risk factors), symptomatic/asymptomatic status, and combinations thereof.
• a blood-derived sample from a subject in need thereof shows an elevated level of target biomarker signature-expressing extracellular vesicles relative to a reference threshold level
• the subject is classified as having or being susceptible to lung cancer.
• a subject in need thereof is classified as having or being susceptible to lung cancer when his/her blood-derived sample shows a level of target biomarker signature-expressing extracellular vesicles that is at least 30% or higher, including, e.g, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or higher, as compared to a reference threshold level.
• a subject in need thereof is classified as having or being susceptible to lung cancer when his/her blood-derived sample shows a level of target biomarker signature-expressing extracellular vesicles that is at least 2-fold or higher, including, e.g, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7- fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40- fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1000-fold, or higher, as compared to a reference threshold level.
• a reference threshold level corresponds to a level of extracellular vesicles that express a target biomarker signature in comparable samples from a population of reference subjects, e.g, non-cancer subjects.
• exemplary non-cancer subjects include healthy subjects (e.g, healthy subjects of specified age ranges, such as e.g, below age 55 or above age 55), subjects with non-lung related health diseases, disorders, or conditions (including, e.g, subjects having non-lung cancer such as pancreatic cancer, colorectal cancer, etc., or subjects having symptoms of chronic obstructive pulmonary disease or chronic lung infections), subjects having benign lung tumors (e.g, a benign mass observed in the lung through imaging such as chest X-ray or low-dose CT scan), and combinations thereof.
• healthy subjects e.g, healthy subjects of specified age ranges, such as e.g, below age 55 or above age 55
• subjects with non-lung related health diseases, disorders, or conditions including, e.g, subjects having non-lung cancer such as pancreatic cancer, colorectal cancer, etc., or subjects having symptoms of chronic obstructive pulmonary disease or chronic lung infections
• subjects having benign lung tumors e.g, a benign mass observed in the lung through
• assays for profiling individual extracellular vesicles can be used to detect one or more provided biomarkers of one or more target biomarker signatures for lung cancer.
• an assay may involve (i) a capture assay through targeting one or more provided markers of a target biomarker signature for lung cancer and (ii) a detection assay for at least one or more additional provided markers of such a target biomarker signature for lung cancer, wherein such a capture assay is performed prior to such a detection assay.
• a capture assay is performed to selectively capture tumor- associated extracellular vesicles (e.g, lung tumor-associated extracellular vesicles) from a blood or blood-derived sample (e.g, plasma sample) of a subject in need thereof.
• a capture assay is performed to selectively capture extracellular vesicles of a certain size range, and/or certain characteristic(s), for example, extracellular vesicles associated with lung cancer.
• a blood or blood-derived sample may be pre-processed to remove non-extracellular vesicles, including, e.g, but not limited to soluble proteins and interfering entities such as, e.g, cell debris.
• extracellular vesicles are purified from a blood or blood-derived sample of a subject using size exclusion chromatography.
• extracellular vesicles can be directly purified from a blood or blood-derived sample using size exclusion chromatography, which in some embodiments may remove at least 90% or higher (including, e.g, at least 93%, 95%, 97%, 99% or higher) of soluble proteins and other interfering agents such as, e.g, cell debris.
• a capture assay comprises a step of contacting a blood or blood- derived sample with at least one capture agent comprising a target-capture moiety that binds to at least one or more provided biomarkers of a target biomarker signature for lung cancer.
• a capture assay may be multiplexed, which comprises a step of contacting a blood or blood-derived sample with a set of capture agents, each capture agent comprising a target- capture moiety that binds to a distinct provided biomarker of a target biomarker signature for lung cancer.
• a target-capture moiety is directed to an extracellular vesicle- associated membrane-bound polypeptide ( e.g ., ones as described and/or utilized herein).
• a target-capture moiety may be immobilized on a solid substrate.
• a capture agent employed in a capture assay is or comprises a solid substrate comprising at least one or more (e.g., 1, 2, 3, 4, 5, or more) target- capture moiety conjugated thereto, each target-capture moiety directed to an extracellular vesicle-associated membrane-bound polypeptide (e.g, ones as described and/or utilized herein).
• a solid substrate may be provided in a form that is suitable for capturing extracellular vesicles and does not interfere with downstream handling, processing, and/or detection.
• a solid substrate may be or comprise a bead (e.g, a magnetic bead).
• a solid substrate may be or comprise a surface.
• a surface may be a capture surface of an assay chamber (including, e.g, a tube, a well, a microwell, a plate, a filter, a membrane, a matrix, etc.).
• a capture agent is or comprises a magnetic bead comprising a target-capture moiety conjugated thereto.
• a detection assay is performed to detect one or more provided biomarkers of a target biomarker signature for lung cancer (e.g, ones that are different from ones targeted in a capture assay) in extracellular vesicles that are captured by a capture assay (e.g, as described above).
• a detection assay comprises immuno-PCR.
• an immuno-PCR may involve at least one probe targeting a single provided biomarker (e.g, ones described herein) of a target biomarker signature for lung cancer.
• an immuno-PCR may involve a plurality of (e.g., at least two, at least three, at least four, or more) probes directed to different epitopes of the same biomarker (e.g, ones described herein) of a target biomarker signature.
• an immuno-PCR may involve a plurality of (e.g., at least two, at least three, at least four, or more) probes, each directed to a different provided biomarker described herein.
• a detection assay comprises reverse transcription polymerase chain reaction (RT-PCR).
• RT-PCR reverse transcription polymerase chain reaction
• an RT-PCR may involve at least one primer/probe set targeting a single provided biomarker described herein.
• an RT-PCR may involve a plurality of ( e.g ., at least two, at least three, at least four, or more) primer/probe sets, each set directed to a different provided biomarker described herein.
• a detection assay comprises a proximity-ligation-immuno quantitative polymerase chain reaction (pliq-PCR), for example, to determine co-localization of one or more provided biomarkers of a target biomarker signature for lung cancer within extracellular vesicles (e.g., captured extracellular vesicles that express at least one extracellular vesicle-associated membrane-bound polypeptide).
• pliq-PCR proximity-ligation-immuno quantitative polymerase chain reaction
• a detection assay employs a target entity detection system that was developed by Applicant and described in U.S. Application No. 16/805,637, and International Application PCT/US2020/020529, both filed February 28, 2020 and entitled “Systems, Compositions, and Methods for Target Entity Detection” (the “’637 application” and the “’529 application”; both of which are incorporated herein by reference in their entirety) which are, in part, based on interaction and/or co-localization of a target biomarker signature in individual extracellular vesicles.
• a target entity detection system as described in the ’637 application and ‘529 application and also further described below in the section entitled
• Provided Target Entity Detection Systems and Methods Involving the Same can detect in a sample (e.g, in a biological, environmental, or other sample), in some embodiments at a single entity level, entities of interest (e.g, biological or chemical entities of interest, such as extracellular vesicles or analytes) comprising at least one or more (e.g, at least two or more) targets (e.g, molecular targets).
• entities of interest e.g, biological or chemical entities of interest, such as extracellular vesicles or analytes
• targets e.g, molecular targets.
• provided target entity detection systems are useful for a wide variety of applications and/or purposes, including, e.g, for detection of lung cancer.
• provided target entity detection systems may be useful for medical applications and/or purposes.
• provided target entity detection systems may be useful to screen (e.g, regularly screen) individuals (e.g, in some embodiments which may be asymptomatic individuals, or in some embodiments which may be individuals experiencing one or more symptoms associated with lung cancer, or in some embodiments which may be individuals at risk for lung cancer such as, e.g, individuals with a hereditary risk for lung cancer and/or life-history-associated risk factor, or smoking individuals) for a disease or condition (e.g, lung cancer).
• individuals e.g, in some embodiments which may be asymptomatic individuals, or in some embodiments which may be individuals experiencing one or more symptoms associated with lung cancer, or in some embodiments which may be individuals at risk for lung cancer such as, e.g, individuals with a hereditary risk for lung cancer and/or life-history-associated risk factor, or smoking individuals
• a disease or condition e.g, lung cancer
• provided target entity detection systems may be useful to screen (e.g, regularly screen) individuals (e.g, in some embodiments which may be asymptomatic individuals, or in some embodiments which may be individuals experiencing one or more symptoms associated with lung cancer, or in some embodiments which may be individuals at risk for lung cancer such as, e.g ., individuals with a hereditary risk for lung cancer and/or life-history-associated risk factor, or smoking individuals) for different types of cancer (e.g, for a plurality of different cancers, one of which may be lung cancer).
• individuals e.g, in some embodiments which may be asymptomatic individuals, or in some embodiments which may be individuals experiencing one or more symptoms associated with lung cancer, or in some embodiments which may be individuals at risk for lung cancer such as, e.g ., individuals with a hereditary risk for lung cancer and/or life-history-associated risk factor, or smoking individuals
• different types of cancer e.g, for a plurality of different cancers, one of which may be lung cancer.
• provided target entity detection systems are effective even when applied to populations comprising or consisting of asymptomatic individuals (e.g, due to sufficiently high sensitivity and/or low rates of false positive and/or false negative results).
• provided target entity detection systems may be useful as a companion diagnostic in conjunction with a disease treatment (e.g, treatment of lung cancer).
• a plurality of (e.g, at least two or more) detection assays may be performed to detect a plurality of biomarkers (e.g, at least two or more) of one or more target biomarker signatures for lung cancer (e.g, ones that are different from ones targeted in a capture assay) in extracellular vesicles, e.g, ones that are captured by a capture assay (e.g, as described above).
• a plurality of detection assays comprises (i) a provided target entity detection system or a system described in the ’637 application and ‘529 application; and (ii) immuno-PCR.
• a plurality of detection assays comprises (i) a provided target entity detection system or a system described in the ’637 application and ‘529 application; and (ii) RT-PCR.
• a target entity detection system that can be useful in a detection assay for one or more provided biomarkers of one or more target biomarker signatures for lung cancer includes a plurality of detection probes each for a specific target (e.g, a provided biomarker of a target biomarker signature).
• a specific target e.g, a provided biomarker of a target biomarker signature
• such a system comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, or more detection probes each for a specific target (e.g, a provided biomarker of a target biomarker signature).
• such a system comprises 2-50 detection probes each for a specific target (e.g, a provided biomarker of a target biomarker signature). In some embodiments, such a system comprises 2-30 detection probes each for a specific target (e.g, a provided biomarker of a target biomarker signature). In some embodiments, such a system comprises 2-25 detection probes each for a specific target ( e.g ., a provided biomarker of a target biomarker signature). In some embodiments, such a system comprises 5-30 detection probes each for a specific target (e.g., a provided biomarker of a target biomarker signature).
• such a system comprises 5-25 detection probes each for a specific target (e.g, a provided biomarker of a target biomarker signature).
• at least two of such detection probes in a set may be directed to the same biomarker of a target biomarker signature.
• at least two of such detection probes in a set may be directed to the same epitope of the same biomarker of a target biomarker signature.
• at least two of such detection probes in a set may be directed to different epitopes of the same biomarker of a target biomarker signature.
• detection probes appropriate for use in a target entity detection system provided herein may be used for detection of a single disease or condition, e.g, lung cancer. In some embodiments, detection probes appropriate for use in a target entity detection system provided herein may permit detection of at least two or more diseases or conditions, e.g, one of which is lung cancer.
• detection probes appropriate for use in a target entity detection system provided herein may permit detection of lung cancer of certain subtypes including, e.g, lung adenocarcinoma lung cancer, small cell lung cancer, squamous and transitional cell lung cancer, large cell lung cancer, non-small cell carcinoma lung cancer, other specified carcinoma lung cancer, sarcoma lung cancer, and other specified types of lung cancer as known in the art (SEER Cancer Statistics Review 1975-2017).
• detection probes appropriate for use in a target entity detection system provided herein may permit detection of lung cancer of certain stages, including, e.g., stage I, stage II, stage III, and/or stage IV.
• detection probes appropriate for use in a target entity detection system comprises a plurality (e.g, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more) of sets of detection probes, wherein each set is directed to detection of a different disease or a different type of disease or condition.
• detection probes appropriate for use in a target entity detection system comprises a plurality (e.g, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more) of sets of detection probes, wherein in some embodiments, each set is directed to detection of a different type of cancer, one of which is lung cancer, or in some embodiments, each set is directed to detection of lung cancer of various subtypes and/or stages.
• a detection probe as provided and/or utilized herein comprises a target-binding moiety and an oligonucleotide domain coupled to the target-binding moiety.
• an oligonucleotide domain coupled to a target-binding moiety comprises a double-stranded portion and a single-stranded overhang extended from at least one end of the oligonucleotide domain.
• an oligonucleotide domain coupled to a target- binding moiety comprises a double-stranded portion and a single-stranded overhang extended from each end of the oligonucleotide domain.
• detection probes may be suitable for proximity -ligation-immuno quantitative polymerase chain reaction (pliq-PCR) and be referred to as pliq-PCR detection probes.
• a target-binding moiety that is coupled to an oligonucleotide domain is an entity or an agent that specifically binds to a target (e.g. , a provided biomarker of a target biomarker signature; those skilled in the art will appreciate that, where the target biomarker is a particular form or moiety/component, the target-binding moiety specifically binds to that form or moiety/component).
• a target e.g. , a provided biomarker of a target biomarker signature; those skilled in the art will appreciate that, where the target biomarker is a particular form or moiety/component, the target-binding moiety specifically binds to that form or moiety/component).
• a target-binding moiety may have a binding affinity (e.g, as measured by a dissociation constant) for a target (e.g, molecular target) of at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or lower.
• a target e.g, molecular target
• binding affinity e.g, as measured by a dissociation constant
• binding affinity may be influenced by non-covalent intermolecular interactions such as hydrogen bonding, electrostatic interactions, hydrophobic and Van der Waals forces between the two molecules.
• binding affinity between a ligand and its target molecule may be affected by the presence of other molecules.
• Those skilled in the art will be familiar with a variety of technologies for measuring binding affinity and/or dissociation constants in accordance with the present disclosure, including, e.g, but not limited to ELISAs, gel-shift assays, pull-down assays, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), bio-layer interferometry, grating-coupled interferometry, and spectroscopic assays.
• a target-binding moiety may be or comprise an agent of any chemical class such as, for example, a carbohydrate, a nucleic acid, a lipid, a metal, a polypeptide, a small molecule, etc ., and/or a combination thereof.
• a target-binding moiety may be or comprise an antibody agent and/or an aptamer.
• a target-binding moiety is or comprises an antibody agent, e.g ., an antibody agent that specifically binds to a target or an epitope thereof, e.g. , a provided biomarker of a target biomarker signature for lung cancer or an epitope thereof.
• a targetbinding moiety for a provided biomarker may be a commercially available. In some embodiments, a target-binding moiety for a provided biomarker may be designed and created for the purpose of use in assays as described herein. In some embodiments, a target-binding moiety is or comprises an aptamer, e.g. , an aptamer that specifically binds to a target or an epitope thereof, e.g. , a provided biomarker of a target biomarker signature for lung cancer or an epitope thereof. In some embodiments, a target-binding moiety is or comprises an affimer molecule that specifically binds to a target or an epitope thereof, e.g.
• a provided biomarker of a target biomarker signature for lung cancer or an epitope thereof can be or comprise a peptide or polypeptide that binds to a target or an epitope thereof (e.g, as described herein) with similar specificity and affinity to that of a corresponding antibody.
• a target may be or comprise a target that is associated with lung cancer.
• a cancer-associated target can be or comprise a target is associated with more than one cancer (i.e., at least two or more cancers).
• a cancer-associated target can be or comprise a target that is typically associated with cancers.
• a cancer-associated target can be or comprise a target that is associated with cancers of a specific tissue, e.g, lung cancer.
• a cancer- associated target can be or comprise a target that is specific to a particular cancer, e.g, a particular lung cancer.
• a target-binding moiety recognizes and specifically binds to a target present in a biological entity (including, e.g, but not limited to cells and/or extracellular vesicles).
• a target-binding moiety may recognize and specifically bind to a tumor-associated antigen or epitope thereof.
• a tumor-associated antigen may be or comprise an antigen that is associated with a cancer such as, for example, skin cancer, brain cancer (including, e.g ., glioblastoma), breast cancer, colorectal cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, and skin cancer.
• a target-binding moiety may recognize a tumor antigen associated with lung cancer (e.g, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, etc.). In some embodiments, a target-binding moiety may recognize a tumor antigen associated with lung adenocarcinoma.
• lung cancer e.g, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, etc.
• a target-binding moiety may recognize a tumor antigen associated with lung adenocarcinoma.
• a target-binding moiety may specifically bind to an intravesicular target, e.g, a provided intravesicular protein or RNA (e.g, mRNA).
• a target-binding moiety may specifically bind to a surface target that is present on/within extracellular vesicles, e.g, a membrane-bound polypeptide present on lung cancer-associated extracellular vesicles.
• a target-binding moiety is directed to a biomarker for a specific condition or disease (e.g, cancer), which biomarker is or has been determined, for example, by analyzing a population or library (e.g. tens, hundreds, thousands, tens of thousands, hundreds of thousands, or more) of patient biopsies and/or patient data to identify such a biomarker (e.g, a predictive biomarker).
• a population or library e.g. tens, hundreds, thousands, tens of thousands, hundreds of thousands, or more
• a relevant biomarker may be one identified and/or characterized, for example, via data analysis.
• a diverse set of data e.g, in some embodiments comprising one or more of bulk RNA sequencing, single-cell RNA (scRNA) sequencing, mass spectrometry, histology, post-translational modification data, in vitro and/or in vivo experimental data
• biomarkers e.g, predictive markers
• a disease or condition e.g, cancer
• a target-binding moiety is directed to a tissue-specific target, for example, a target that is associated with a specific tissue such as, for example, brain, breast, colon, ovary and/or other tissues associated with a female reproductive system, pancreas, prostate and/or other tissues associated with a male reproductive system, liver, lung, and skin.
• tissue-specific target may be associated with a normal healthy tissue and/or a diseased tissue, such as a tumor.
• a target-binding moiety is directed to a target that is specifically associated with a normal healthy condition of a subject.
• individual target binding entities utilized in a plurality of detection probes are directed to different targets.
• such different targets may represent different marker proteins or polypeptides.
• such different targets may represent different epitopes of the same marker proteins or polypeptides.
• two or more individual target binding entities utilized in a plurality of detection probes may be directed to the same target.
• individual target binding entities utilized in a plurality of detection probes for detection of lung cancer may be directed to different target biomarkers of a target biomarker signature for lung cancer (e.g, ones as described in the section entitled “ Provided Biomarkers and/or Target Biomarker Signatures for Detection of Lung Cancer ” above).
• a target biomarker signature for lung cancer e.g, ones as described in the section entitled “ Provided Biomarkers and/or Target Biomarker Signatures for Detection of Lung Cancer ” above.
• at least two detection probes in a plurality may have their target binding entities directed to CEACAM6 and EpCAM, respectively.
• at least two detection probes in a plurality may have their target binding entities directed to CEACAM6 and SLC34A2, respectively.
• at least two detection probes in a plurality may have their target binding entities directed to MUC1 and CEACAM6 respectively.
• individual target binding entities utilized in a plurality of detection probes for detection of lung cancer may be directed to the same target biomarker of a target biomarker signature for lung cancer (e.g, ones as described in the section entitled “ Provided Biomarkers and/or Target Biomarker Signatures for Detection of Lung Cancer ” above).
• target binding entities may be directed to the same or different epitopes of the same target biomarker of such a target biomarker signature for lung cancer.
• At least two detection probes in a plurality may have their target binding entities each directed to CEACAM6 (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• at least two detection probes in a plurality may have their target binding entities each directed to ALCAM (e.g. , in its intact protein form or a fragment thereof, e.g. , an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• At least two detection probes in a plurality may have their target binding entities each directed to CD55 (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes). In some embodiments, at least two detection probes in a plurality may have their target binding entities each directed to CD1 ( e.g ., in its intact protein form or a fragment thereof, e.g. , an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• At least two detection probes in a plurality may have their target binding entities each directed to CDH3 (e.g., in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• at least two detection probes in a plurality may have their target binding entities each directed to CD274 (PD-L1) (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• At least two detection probes in a plurality may have their target binding entities each directed to CEACAM5 (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes). In some embodiments, at least two detection probes in a plurality may have their target binding entities each directed to DSG2 (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• At least two detection probes in a plurality may have their target binding entities each directed to EGFR (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes). In some embodiments, at least two detection probes in a plurality may have their target binding entities each directed to EPCAM (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• At least two detection probes in a plurality may have their target binding entities each directed to FOLR1 (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes). In some embodiments, at least two detection probes in a plurality may have their target binding entities each directed to IG1FR (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• At least two detection probes in a plurality may have their target binding entities each directed to MET (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• at least two detection probes in a plurality may have their target binding entities each directed to MSLN (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• at least two detection probes in a plurality may have their target binding entities each directed to MUC1 (e.g, in its intact protein form or a fragment thereof, e.g.
• At least two detection probes in a plurality may have their target binding entities each directed to SLC34A2 (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes). In some embodiments, at least two detection probes in a plurality may have their target binding entities each directed to sTn antigen (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• At least two detection probes in a plurality may have their target binding entities each directed to Tn antigen (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes). In some embodiments, at least two detection probes in a plurality may have their target binding entities each directed to T antigen (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• At least two detection probes in a plurality may have their target binding entities each directed to TACSTD (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• at least two detection probes in a plurality may have their target binding entities each directed to TNFRSF10B (e.g, in its intact protein form or a fragment thereof, e.g, an extracellular domain thereof, and/or at the same epitope or at different epitopes).
• an oligonucleotide domain for use in accordance with the present disclosure (e.g, that may be coupled to a target-binding moiety) comprises a double-stranded portion and a single-stranded overhang extended from one or both ends of the oligonucleotide domain.
• an oligonucleotide domain comprises a single-stranded overhang extended from each end, a single-stranded overhang is extended from a different strand of a double-stranded portion.
• an oligonucleotide domain comprises a single-stranded overhang extended from one end of the oligonucleotide domain
• the other end of the oligonucleotide domain may be a blunt end.
• an oligonucleotide domain comprises ribonucleotides, deoxyribonucleotides, synthetic nucleotide residues that are capable of participating in Watson- Crick type or analogous base pair interactions, and any combinations thereof.
• an oligonucleotide domain is or comprises DNA.
• an oligonucleotide domain is or comprises peptide nucleic acid (PNA).
• an oligonucleotide may have a length that is determined, at least in part, for example, by, e.g ., the physical characteristics of an entity of interest (e.g, biological entity such as extracellular vesicles) to be detected, and/or selection and localization of molecular targets in an entity of interest (e.g, biological entity such as extracellular vesicles) to be detected.
• entity of interest e.g, biological entity such as extracellular vesicles
• an oligonucleotide domain of a detection probe is configured to have a length such that when a first detection probe and a second detection probe bind to an entity of interest (e.g, biological entity such as extracellular vesicles), the first single-stranded overhang and the second single-stranded overhang are in sufficiently close proximity to permit interaction (e.g, hybridization) between the single-stranded overhangs.
• entity of interest e.g, biological entity such as extracellular vesicles
• oligonucleotide domains of detection probes can each independently have a length such that their respective single-stranded overhangs are in sufficiently close proximity to anneal or interact with each other when the corresponding detection probes are bound to the same extracellular vesicle.
• oligonucleotide domains of detection probes for use in detecting extracellular vesicles may each independently have a length of about 20 nm to about 200 nm, about 40 nm to about 500 nm, about 40 nm to about 300 nm, or about 50 nm to about 150 nm.
• oligonucleotide domains of detection probes for use in detecting extracellular vesicles may each independently have a length of about 20 nm to about 200 nm.
• lengths of oligonucleotide domains of detection probes in a set can each independently vary to increase and/or maximize the probability of them finding each other when they simultaneously bind to the same entity of interest.
• an oligonucleotide domain for use in technologies provided herein may have a length in the range of about 20 up to about 1000 nucleotides. In some embodiments, an oligonucleotide domain may have a length in the range of about 30 up to about 1000 nucleotides.
• an oligonucleotide domain may have a length in the range of about 30 to about 500 nucleotides, from about 30 to about 250 nucleotides, from about 30 to about 200 nucleotides, from about 30 to about 150 nucleotides, from about 40 to about 150 nucleotides, from about 40 to about 125 nucleotides, from about 40 to about 100 nucleotides, from about 40 to about 60 nucleotides, from about 50 to about 90 nucleotides, from about 50 to about 80 nucleotides.
• an oligonucleotide domain may have a length of at least 20 or more nucleotides, including, e.g ., at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 250, at least 500, at least 750, at least 1000 nucleotides or more.
• an oligonucleotide domain may have a length of no more than 1000 nucleotides or lower, including, e.g., no more than 900, no more than 800, no more than 700, no more than 600, no more than 500, no more than 400, no more than 300, no more than 200, no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40 nucleotides, no more than 30 nucleotides, no more than 20 nucleotides, or lower.
• an oligonucleotide domain may have a length of about 20 nm to about 500 nm. In some embodiments, an oligonucleotide domain may have a length of about 20 nm to about 400 nm, about 30 nm to about 200 nm, about 50 nm to about 100 nm, about 30 nm to about 70 nm, or about 40 nm to about 60 nm.
• an oligonucleotide domain may have a length of at least about 20 nm or more, including, e.g, at least about 30 nm, at least about 40 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, at least about 200 nm, at least about 300 nm, at least about 400 nm or more.
• an oligonucleotide domain may have a length of no more than 1000 nm or lower, including, e.g., no more than 900 nm, no more than 800 nm, no more than 700 nm, no more than 600 nm, no more than 500 nm, no more than 400 nm, no more than 300 nm, no more than 200 nm, no more than 100 nm or lower.
• a double-stranded portion of an oligonucleotide domain for use in technologies provided herein may have a length in the range of about 30 up to about 1000 nucleotides.
• a double-stranded portion of an oligonucleotide domain may have a length in the range of about 30 to about 500 nucleotides, from about 30 to about 250 nucleotides, from about 30 to about 200 nucleotides, from about 30 to about 150 nucleotides, from about 40 to about 150 nucleotides, from about 40 to about 125 nucleotides, from about 40 to about 100 nucleotides, from about 50 to about 90 nucleotides, from about 50 to about 80 nucleotides.
• a double-stranded portion of an oligonucleotide domain may have a length of at least 30 or more nucleotides, including, e.g ., at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 250, at least 500, at least 750, at least 1000 nucleotides or more.
• a double-stranded portion of an oligonucleotide domain may have a length of no more than 1000 nucleotides or lower, including, e.g., no more than 900, no more than 800, no more than 700, no more than 600, no more than 500, no more than 400, no more than 300, no more than 200, no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40 nucleotides or lower.
• a double-stranded portion of an oligonucleotide domain may have a length of about 20 nm to about 500 nm. In some embodiments, a double-stranded portion of an oligonucleotide domain may have a length of about 20 nm to about 400 nm, about 30 nm to about 200 nm, about 50 nm to about 100 nm, about 30 nm to about 70 nm, or about 40 nm to about 60 nm.
• a double-stranded portion of an oligonucleotide domain may have a length of at least about 20 nm or more, including, e.g, at least about 30 nm, at least about 40 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, at least about 200 nm, at least about 300 nm, at least about 400 nm or more.
• a double-stranded portion of an oligonucleotide domain may have a length of no more than 1000 nm or lower, including, e.g, no more than 900 nm, no more than 800 nm, no more than 700 nm, no more than 600 nm, no more than 500 nm, no more than 400 nm, no more than 300 nm, no more than 200 nm, no more than 100 nm or lower.
• a double-stranded portion of an oligonucleotide domain is characterized in that when detection probes are connected to each other through hybridization of respective complementary single-stranded overhangs (e.g, as described and/or utilized herein), the combined length of the respective oligonucleotide domains (including, if any, a linker that links a target-binding moiety to an oligonucleotide domain) is long enough to allow respective target binding entities to substantially span the full characteristic length (e.g., diameter) of an entity of interest (e.g, an extracellular vesicle).
• the full characteristic length e.g., diameter
• a combined length of oligonucleotide domains (including, if any, a linker that links a target-binding moiety to an oligonucleotide domain) of detection probes may be approximately 50 to 200 nm, when the detection probes are fully connected to each other.
• a double-stranded portion of an oligonucleotide domain comprises a binding site for a primer.
• a binding site for a primer comprises a nucleotide sequence that is designed to reduce or minimize the likelihood for miss-priming or primer dimers. Such a feature, in some embodiments, can decrease the lower limit of detection and thus increase the sensitivity of systems provided herein.
• a binding site for a primer comprises a nucleotide sequence that is designed to have a similar annealing temperature as another primer binding site.
• a double-stranded portion of an oligonucleotide domain comprises a nucleotide sequence designed to reduce or minimize overlap with nucleic acid sequences (e.g ., DNA and/or RNA sequences) typically associated with genome and/or gene transcripts (e.g., genomic DNA and/or RNA, such as mRNA of genes) of a subject (e.g, a human subject).
• nucleic acid sequences e.g ., DNA and/or RNA sequences
• genomic DNA and/or RNA such as mRNA of genes
• a subject e.g, a human subject.
• Such a feature may reduce or minimize interference of any genomic DNA and/or mRNA transcripts of a subject that may be present (e.g, as contaminants) in a sample during detection.
• a double-stranded portion of an oligonucleotide domain may have a nucleotide sequence designed to reduce or minimize formation of self-dimers, homo-dimers, or hetero-dimers.
• a single-stranded overhang of an oligonucleotide domain for use in technologies provided herein may have a length of about 2 to about 20 nucleotides. In some embodiments, a single-stranded overhang of an oligonucleotide domain may have a length of about 2 to about 15 nucleotides, from about 2 to about 10 nucleotides, from about 3 to about 20 nucleotides, from about 3 to about 15 nucleotides, from about 3 to about 10 nucleotides. In some embodiments, a single-stranded overhang can have at least 1 to 5 nucleotides in length.
• a single-stranded overhang of an oligonucleotide domain may have a length of at least 2 or more nucleotides, including, e.g, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20 nucleotides, or more.
• a single-stranded overhang of an oligonucleotide domain may have a length of no more than 20 nucleotides or lower, including, e.g, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4 nucleotides or lower.
• a single-stranded overhang of an oligonucleotide domain may have a length of about 1 nm to about 10 nm. In some embodiments, a single-stranded overhang of an oligonucleotide domain may have a length of about 1 nm to about 5 nm.
• a single-stranded overhang of an oligonucleotide domain may have a length of at least about 0.5 nm or more, including, e.g ., at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm or more.
• a single-stranded overhang of an oligonucleotide domain may have a length of no more than 10 nm or lower, including, e.g., no more than 9 nm, no more than 8 nm, no more than 7 nm, no more than 6 nm, no more than 5 nm, no more than 4 nm, no more than 3 nm, no more than 2 nm, no more than 1 nm or lower.
• a single-stranded overhang of an oligonucleotide domain is designed to comprise a nucleotide sequence that is complementary to at least a portion of a single-stranded overhang of a second detection probe such that a double-stranded complex comprising a first detection probe and a second detection probe can be formed through hybridization of the complementary single- stranded overhangs.
• nucleotide sequences of complementary single- stranded overhangs are selected for optimal ligation efficiency in the presence of an appropriate nucleic acid ligase.
• a single-stranded overhang has a nucleotide sequence preferentially selected for efficient ligation by a specific nucleic acid ligase of interest (e.g, a DNA ligase such as a T4 or T7 ligase).
• a specific nucleic acid ligase of interest e.g, a DNA ligase such as a T4 or T7 ligase.
• such a single-stranded overhang may have a nucleotide sequence of GAGT, e.g, as described in Song el al., “Enzyme-guided DNA sewing architecture” Scientific Reports 5: 17722 (2015), which is incorporated herein by reference for the purpose described herein.
• the respective oligonucleotide domains comprising the hybridized single-stranded overhangs can, in some embodiments, have a combined length of about 90%-l 10% or about 95%-105% of a characteristic length (e.g, diameter) of an entity of interest (e.g, a biological entity).
• a characteristic length e.g, diameter
• the combined length can be about 50 nm to about 200 nm, or about 75 nm to about 150 nm, or about 80 nm to about 120 nm.
• An oligonucleotide domain and a target-binding moiety can be coupled together in a detection probe by a covalent linkage, and/or by a non-covalent association (such as, e.g. , a protein-protein interaction such as streptavidin-biotin interaction and/or an ionic interaction).
• a detection probe appropriate for use in accordance with the present disclosure is a conjugate molecule comprising a target-binding moiety and an oligonucleotide domain, where the two components are typically covalently coupled to each other, e.g. , directly through a bond, or indirectly through one or more linkers.
• a targetbinding moiety is coupled to one of two strands of an oligonucleotide domain by a covalent linkage (e.g, directly through a bond or indirectly through one or more linkers) and/or by a non- covalent association (such as, e.g, a protein-protein interaction such as streptavidin-biotin interaction and/or ionic interaction).
• a covalent linkage e.g, directly through a bond or indirectly through one or more linkers
• a non- covalent association such as, e.g, a protein-protein interaction such as streptavidin-biotin interaction and/or ionic interaction.
• linkers are chosen to provide for covalent attachment of a target-binding moiety to one or both strands of an oligonucleotide domain through selected linkers. In some embodiments, linkers are chosen such that the resulting covalent attachment of a target-binding moiety to one or both strands of an oligonucleotide domain maintains the desired binding affinity of the target-binding moiety for its target. In some embodiments, linkers are chosen to enhance binding specificity of a target-binding moiety for its target. Linkers and/or conjugation methods of interest may vary widely depending on a targetbinding moiety, e.g, its size and/or charges. In some embodiments, linkers are biologically inert.
• a linker can comprise a spacer group at either end with a reactive functional group capable of covalent attachment to a target-binding moiety.
• spacer groups that can be used in linkers include, but are not limited to, aliphatic and unsaturated hydrocarbon chains (including, e.g., C4, C5, C6, C7, C8, C9, CI0, Cll, C12, C13, C14, C15, C16, C17, C18, C19, C20, or longer), spacers containing heteroatoms such as oxygen (e.g, ethers such as polyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic or acyclic systems that may contain heteroatoms.
• aliphatic and unsaturated hydrocarbon chains including, e.g., C4, C5, C6, C7, C8, C9, CI0, Cll, C12, C13, C14, C15, C16, C17, C18, C19, C20, or longer
• spacers containing heteroatoms such as oxygen (e.g, ethers such as polyethylene glycol) or nitrogen (polyamines),
• Non-limiting examples of a reactive functional group to facilitate covalent attachment include nucleophilic functional groups (e.g ., amines, alcohols, thiols, and/or hydrazides), electrophilic functional groups (e.g, aldehydes, esters, vinyl ketones, epoxides, isocyanates, and/or maleimides), functional groups capable of cycloaddition reactions, forming disulfide bonds, or binding to metals.
• nucleophilic functional groups e.g amines, alcohols, thiols, and/or hydrazides
• electrophilic functional groups e.g, aldehydes, esters, vinyl ketones, epoxides, isocyanates, and/or maleimides
• functional groups capable of cycloaddition reactions forming disulfide bonds, or binding to metals.
• exemplary reactive functional groups are not limited to, primary and secondary amines, hydroxamic acids, N- hydroxysuccinimidyl (NHS) esters, dibenzocyclooctyne (DBCO)-NHS esters, azido- NHS esters, azidoacetic acid NHS ester, propargyl-NHS ester, trans-cyclooctene-NHS esters, N-hydroxysuccinimidyl carbonates, oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters, glycidyl ethers, vinylsulfones, maleimides, azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'- pyridyldithiojpropionamid), bis-sulfosuccinimidyl suberate, dimethyladipimidate, dis
• a target-binding moiety e.g, a target binding antibody agent
• a target-binding moiety is coupled or conjugated to one or both strands of an oligonucleotide domain using N- hydrosysuccinimide (NHS) ester chemistry.
• NHS esters react with free primary amines and result in stable covalent attachment.
• a primary amino group can be positioned at a terminal end with a spacer group, e.g, but not limited to an aliphatic and unsaturated hydrocarbon chain (e.g, a C6 or C12 spacer group).
• a target-binding moiety e.g, a target binding antibody agent
• a site-specific conjugation method known in the art, e.g, to enhance the binding specificity of conjugated target-binding moiety (e.g, conjugated target binding antibody agent).
• a site-specific conjugation method include, but are not limited to coupling or conjugation through a disulfide bond, C-terminus, carbohydrate residue or glycan, and/or unnatural amino acid labeling.
• an oligonucleotide can be coupled or conjugated to the target-binding moiety via at least one or more free amine groups present in the target-binding moiety.
• an oligonucleotide can be coupled or conjugated to a target-binding moiety that is or comprises an antibody agent or a peptide aptamer via at least one or more reactive thiol groups present in the target-binding moiety.
• an oligonucleotide can be coupled or conjugated to a target-binding moiety that is or comprises an antibody agent or a peptide aptamer via at least one or more carbohydrate residues present in the target-binding moiety.
• a plurality of oligonucleotides e.g ., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least ten, or more
• a target-binding moiety e.g., a target binding antibody agent
• a target entity detection system as provided by the present disclosure (and useful, for example, for detecting, e.g, at a single entity level, extracellular vesicles associated with lung cancer) comprises a first population of first detection probes (e.g, as described and/or utilized herein) for a provided target biomarker (e.g, ones described herein) and a second population of second detection probes (e.g, as described and/or utilized herein) for a provided target biomarker (e.g, ones described herein).
• the first detection probes and the second detection probes are directed to the same provided target biomarker.
• the first detection probes and the second detection probes are directed to different provided target biomarkers.
• Figure 2 illustrates an exemplary duplex target entity detection system for detecting, at a single entity level, an entity of interest (e.g, biological entity such as an extracellular vesicle) comprising (i) at least one target (e.g, a provided biomarker of a target biomarker signature for lung cancer) which expression level is high enough such that two molecules of the same target (e.g, a provided biomarker of a target biomarker signature for lung cancer) are found in close proximity, or (ii) at least two or more distinct targets (e.g.,. provided biomarkers of a target biomarker signature for lung cancer).
• an entity of interest e.g, biological entity such as an extracellular vesicle
• a target e.g, a provided biomarker of a target biomarker signature for lung cancer
• a first detection probe comprises a first target-binding moiety (e.g, directed to a target cancer marker 1) and a first oligonucleotide domain coupled to the first target-binding moiety, the first oligonucleotide domain comprising a first double- stranded portion and a first single-stranded overhang extended from one end of the first oligonucleotide domain.
• a first oligonucleotide domain may be resulted from hybridization of a longer strand (strand 3) and a shorter strand (strand 1), thereby forming a double-stranded portion and a single-stranded overhang at one end.
• a first target-binding moiety (e.g ., directed to target cancer marker 1) is coupled (e.g, covalently coupled) to a5'end or 3' end of a strand of a first oligonucleotide domain (e.g, strand 1).
• a 5' end or 3' end of a strand that is coupled to a first target-binding moiety may be modified with a linker (e.g, as described and/or utilized herein with or without a spacer group).
• a 5' end of another strand of a first oligonucleotide domain (e.g, strand 3) has a free phosphate group.
• a second detection probe comprises a second target-binding moiety (e.g, directed to a target cancer marker 2) and a second oligonucleotide domain coupled to the second target-binding moiety, the second oligonucleotide domain comprising a second double-stranded portion and a second single-stranded overhang extended from one end of the second oligonucleotide domain.
• a second oligonucleotide domain may be resulted from hybridization of a longer strand (strand 4) and a shorter strand (strand 2), thereby forming a double-stranded portion and a single-stranded overhang at one end.
• a second target-binding moiety (e.g, directed to a target cancer marker 2) is coupled (e.g, covalently coupled) to a 5' end of a strand of a second oligonucleotide domain (e.g, strand 2).
• a 5' end of a strand that is coupled to a second target-binding moiety may be modified with a linker (e.g, as described and/or utilized herein with or without a spacer group).
• a 5' end of another strand of a second oligonucleotide domain (e.g, strand 4) has a free phosphate group.
• At least portions of a first single-stranded overhang and a second single-stranded overhang are complementary to each other such that they can hybridize to form a double- stranded complex when they are in sufficiently close proximity, e.g, when a first detection probe and a second detection probe simultaneously bind to the same entity of interest (e.g, biological entity such as extracellular vesicle).
• entity of interest e.g, biological entity such as extracellular vesicle
• a first single-stranded overhang and a second single-stranded overhang have equal lengths such that when they hybridize to form a double-stranded complex, there is no gap (other than a nick to be ligated) between their respective oligonucleotide domains and each respective target-binding moiety is located at an opposing end of the double-stranded complex.
• a double- stranded complex forms before ligation occurs, wherein the double-stranded complex comprises a first detection probe and a second detection probe coupled to each other through direct hybridization of their respective single-stranded overhangs ( e.g ., having 4 nucleotides in length), wherein each respective target-binding moiety (e.g., directed to a target cancer marker 1 and a target cancer marker 2, respectively) is present at opposing ends of the double-stranded complex.
• both strands of the double-stranded complex (comprising a nick between respective oligonucleotide domains) are ligatable, e.g, for amplification and detection.
• a double-stranded complex (e.g, before ligation occurs) can comprise an entity of interest (e.g, a biological entity such as an extracellular vesicle), wherein a first target-binding moiety (e.g, directed to a target cancer marker 1) and a second target-binding moiety (e.g, directed to a target cancer marker 2) are simultaneously bound to the entity of interest.
• entity of interest e.g, a biological entity such as an extracellular vesicle
• a first target-binding moiety of a first detection probe may be directed to a first target surface protein biomarker (e.g, ones provided in the section entitled “Provided Biomarkers and/or Target Biomarker Signatures for Detection of Lung Cancer”), while a second targetbinding moiety of a second detection probe may be directed to a second target surface protein biomarker (e.g, ones provided in the section entitled “Provided Biomarkers and/or Target Biomarker Signatures for Detection of Lung Cancer”).
• a first target surface protein biomarker e.g, ones provided in the section entitled “Provided Biomarkers and/or Target Biomarker Signatures for Detection of Lung Cancer”
• a second targetbinding moiety of a second detection probe may be directed to a second target surface protein biomarker (e.g, ones provided in the section entitled “Provided Biomarkers and/or Target Biomarker Signatures for Detection of Lung Cancer”).
• a first targetbinding moiety of a first detection probe may be directed to a first target intravesicular protein biomarker (e.g, ones provided in the section entitled “Provided Biomarkers and/or Target Biomarker Signatures for Detection of Lung Cancer”), while a second target-binding moiety of a second detection probe may be directed to a second target intravesicular protein biomarker (e.g, ones provided in the section entitled “Provided Biomarkers and/or Target Biomarker Signatures for Detection of Lung Cancer”).
• a first target intravesicular protein biomarker e.g, ones provided in the section entitled “Provided Biomarkers and/or Target Biomarker Signatures for Detection of Lung Cancer”
• the first target-binding moiety and the second target-binding moiety may be directed to the same or different epitopes of the same target surface protein biomarker or of the same target intravesicular protein biomarker. In some embodiments, the first target-binding moiety and the second target-binding moiety may be directed to the different target surface protein biomarkers or different target intravesicular protein biomarkers.
• a first detection probe comprises a first target-binding moiety directed to CEACAM6 (e.g ., in intact protein form or a fragment thereof, such as an extracellular domain thereof) conjugated to a first oligonucleotide domain; whereas a second detection probe comprises a second target-binding moiety detected to CEACAM6 (e.g., in intact protein form or a fragment thereof, such as an extracellular domain thereof) conjugated to a second oligonucleotide domain.
• the first target-binding moiety and the second target-binding moiety can be directed to the same or different epitope(s) of CEACAM6 (e.g, in intact protein form or a fragment thereof, such as an extracellular domain thereof).
• the double stranded portion of a first oligonucleotide domain and a second oligonucleotide domain may be the same.
• the double stranded portion of a first oligonucleotide domain and a second oligonucleotide domain may be different.
• a first detection probe comprises a first target-binding moiety directed to CEACAM6 polypeptide conjugated to a first oligonucleotide domain; whereas a second detection probe comprises a second target-binding moiety directed to EpCAM polypeptide conjugated to a second oligonucleotide domain.
• the double stranded portion of a first oligonucleotide domain and a second oligonucleotide domain may be the same.
• the double stranded portion of a first oligonucleotide domain and a second oligonucleotide domain may be different.
• a first detection probe comprises a first target-binding moiety directed to CEACAM6 polypeptide conjugated to a first oligonucleotide domain; whereas a second detection probe comprises a second target-binding moiety directed to SLC34A2 polypeptide conjugated to a second oligonucleotide domain.
• the double stranded portion of a first oligonucleotide domain and a second oligonucleotide domain may be the same.
• the double stranded portion of a first oligonucleotide domain and a second oligonucleotide domain may be different.
• a duplex target entity detection system for detection of lung cancer comprises at least two distinct sets of detection probes.
• each set may be directed to a distinct target biomarker signature comprising one or more target biomarkers ( e.g ., ones described herein).
• a duplex target entity detection system comprising at least two distinct sets of detection probes may also comprise a capture assay comprising a capture agent directed to an extracellular vesicle-associated membrane-bound polypeptide.
• any combination of biomarker probes including capture probes or detection probes as described herein may be utilized in combination with any other set of biomarker probes (e.g, a biomarker signature) including capture probes or detection probes as described herein.
• a target entity detection system as provided by the present disclosure (and useful, for example, for detecting, e.g, at a single entity level, extracellular vesicles associated with lung cancer) comprises n populations of distinct detection probes (e.g, as described and/or utilized herein), wherein n >3.
• a target entity detection system comprises a first detection probe (e.g, as described and/or utilized herein) for a first target, a population of a second detection probe (e.g, as described and/or utilized herein) for a second target, and a population of a third detection probe (e.g, as described and/or utilized herein) for a third target.
• Figure 13 illustrates an exemplary triplex target entity detection system for detecting, at a single entity level, an entity of interest (e.g, a biological entity such as an extracellular vesicle) comprising three distinct molecular targets.
• a first detection probe comprises a first targetbinding moiety (e.g, anti-cancer marker 1 antibody agent) and a first oligonucleotide domain coupled to the first target-binding moiety, the first oligonucleotide domain comprising a first double-stranded portion and a first single-stranded overhang extended from one end of the first oligonucleotide domain.
• a first targetbinding moiety e.g, anti-cancer marker 1 antibody agent
• a first oligonucleotide domain may be resulted from hybridization of a longer strand (strand 8) and a shorter strand (strand 1), thereby forming a double-stranded portion and a single-stranded overhang at one end.
• a first target-binding moiety e.g, anti-cancer marker 1 antibody agent
• is coupled e.g, covalently coupled
• a 5' end of a strand that is coupled to a first target-binding moiety may be modified with a linker (e.g ., as described and/or utilized herein with or without a spacer group).
• a 5' end of another strand of a first oligonucleotide domain (e.g., strand 8) has a free phosphate group.
• a second detection probe comprises a second target-binding moiety (e.g, anti-cancer marker 3 antibody agent) and a second oligonucleotide domain coupled to the second target-binding moiety, the second oligonucleotide domain comprising a second double-stranded portion and a second single-stranded overhang extended from one end of the second oligonucleotide domain.
• a second oligonucleotide domain may be resulted from hybridization of a longer strand (strand 4) and a shorter strand (strand 2), thereby forming a double-stranded portion and a single-stranded overhang at one end.
• a second target-binding moiety e.g, anti-cancer marker 3 antibody agent
• a second target-binding moiety is coupled (e.g, covalently coupled) to a 5' end of a strand of a second oligonucleotide domain (e.g, strand 2).
• a 5' end of a strand that is coupled to a second target-binding moiety may be modified with a linker (e.g, as described and/or utilized herein with or without a spacer group).
• a 5' end of another strand of a second oligonucleotide domain e.g, strand 4 has no free phosphate group.
• a third detection probe comprises a third target-binding moiety (e.g, anti-cancer marker 2 antibody agent) and a third oligonucleotide domain coupled to the third target-binding moiety, the third oligonucleotide domain comprising a third double-stranded portion and a single- stranded overhang extended from each end of the third oligonucleotide domain.
• a single-stranded overhang is extended from one end of a strand of a third oligonucleotide domain while another single-stranded overhang is extended from an opposing end of a different strand of the third oligonucleotide domain.
• a third oligonucleotide domain may be resulted from hybridization of portions of two strands (e.g, strands 9 and 10), thereby forming a double-stranded portion and a single-stranded overhang at each end.
• a single- stranded overhang (3 A) is formed at a 5' end of strand 9 of a third detection probe, wherein the 5' end of strand 9 has a free phosphate group.
• a single-stranded overhang (3B) is formed at a 5' end of strand 10 of the same third detection probe and a third target-binding moiety (e.g, anti-target 2 antibody agent) is also coupled (e.g, covalently coupled) to the 5' end of strand 10.
• a 5' end of a strand (e.g, strand 10) that is coupled to a third target-binding moiety may be modified with a linker ( e.g ., as described and/or utilized herein with or without a spacer group).
• At least (n >3) detection probes when single-stranded overhangs of detection probes anneal to each respective partner(s) to form a double-stranded complex, at least (n-2) target-binding moiety/moieties is/are present at internal position(s) of the double-stranded complex.
• a strand of a double-stranded complex comprises at least one or more internal target binding moieties
• the strand comprises a gap between an end of an oligonucleotide strand of a detection probe to which the internal target-binding moiety is coupled and an end of an oligonucleotide strand of another detection probe.
• the size of the gap is large enough such that the strand becomes non-ligatable in the presence of a nucleic acid ligase.
• the gap may be 2 - 8 nucleotides in size or 2-6 nucleotides in size. In some embodiments, the gap is 6 nucleotides in size.
• the overlap (hybridization region between single-stranded overhangs) can be 2 - 15 nucleotides in length or 4-10 nucleotides in length. In some embodiments, the overlap (hybridization region between single- stranded overhangs) is 8 nucleotides in length.
• the size of the gap and/or hybridization region are selected to provide an optimum signal separation from a ligated template (comprising no internal target binding moieties) and non-ligated template (comprising at least one internal target-binding moiety).
• a double-stranded complex e.g ., before ligation occurs
• an entity of interest e.g., a biological entity
• a double-stranded complex e.g ., before ligation occurs
• an entity of interest e.g., a biological entity such as extracellular vesicles
• at least three or more target binding moieties are simultaneously bound to the entity of interest.
• selection of a combination e.g, a set
• detection probes e.g, number of detection probes and/or specific biomarkers
• a target entity detection system e.g, a duplex, triplex or multiplex target entity detection system described herein
• selection of a combination is based on, for example, a desired specificity and/or a desired sensitivity that is deemed to be optimal for a particular application.
• a combination of detection probes is selected for detection of lung cancer (e.g, for stage I, II, III, or IV) such that it provides a specificity of at least 95% or higher, including, e.g, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8% or higher.
• a combination of detection probes is selected for detection of lung cancer (e.g, for stage I, II, III, or IV) such that it provides a sensitivity of at least 30% or higher, including, e.g, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or higher.
• a combination of detection probes is selected for detection of lung cancer (e.g, for stage I, II, III, or IV) such that it provides a positive predictive value of at least 8% or higher, including, e.g, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or higher.
• a combination of detection probes is selected for detection of lung cancer (e.g, for stage I, II, III, or IV) such that it provides a positive predictive value of at least 2% or higher, including, e.g, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or higher.
• a combination of detection probes is selected for detection of lung cancer (e.g, for stage I, II, III, or IV) such that it provides a limit of detection (LOD) below lxlO 7 EV/mL sample or lower, including, e.g, below 7xl0 6 EV/mL sample, below 6xl0 6 EV/mL sample, below 5xl0 6 EV/mL sample, below 4xl0 6 EV/mL sample, below 3xl0 6 EV/mL sample, below 2xl0 6 EV/mL sample, below lxlO 6 EV/mL sample, or lower.
• LOD limit of detection
• such lung cancer detection assay may be used to detect different subtypes of lung cancer including, e.g, lung adenocarcinoma, small cell lung cancer, squamous and transitional cell lung cancer, large cell lung cancer, non-small cell carcinoma lung cancer, other specified carcinoma lung cancer, sarcoma lung cancer, and other specified types of lung cancer as known in the art (SEER Cancer Statistics Review 1975-2017).
• lung cancer detection assay may be used to detect lung cancer of an epithelial origin.
• such lung cancer detection assay may be used to detect non-small cell lung cancer (e.g ., lung adenocarcinoma and/or lung squamous cell carcinoma).
• such lung cancer detection assay may be used to detect lung adenocarcinoma.
• such lung cancer detection assay may be used to detect lung squamous cell carcinoma.
• a combination e.g., a set
• individual detection probes confers specificity to detection of a disease, disorder, or condition (e.g, a particular lung cancer and/or a stage of lung cancer as described herein), for example, one or more individual probes may be directed to a target that itself is not specific to lung cancer.
• a useful combination of detection probes in a target entity detection system comprises at least one detection probe directed to a target specific for the relevant disease, disorder, or condition (i.e., a target that is specific to the relevant disease, disorder, or condition), and may further comprise at least one detection probe directed to a target that is not necessarily or completely specific for the relevant disease, disorder, or condition (e.g, that may also be found on some or all cells that are healthy, are not of the particular disease, disorder, or condition, and/or are not of the particular disease stage of interest).
• a target specific for the relevant disease, disorder, or condition i.e., a target that is specific to the relevant disease, disorder, or condition
• a target that is not necessarily or completely specific for the relevant disease, disorder, or condition e.g, that may also be found on some or all cells that are healthy, are not of the particular disease, disorder, or condition, and/or are not of the particular disease stage of interest.
• the set of detection probes utilized in accordance with the present invention is or comprises a plurality of individual detection probes that together are specific for detection of the relevant disease, disorder, or condition (i.e., sufficiently distinguish biological entities for detection that are associated with the relevant disease, disorder, or condition from other biological entities not of interest for detection), the set is useful in accordance with certain embodiments of the present disclosure.
• a target entity detection system e.g, a duplex, triplex or multiplex target entity detection system described herein
• a control probe is designed such that its binding to an entity of interest (e.g ., a biological entity) inhibits (completely or partially) generation of a detection signal.
• a control probe comprises a control binding moiety and an oligonucleotide domain (e.g., as described and/or utilized herein) coupled to the control binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single-stranded overhang extended from one end of the oligonucleotide domain.
• a control binding moiety is an entity or moiety that bind to a control reference.
• a control reference can be or comprise a biomarker that is preferentially associated with a normal healthy cell.
• a control reference can be or comprise a biomarker preferentially associated from a non-target tissue.
• inclusion of a control probe can selectively remove or minimize detectable signals generated from false positives (e.g, entities of interest comprising a control reference, optionally in combination with one or more targets to be detected).
• false positives e.g, entities of interest comprising a control reference, optionally in combination with one or more targets to be detected.
• the present disclosure provides insights, among other things, that detection probes as described or utilized herein may non-specifically bind to a solid substrate surface and some of them may remain in an assay sample even after multiple washes to remove any excess or unbound detection probes; and that such non-specifically bound detection probes may come off from the solid substrate surface and become free-floating in a ligation reaction, thus allowing them to interact with one another to generate a non-specific ligated template that produces an undesirable background signal.
• a target entity detection system e.g, a duplex, triplex, or multiplex target entity detection described herein
• a target entity detection system can comprise at least one or more (e.g, at least 2 or more) inhibitor oligonucleotides that are designed to capture residual detection probes that are not bound to an entity of interest but remain as free agents in a ligation reaction, thereby preventing such free- floating detection probes from interacting with other free-floating complementary detection probes to produce an undesirable background signal.
• an inhibitor oligonucleotide may be or comprise a single-stranded or double-stranded oligonucleotide comprising a binding domain for a single-stranded overhang of a detection probe ( e.g ., as described or utilized herein), wherein the inhibitor oligonucleotide does not comprise a primer binding site.
• the absence of such a primer binding site in an inhibitor oligonucleotide prevents a primer from binding to a non-specific ligated template resulting from ligation of a detectable probe to an inhibitor oligonucleotide, thereby reducing or inhibiting the non-specific ligated template from amplification and/or detection, e.g., by polymerase chain reaction.
• an inhibitor oligonucleotide comprises a binding domain for a single-stranded overhang of a detection probe (e.g, as described or utilized herein), wherein the binding domain is or comprises a nucleotide sequence that is substantially complementary to the single-stranded overhang of the detection probe such that a free, unbound detection probe having a complementary single-stranded overhang can bind to the binding domain of the inhibitor oligonucleotide.
• an inhibitor oligonucleotide may have a hairpin at one end.
• an inhibitor oligonucleotide may be a single-stranded oligonucleotide comprising at one end a binding domain for a single-stranded overhang of a detection probe, wherein a portion of the single-stranded oligonucleotide can self-hybridize to form a hairpin at another end.
• a target entity detection system does not comprise a connector oligonucleotide that associates an oligonucleotide domain of a detection probe with an oligonucleotide domain of another detection probe.
• a connector oligonucleotide is designed to bridge oligonucleotide domains of any two detection probes that would not otherwise interact with each other when they bind to an entity of interest.
• a connector oligonucleotide is designed to hybridize with at least a portion of an oligonucleotide domain of a detection probe and at least a portion of an oligonucleotide domain of another detection probe.
• a connector oligonucleotide can be single-stranded, double-stranded, or a combination thereof.
• a connector oligonucleotide is free of any target-binding moiety (e.g, as described and/or utilized herein) or control binding moiety.
• no connector oligonucleotides are necessary to indirectly connect oligonucleotide domains of detection probes; in some embodiments, such connector oligonucleotides are not utilized, in part because detection probes as provided and/or utilized herein are designed such that their respective oligonucleotide domains have a sufficient length to reach and interact with each other when they are in sufficiently close proximity, e.g ., when the detection probes simultaneously bind to an entity of interest (e.g, a biological entity such as an extracellular vesicle).
• an entity of interest e.g, a biological entity such as an extracellular vesicle.
• Provided target entity detection systems are useful in detecting an entity of interest (e.g, a biological entity such as extracellular vesicles) in a sample (e.g, in a biological, environmental, or other sample) for various applications and/or purposes associated with detection of lung cancer. Accordingly, some aspects provided herein relate to methods of using a plurality of (e.g, at least 2, at least 3, or more) detection probes appropriate for use in accordance with the present disclosure.
• a method comprises contacting an entity of interest (e.g, a biological entity such as extracellular vesicles) in a sample (e.g, a blood or blood-derived sample from a human subject) with a set of detection probes comprising at least 2 or more (including, e.g, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more) detection probes as described and/or utilized herein.
• an entity of interest e.g, a biological entity such as extracellular vesicles
• a sample e.g, a blood or blood-derived sample from a human subject
• detection probes comprising at least 2 or more (including, e.g, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at
• a method comprises subjecting a sample comprising an entity of interest (e.g, a biological entity such as extracellular vesicles) to a target entity detection system (e.g, as provided herein).
• a plurality of detection probes e.g, at least two or more
• a method comprises, prior to contacting with a plurality of detection probes, contacting a sample comprising an entity of interest with at least one capture agent directed to an extracellular vesicle-associated membrane-bound polypeptide.
• a method comprises contacting an entity of interest (e.g, a biological entity such as extracellular vesicles) in a sample (e.g, a blood or blood-derived sample from a human subject) with a plurality of sets of detection probes, wherein each set comprises at least 2 or more (including, e.g, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more) detection probes as described and/or utilized herein.
• an entity of interest e.g, a biological entity such as extracellular vesicles
• a sample e.g, a blood or blood-derived sample from a human subject
• each set comprises at least 2 or more (including, e.g, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least
• a method comprises subjecting a sample comprising an entity of interest (e.g, a biological entity such as extracellular vesicles) to a target entity detection system (e.g, as provided herein).
• a plurality of detection probes and/or detection probe combinations e.g, at least two or more can be added to a sample comprising an entity of interest (e.g, a biological entity such as extracellular vesicles) at the same time or at different times (e.g, sequentially).
• a method comprises, prior to contacting with a plurality of detection probes, contacting a sample comprising an entity of interest with at least one capture agent directed to an extracellular vesicle-associated membrane-bound polypeptide.
• the relationship between results (e.g, Ct values and/or relative number of ligated nucleic acid templates (e.g, ligated DNA templates)) from profiling one or more biomarker combinations in a sample can be combined with clinical information (including, e.g, but not limited to patient age, past medical history, X-ray results, smoking history, etc.) and/or other information to better classify patients with or at risk for lung cancer.
• clinical information including, e.g, but not limited to patient age, past medical history, X-ray results, smoking history, etc.
• Various classification algorithms can be used to interpret the relationship between multiple variables to increase an assay’s sensitivity and/or specificity.
• such algorithms include, but are not limited to, logistic regression models, support vector machines, gradient boosting machines, random forest algorithms, Naive Bayes algorithms, K-nearest neighborhood algorithms, and combinations thereof.
• performance (e.g, accuracy) of assays described herein can be improved, e.g, by selection of biomarker combinations (e.g, as described herein), selection of other factors or variables (e.g, clinical information and/or lifestyle information, e.g, smoking history) to include an algorithm, and/or selection of the type of algorithm itself.
• technologies described herein utilize a predictive algorithm that is trained and validated using data sets as described herein.
• technologies described herein are utilized to generate a risk score using an algorithm created from training samples which is designed to take into account results from at least two, e.g, at least two, at least 3, at least 4, at least 5, or more than 5 separate assays comprising biomarker signatures ( e.g ., as described herein).
• an algorithm-generated risk score can be generated at least in part using diagnostic data (e.g., raw and/or normalized Ct values) from at least one individual assay (e.g, individual biomarker signature).
• a reference threshold can be included within a risk score.
• multiple threshold levels denoting multiple different degrees of lung cancer risk may be included in a risk score.
• separate target biomarker signature assays may be performed as individual assays in a series of assays, and individual assays may be weighted equally or differently in a predictive algorithm.
• weighting of individual assays combined in an algorithm may be determined by a number of factors including but not limited to the sensitivity of an individual assay, the specificity of an individual assay, the reproducibility of an individual assay, the variability of an individual assay, the positive predictive value of an individual assay, and/or the lowest limit of detection of a specific assay.
• a cohort of biomarker assays may be ranked according to a characteristic (e.g, sensitivity, specificity, lowest limit of detection etc) and the biomarker assays may then be weighted based upon their relative rank.
• a risk score generated by an algorithm can be presented in a suitable manner, e.g, on a nominal scale, e.g, on a scale of 0-100 reflecting a number of likelihoods, e.g, including but not limited to the likelihood a subject has lung cancer, the likelihood a subject will develop lung cancer, and/or the likely stage of lung cancer.
• a higher risk score can demonstrate that there is an increasing likelihood of disease pathology, e.g, lower to higher values may reflect healthy controls, benign controls, stage I, stage II, stage III, and stage IV lung cancers.
• a risk score can be utilized to reduce the potential of cross reactivity of technologies as described herein when compared with other cancer types.
• a risk score may be generated from a combination of data derived from assays as described herein coupled with other applicable diagnostic data such as age, life history, X-ray results, MRI results, low-dose CT scanning, or any combination thereof.
• a risk score provides predictive value above and beyond that of conventional standard of care diagnostic assay predictive values, e.g, higher than predictive values provided low-dose CT scanning assays utilized in isolation or in combination with another diagnostic assay.
• a risk score may be generated that has high specificity for lung cancers (e.g ., lung adenocarcinoma and/or lung squamous cell carcinoma) and has low sensitivity for other cancers.
• a risk score may have an associated clinical cutoff for detection of lung cancer.
• a risk score clinical cutoff for detection may require an assay that yields at least 40%, e.g. , at least 50%, at least 60%, or greater sensitivity for detection of both early and late stage lung cancer and has a minimum of 95% specificity, e.g., at least 96%, at least 97%, at least 98%, at least 99% or greater specificity in a generally healthy population of subjects aged 20 to 89 years of age.
• sensitivity and specificity targets are the approximate lower bounds of the two-sided 95% confidence interval for the targeted 77% sensitivity and 99.5% specificity.
• a training study is performed to provide the necessary data required to program a risk score algorithm.
• a training study comprises a cohort of samples from a range of suppliers, including at least commercial suppliers, purpose driven studies, and/or physicians.
• a training study comprises positive samples from lung adenocarcinoma and/or lung squamous cell carcinoma cancer patients (e.g, stage I, stage II, stage III, and/or stage IV), positive control samples from certain lung cancer cell lines, negative samples from benign lung tumor patients, negative samples from non-lung cancer patients (e.g, brain cancer, breast cancer, colorectal cancer, endometrial cancer, ovarian cancer, melanoma, non-Hodgkin’s lymphoma, pancreatic cancer, skin cancer, etc.), negative samples from inflammatory condition patients (e.g, Crohn’s disease, endometriosis, diabetes type II, lupus, pancreatitis, rheumatoid arthritis, ulcerative colitis, chronic obstructive pulmonary disease, etc.), negative samples from healthy patients, or any combination thereof.
• lung adenocarcinoma and/or lung squamous cell carcinoma cancer patients e.g, stage I, stage II, stage III, and/or
• a training study comprises samples from patients of any appropriate age range, e.g, ⁇ 31 years old, 31-40 years old, 41-50 years old, 51-60 years old, 61-70 years old, 71- 80 years old, or >80 years old.
• a training study comprises samples from patients of any race/ethnicity/decent, (e.g, Caucasians, Africans, Asians etc.).
• a validation study is performed to provide the necessary data required to confirm a risk score algorithm’s utility.
• a validation study comprises a cohort of samples from a range of suppliers, including at least commercial suppliers, purpose driven studies, and/or physicians.
• a validation study comprises positive samples from lung adenocarcinoma and/or lung squamous cell carcinoma cancer patients (e.g ., stage I, stage II, stage III, and/or stage IV), positive control samples from certain lung cancer cell lines, negative samples from benign lung tumor patients, negative samples from non-lung cancer patients (e.g., brain cancer, breast cancer, colorectal cancer, endometrial cancer, ovarian cancer, melanoma, non-Hodgkin’s lymphoma, pancreatic cancer, skin cancer, etc.), negative samples from inflammatory condition patients (e.g, Crohn’s disease, endometriosis, diabetes type II, lupus, pancreatitis, rheumatoid arthritis, ulcerative colitis, chronic obstructive pulmonary disease, etc.), negative samples from healthy patients, or any combination thereof.
• lung adenocarcinoma and/or lung squamous cell carcinoma cancer patients e.g ., stage I, stage II, stage
• a validation study comprises samples from patients of any appropriate age range, e.g, ⁇ 31 years old, 31-40 years old, 41-50 years old, 51-60 years old, 61-70 years old, 71-80 years old, or >80 years old.
• a validation study comprises samples from patients of any race/ethnicity/decent, (e.g, Caucasians, Africans, Asians etc).
• At least one target biomarker signature comprising at least one extracellular vesicle-associated membrane-bound polypeptide and at least one (including, e.g, at least two, or more) target biomarker (which may be selected from any of surface protein biomarkers described herein, intravesicular protein biomarkers described herein, and/or intravesicular RNA biomarkers described herein) may be embodied in a lung cancer detection assay.
• at least one capture agent is directed to the extracellular vesicle-associated membrane-bound polypeptide, and at least one set of detection probes is directed to one or more of such target biomarkers described herein.
• Figures 5-7 disclose certain examples of target biomarker signatures, each of which may be embodied in a lung cancer detection assay (e.g, ones described herein).
• each distinct target biomarker signature may have a different pre-determined cutoff value for individually determining whether a sample is positive for lung cancer.
• a sample is determined to be positive for lung cancer if assay readout is above at least one of cutoff values for a plurality of ( e.g ., at least 2 or more) target biomarker signatures.
• a combination of cutoff values e.g., at least 2, at least 3, or more can be utilized to create a diagnostic value with corollarily improved sensitivity and/or specificity.
• a sample can be divided into aliquots such that a different capture agent and/or a different set of detection probes (e.g, each directed to detection of a distinct disease or condition) can be added to a different aliquot.
• a different capture agent and/or a different set of detection probes e.g, each directed to detection of a distinct disease or condition
• provided technologies can be implemented with one aliquot at a time or multiple aliquots at a time (e.g, for parallel assays to increase throughput).
• amount of detection probes that is added to a sample provides a sufficiently low concentration of detection probes in a mixture to ensure that the detection probes will not randomly come into close proximity with one another in the absence of binding to an entity of interest (e.g, biological entity), at least not to any great or substantial degree.
• an entity of interest e.g, biological entity
• the detection probes when detection probes simultaneously bind to the same entity of interest (e.g, biological entity) through the binding interaction between respective targeting binding moieties of the detection probes and the binding sites of an entity of interest (e.g, a biological entity), the detection probes come into sufficiently close proximity to one another to form double-stranded complex (e.g, as described herein).
• the concentration of detection probes in a mixture following combination with a sample may range from about 1 fM to 1 mM, such as from about lpM to about 1 nM, including from about 1 pM to about 100 nM.
• the concentration of an entity of interest (e.g, a biological entity) in a sample is sufficiently low such that a detection probe binding to one entity of interest (e.g, a biological entity) will not randomly come into close proximity with another detection probe binding to another entity of interest (e.g, biological entity) in the absence of respective detection probes binding to the same entity of interest (e.g, biological entity), at least not to any great or substantial degree.
• the concentration of an entity of interest (e.g, biological entity) in a sample is sufficiently low such that a first target detection probe binding to a non-target entity of interest (e.g, a non-cancerous biological entity such as an extracellular vesicle comprising a first target) will not randomly come into close proximity with another different target detection probe that is bound to another non-target entity of interest (e.g, a non- cancerous biological entity such as an extracellular vesicle), at least not to any great or substantial degree, to generate a false positive detectable signal.
• a non-target entity of interest e.g, a non-cancerous biological entity such as an extracellular vesicle comprising a first target
• another non-target entity of interest e.g, a non- cancerous biological entity such as an extracellular vesicle
• such a mixture may be incubated for a period of time sufficient for the detection probes to bind corresponding targets (e.g., molecular targets), if present, in the entity of interest to form a double-stranded complex (e.g, as described herein).
• targets e.g., molecular targets
• such a mixture is incubated for a period of time ranging from about 5 min to about 5 hours, including from about 30 min to about 2 hours, at a temperature ranging from about 10 to about 50 °C, including from about 20 °C to about 37 °C.
• a double-stranded complex (resulted from contacting an entity of interest such as a biological entity with detection probes) can then be subsequently contacted with a nucleic acid ligase to perform nucleic acid ligation of a free 3' end hydroxyl and 5' end phosphate end of oligonucleotide strands of detection probes, thereby generating a ligated template comprising oligonucleotide strands of at least two or more detection probes.
• At least one or more inhibitor oligonucleotide can be added to the assay sample such that the inhibitor oligonucleotide can capture any residual free-floating detection probes that may otherwise interact with each other during a ligation reaction.
• ligases catalyze the formation of a phosphodiester bond between juxtaposed 3'-hydroxyl and 5'-phosphate termini of two immediately adjacent nucleic acids when they are annealed or hybridized to a third nucleic acid sequence to which they are complementary.
• Any known nucleic acid ligase e.g, DNA ligases
• temperature sensitive ligases include bacteriophage T4 DNA ligase, bacteriophage T7 ligase, and E. coli ligase.
• thermostable ligases include Taq ligase, Tth ligase, and Pfu ligase.
• Thermostable ligase may be obtained from thermophilic or hyperthermophilic organisms, including but not limited to, prokaryotic, eukaryotic, or archael organisms.
• a nucleic acid ligase is a DNA ligase.
• a nucleic acid ligase can be a RNA ligase.
• a suitable nucleic acid ligase e.g., a DNA ligase
• any reagents that are necessary and/or desirable are combined with the reaction mixture and maintained under conditions sufficient for ligation of the hybridized ligation oligonucleotides to occur.
• Ligation reaction conditions are well known to those of skill in the art.
• a reaction mixture in some embodiments, may be maintained at a temperature ranging from about 20° C to about 45° C, such as from about 25° C to about 37° C for a period of time ranging from about 5 minutes to about 16 hours, such as from about 1 hour to about 4 hours.
• a reaction mixture may be maintained at a temperature ranging from about 35° C to about 45° C, such as from about 37° C to about 42° C, e.g, at or about 38 ° C, 39° C, 40° C or 41° C, for a period of time ranging from about 5 minutes to about 16 hours, such as from about 1 hour to about 10 hours, including from about 2 to about 8 hours.
• Detection of such a ligated template can provide information as to whether an entity of interest (e.g, a biological entity) in a sample is positive or negative for targets to which detection probes are directed.
• a detectable level of such a ligated template is indicative of a tested entity of interest (e.g, a biological entity) comprising targets (e.g, molecular targets) of interest.
• a detectable level is a level that is above a reference level, e.g, by at least 10% or more, including, e.g, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more.
• a reference level may be a level observed in a negative control sample, such as a sample in which an entity of interest comprising such targets is absent.
• a non-detectable level e.g, a level that is below the threshold of a detectable level
• targets e.g, molecular targets
• a threshold that separates a detectable level from a non-detectable level may be determined based on, for example, a desired sensitivity level, and/or a desired specificity level that is deemed to be optimal for each application and/or purpose.
• a specificity of 99.7% may be achieved using a system provided herein, for example by setting a threshold that is three standard deviations above a reference level (e.g, a level observed in a negative control sample, such as, e.g, a sample derived from one or more normal healthy individuals).
• a threshold of a detectable level e.g, as reflected by a detection signal intensity
• a method provided herein comprises, following ligation, detecting a ligated template, e.g, as a measure of the presence and/or amount of an entity of interest in a sample.
• detection of a ligated template may be qualitative or quantitative.
• a method provides a reading or evaluation, e.g., assessment, of whether or not an entity of interest (e.g, a biological entity) comprising at least two or more targets (e.g, molecular targets) is present in a sample being assayed.
• a method provides a quantitative detection of whether an entity of interest (e.g, a biological entity) comprising at least two or more targets (e.g., molecular targets) is present in a sample being assayed, e.g, an evaluation or assessment of the actual amount of an entity of interest (e.g, a biological entity) comprising at least two or more targets (e.g, molecular targets) in a sample being assayed.
• an evaluation or assessment of the actual amount of an entity of interest e.g, a biological entity
• targets e.g., molecular targets
• such quantitative detection may be absolute or relative.
• a ligated template formed by using technologies provided herein may be detected by an appropriate method known in the art. Those of skill in the art will appreciate that appropriate detection methods may be selected based on, for example, a desired sensitivity level and/or an application in which a method is being practiced.
• a ligated template can be directly detected without any amplification, while in other embodiments, ligated template may be amplified such that the copy number of the ligated template is increased, e.g, to enhance sensitivity of a particular assay.
• a ligated template may be detected in a number of different ways.
• oligonucleotide domains of detection probes may have been directly labeled, e.g, fluorescently or radioisotopically labeled, such that a ligated template is directly labeled.
• an oligonucleotide domain of a detection probe can comprise a detectable label.
• a detectable label may be a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
• Such labels include biotin for staining with labeled Streptavidin conjugate, magnetic beads (e.g, Dynabeads®), fluorescent dyes (e.g, fluorescein, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g, 3 H, 125 1, 34 S, 14 C, or 32 P), enzymes (e.g, horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g, polystyrene, polypropylene, latex, etc.) beads.
• a directly labeled ligated template may be size separated from the remainder of the reaction mixture, including unligated directly labeled ligation oligonucleotides, in order to detect the ligated template.
• detection of a ligated template can include an amplification step, where the copy number of ligated nucleic acids is increased, e.g., in order to enhance sensitivity of the assay.
• the amplification may be linear or exponential, as desired, where amplification can include, but are not limited to polymerase chain reaction (PCR); quantitative PCR, isothermal amplification, NASB A, digital droplet PCR, etc.
• PCR primers such as forward and reverse primers employed in geometric (or exponential) amplification or a single primer employed in a linear amplification.
• Oligonucleotide primers with which one or more ligated templates are contacted should be of sufficient length to provide for hybridization to complementary template DNA under appropriate annealing conditions.
• Primers are typically at least 10 bp in length, including, e.g. , at least 15 bp in length, at least 20 bp in length, at least 25 bp in length, at least 30 bp in length or longer.
• the length of primers can typically range from about 15 to 50 bp in length, from about 18 to 30 bp, or about 20 to 35 bp in length.
• Ligated templates may be contacted with a single primer or a set of two primers (forward and reverse primers), depending on whether primer extension, linear, or exponential amplification of the template DNA is desired.
• a reaction mixture comprising a ligated template typically includes a polymerase and deoxyribonucleoside triphosphates (dNTPs).
• the desired polymerase activity may be provided by one or more distinct polymerase enzymes.
• various constituent components may be combined in any convenient order. For example, an appropriate buffer may be combined with one or more primers, one or more polymerases and a ligated template to be detected, or all of the various constituent components may be combined at the same time to produce the reaction mixture.
• one or more provided biomarkers of one or more target biomarker signatures for lung cancer can be detected in a sample comprising biological entities (including, e.g, cells, circulating tumor cells, cell-free DNA, extracellular vesicles, etc.), for example, using methods of detecting and/or assays as described herein.
• one or more provided biomarkers of one or more target biomarker signatures for lung cancer can be detected in a sample comprising extracellular vesicles, for example, using methods of detecting and/or assays as described herein.
• a sample may be or comprise a biological sample.
• a biological sample can be derived from a blood or blood-derived sample of a subject ( e.g ., a human subject) in need of such an assay.
• a biological sample can be or comprise a primary sample (e.g., a tissue or tumor sample) from a subject (e.g, a human subject) in need of such an assay.
• a biological sample can be processed to separate one or more entities of interest (e.g, biological entity) from non-target entities of interest, and/or to enrich one or more entities of interest (e.g, biological entity).
• an entity of interest present in a sample may be or comprise a biological entity, e.g, a cell or an extracellular vesicle (e.g, an exosome).
• a biological entity e.g, extracellular vesicle
• a chemical reagent e.g, to stabilize and/or crosslink targets (e.g, provided target biomarkers) to be assayed in the biological entity and/or to reduce non-specific binding with detection probes.
• a biological entity is or comprises a cell, which may be optionally processed, e.g, with a chemical reagent for stabilizing and/or crosslinking targets (e.g, molecular targets) and/or for reducing non-specific binding.
• a biological entity is or comprises an extracellular vesicle (e.g, an exosome), which may be optionally processed, e.g, with a chemical reagent for stabilizing and/or crosslinking targets (e.g, molecular targets) and/or for reducing non-specific binding.
• technologies provided herein can be useful for managing patient care, e.g, for one or more individual subjects and/or across a population of subjects.
• provided technologies may be utilized in screening, which for example, may be performed periodically, such as annually, semi-annually, bi-annually, or with some other frequency as deemed to be appropriate by those skilled in the art.
• such a screening may be temporally motivated or incidentally motivated.
• provided technologies may be utilized in temporally-motivated screening for one or more individual subjects or across a population of subjects (e.g, asymptomatic subjects) who are older than a certain age (e.g, over 40, 45, 50, 55, 60, 65, 70, 75, 80, or older).
• a certain age e.g, over 40, 45, 50, 55, 60, 65, 70, 75, 80, or older.
• the screening age and/or frequency may be determined based on, for example, but not limited to prevalence of a disease, disorder, or condition (e.g ., cancer such as lung cancer).
• an incidental motivation relating to determination of one or more indicators of a disease, disorder, or condition (e.g, cancer such as lung cancer) or susceptibility thereto may be or comprise, e.g, an incident based on their family history (e.g, a close relative such as blood-related relative was previously diagnosed for such a disease, disorder, or condition such as lung cancer), identification of one or more life-history associated risk factors for a disease, disorder, or condition (e.g, lung cancer) and/or prior incidental findings from genetic tests (e.g, genome sequencing), and/or imaging diagnostic tests (e.g, chest X-ray, or low-dose CT scanning), development of one or more signs or symptoms characteristic of a particular disease, disorder, or condition (e.g, chronic obstructive pulmonary
• provided technologies for managing patient care can inform treatment and/or payment (e.g, reimbursement for treatment) decisions and/or actions.
• provided technologies can provide determination of whether individual subjects have one or more indicators of risk, incidence, or recurrence of a disease disorder, or condition (e.g, cancer such as lung cancer), thereby informing physicians and/or patients when to provide/receive therapeutic or prophylactic recommendations and/or to initiate such therapy in light of such findings.
• a disease disorder, or condition e.g, cancer such as lung cancer
• such individual subjects may be asymptomatic subjects, who may be temporally-motivated or incidentally-motivated screened at a regular frequency (e.g, annually, semi-annually, bi-annually, or other frequency as deemed to be appropriate by those skilled in the art).
• a regular frequency e.g., annually, semi-annually, bi-annually, or other frequency as deemed to be appropriate by those skilled in the art.
• individual subjects may be experiencing one or more symptoms that may be associated with lung cancer, who may be temporally-motivated or incidentally-motivated screened at a regular frequency (e.g ., annually, semi-annually, bi-annually, or other frequency as deemed to be appropriate by those skilled in the art).
• such individual subjects may be subjects having a benign lung tumor and/or a chronic inflammatory condition, who may be temporally-motivated or incidentally-motivated screened at a regular frequency (e.g., annually, semi-annually, bi- annually, or other frequency as deemed to be appropriate by those skilled in the art).
• a regular frequency e.g., annually, semi-annually, bi- annually, or other frequency as deemed to be appropriate by those skilled in the art.
• such individual subjects may be subjects at hereditary risk for lung cancer, who may be temporally-motivated or incidentally -motivated screened at a regular frequency (e.g, annually, semi-annually, bi-annually, or other frequency as deemed to be appropriate by those skilled in the art).
• such individual subjects may be subjects with life- history associated risk, who may be temporally-motivated or incidentally-motivated screened at a regular frequency (e.g, annually, semi-annually, bi-annually, or other frequency as deemed to be appropriate by those skilled in the art).
• a regular frequency e.g, annually, semi-annually, bi-annually, or other frequency as deemed to be appropriate by those skilled in the art.
• individual subjects may be smoking subjects (e.g, heavy smokers), who may be temporally-motivated or incidentally- motivated screened at a regular frequency (e.g, annually, semi-annually, bi-annually, or other frequency as deemed to be appropriate by those skilled in the art).
• provided technologies can inform physicians and/or patients of treatment selection, e.g., based on findings of specific responsiveness biomarkers (e.g., cancer responsiveness biomarkers).
• provided technologies can provide determination of whether individual subjects are responsive to current treatment, e.g, based on findings of changes in one or more levels of molecular targets associated with a disease, thereby informing physicians and/or patients of efficacy of such therapy and/or decisions to maintain or alter therapy in light of such findings.
• provided technologies can provide determination of whether individual subjects are likely to be responsive to a recommended treatment, e.g., based on findings of molecular targets (e.g, provided biomarkers of one or more target biomarker signatures for lung cancer) that predict therapeutic effects of a recommended treatment on individual subjects, thereby informing physicians and/or patients of potential efficacy of such therapy and/or decisions to administer or alter therapy in light of such findings.
• molecular targets e.g, provided biomarkers of one or more target biomarker signatures for lung cancer
• provided technologies can inform decision making relating to whether health insurance providers reimburse (or not), e.g, for (1) screening itself (e.g., reimbursement available only for periodic/regular screening or available only for temporally - and/or incidentally- motivated screening); and/or for (2) initiating, maintaining, and/or altering therapy in light of findings by provided technologies.
• the present disclosure provides methods relating to (a) receiving results of a screening that employs provided technologies and also receiving a request for reimbursement of the screening and/or of a particular therapeutic regimen; (b) approving reimbursement of the screening if it was performed on a subject according to an appropriate schedule (based on, e.g ., screening age such as older than a certain age, e.g. , over 40, 45, 50, 55, 60, 65, 70, 75, 80, or older, and/or screening frequency such as, e.g.
• a therapeutic regimen is appropriate in light of received screening results if the received screening results detect a biomarker that represents an approved biomarker for the relevant therapeutic regimen (e.g, as may be noted in a prescribing information label and/or via an approved companion diagnostic).
• reporting systems e.g, implemented via appropriate electronic device(s) and/or communications system(s) that permit or facilitate reporting and/or processing of screening results (e.g, as generated in accordance with the present disclosure), and/or of reimbursement decisions as described herein.
• Various reporting systems are known in the art; those skilled in the art will be well familiar with a variety of such embodiments, and will readily be able to select those suitable for implementation.
• the present disclosure recognizes that detection of a single cancer- associated biomarker in a biological entity (e.g, extracellular vesicle) or a plurality of cancer- associated biomarkers based on a bulk sample, rather than at a resolution of a single biological entity (e.g, individual extracellular vesicles), typically does not provide sufficient specificity and/or sensitivity in determination of whether a subject from whom the biological entity is obtained is likely to be suffering from or susceptible to cancer (e.g, lung cancer).
• a biological entity e.g, extracellular vesicle
• a plurality of cancer- associated biomarkers based on a bulk sample rather than at a resolution of a single biological entity (e.g, individual extracellular vesicles)
• a single biological entity e.g, individual extracellular vesicles
• the present disclosure provides technologies, including compositions and/or methods, that solve such problems, including for example by specifically requiring that an entity (e.g ., an extracellular vesicle) for detection be characterized by presence of a combination of at least two or more targets (e.g., at least two or more provided biomarkers of a target biomarker signature for lung cancer).
• an entity e.g ., an extracellular vesicle
• targets e.g., at least two or more provided biomarkers of a target biomarker signature for lung cancer.
• the present disclosure teaches technologies that require such an entity (e.g, an extracellular vesicle) be characterized by presence (e.g, by expression) of a combination of molecular targets that is specific to cancer (i.e., “target biomarker signature” of a relevant cancer, e.g, lung cancer), while biological entities (e.g, extracellular vesicles) that do not comprise the targeted combination (e.g, target biomarker signature) do not produce a detectable signal.
• targets e.g, an extracellular vesicle
• biological entities e.g, extracellular vesicles
• technologies provided herein can be useful for detection of risk, incidence, and/or recurrence of cancer in a subject.
• a combination of two or more provided biomarkers are selected for detection of a specific cancer (e.g, lung cancer) or various cancers (one of which includes lung cancer).
• a specific combination of provided biomarkers for detection of lung cancer can be determined by analyzing a population or library (e.g. tens, hundreds, thousands, tens of thousands, hundreds of thousands, or more) of lung cancer patient biopsies and/or patient data to identify such a predictive combination.
• a relevant combination of biomarkers may be one identified and/or characterized, for example, via data analysis.
• data analysis may comprise a bioinformatic analysis, for example, as described in Examples 7-9.
• a diverse set of lung cancer-associated data e.g, in some embodiments comprising one or more of bulk RNA sequencing, single-cell RNA (scRNA) sequencing, mass spectrometry, histology, post- translational modification data, in vitro and/or in vivo experimental data
• scRNA single-cell RNA
• a combination of predictive markers to distinguish stages of cancer can be determined in silico based on comparing and analyzing diverse data (e.g, in some embodiments comprising bulk RNA sequencing, scRNA sequencing, mass spectrometry, histology, post-translational modification data, in vitro and/or in vivo experimental data) relating to different stages of cancer (e.g, lung cancer).
• diverse data e.g, in some embodiments comprising bulk RNA sequencing, scRNA sequencing, mass spectrometry, histology, post-translational modification data, in vitro and/or in vivo experimental data
• technologies provided herein can be used to distinguish lung cancer subjects from non-lung cancer subjects, including, e.g ., healthy individuals, subjects diagnosed with benign tumors or thoracic masses, and subjects with non- lung-related diseases, disorders, and/or conditions (e.g, subjects with non-lung cancer, or subjects with inflammatory conditions, e.g, chronic obstructive pulmonary disease or chronic lung infections).
• technologies provided herein can be useful for early detection of lung cancer, e.g, detection of lung cancer of stage I or stage II.
• technologies provided herein can be useful for detection of one or more lung cancer subtypes, including, e.g.
• lung adenocarcinoma small cell lung cancer, squamous and transitional cell lung cancer, large cell lung cancer, non-small cell carcinoma, other specified lung carcinoma, lung sarcoma, and other specified types of lung cancer as known in the art (SEER Cancer Statistics Review 1975-2017).
• technologies provided herein can be useful for screening individuals at hereditary risk, life-history associated risk, or average risk for early stage lung cancer (e.g, lung adenocarcinoma and/or lung squamous cell carcinoma).
• technologies provided herein can be useful for screening a subject for risk, incidence, or recurrence of a specific cancer in a single assay.
• technologies provided herein is useful for screening a subject for risk, incidence, or recurrence of lung cancer.
• technologies provided herein can be used to screen a subject for risk or incidence of a specific cancer or a plurality of (e.g, at least 2, at least 3, or more) cancers in a single assay.
• technologies provided herein can be used to screen a subject for a plurality of cancers in a single assay, one of which includes lung cancer and other cancers to be screened can be selected from the group consisting of brain cancer (including, e.g, glioblastoma), breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, liver cancer, ovarian cancer, and skin cancer.
• brain cancer including, e.g, glioblastoma
• breast cancer including, e.g, glioblastoma
• colorectal cancer pancreatic cancer
• prostate cancer pancreatic cancer
• liver cancer ovarian cancer
• skin cancer skin cancer
• provided technologies can be used periodically (e.g, every year, every two years, every three years, etc.) to screen a human subject (e.g, a human subject) for lung cancer (e.g, early-stage lung cancer) or cancer recurrence.
• a human subject amenable to such screening may be an adult or an elderly.
• a human subject amenable to such screening may be older than a specified age, e.g, age 45 and above, age 55 and above, age 65 and above, age 70 and above, age 75 and above, or age 80 and above.
• a human subject amenable to such screening may have an age of about 50 or above.
• a human subject amenable to such screening may have an age of 50 or less.
• a human subject amenable to such screening may have an age over 35.
• a subject that is amenable to provided technologies for detection of incidence or recurrence of lung cancer may be a subject with a smoking history (e.g ., a heavy smoker), who in some embodiments may be experiencing one or more symptoms associated with lung cancer.
• a subject that is amenable to provided technologies for detection of incidence or recurrence of lung cancer may be a human subject who is determined to have a benign lung tumor and/or one or more chronic inflammatory conditions (e.g., COPD).
• a subject that is amenable to provided technologies for detection of incidence or recurrence of lung cancer may be a subject, who has a family history of lung cancer (e.g, subjects having one or more first-degree relatives with a history of lung cancer), who has been previously treated for cancer (e.g, lung cancer), who is at risk of lung cancer recurrence after cancer treatment, who is in remission after lung cancer treatment, and/or who has been previously or periodically screened for lung cancer, e.g, by screening for the presence of at least one lung cancer biomarker (e.g, as described herein).
• a family history of lung cancer e.g, subjects having one or more first-degree relatives with a history of lung cancer
• cancer e.g, lung cancer
• who is at risk of lung cancer recurrence after cancer treatment who is in remission after lung cancer treatment
• who has been previously or periodically screened for lung cancer e.g, by screening for the presence of at least one lung cancer biomarker (e.g, as described here
• the present disclosure provides insights that technologies described and/or utilized herein may be particularly useful for screening certain populations of subjects who are at higher susceptibility to developing lung cancer.
• the present disclosure recognizes that the resulting PPVs of technologies described and/or utilized herein for lung detection may be higher in lung cancer prone or susceptible populations.
• the present disclosure provides insights that screening of smoking individuals, e.g, regular screening prior to or otherwise in absence of developed symptom(s), can be beneficial, and even important for effective management (e.g, successful treatment) of lung cancer.
• the present disclosure provides lung cancer screening systems that can be implemented to detect lung cancer, including early-stage cancer, in some embodiments in smoking individuals (e.g, with or without hereditary and/or life-history risks in lung cancer and/or with or without symptoms associated with lung cancer).
• provided technologies can be implemented to achieve regular screening of smoking individuals (e.g, with or without hereditary and/or life-history risks in lung cancer and/or with or without symptoms associated with lung cancer).
• provided technologies achieve detection (e.g ., early detection, e.g., in symptomatic or asymptomatic individual(s) and/or population(s)) of one or more features (e.g, incidence, progression, responsiveness to therapy, recurrence, etc.) of lung cancer, with sensitivity and/or specificity (e.g, rate of false positive and/or false negative results) appropriate to permit useful application of provided technologies to single-time and/or regular (e.g, periodic) assessment.
• provided technologies are useful in conjunction with a subject’s periodic physical examination (e.g. every year, every other year, or at an interval approved by the attending physician).
• provided technologies are useful in conjunction with treatment regimen(s); in some embodiments, provided technologies may improve one or more characteristics (e.g, rate of success according to an accepted parameter) of such treatment regimen(s).
• a subject that is amenable to provided technologies for detection of incidence or recurrence of lung cancer may be an asymptomatic human subject and/or across an asymptomatic population of subjects.
• Such an asymptomatic subject and/or across an asymptomatic population of subjects may be subject(s) who has/have a family history of cancer such as breast, ovarian, leukemia, and/or lung cancer (e.g, individuals having one or more first- degree relatives with a history of cancers known to be associated with genetic risk factors), who has been previously treated for cancer (e.g, lung cancer), who is at risk of lung cancer recurrence after cancer treatment, who is in remission after lung cancer treatment, and/or who has been previously or periodically screened for lung cancer, e.g, by screening for the presence of at least one lung cancer biomarker or through thoracic imaging (e.g, X-ray imaging, sputum testing, low-dose CT scanning, and/ or molecular tests based on cell-
• an asymptomatic subject may be a subject with a benign lung tumor.
• an asymptomatic subject may be a subject who is susceptible to lung cancer (e.g, at an average population risk, at an elevated life-history associated risk, or with hereditary risk for lung cancer).
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be selected based on one or more characteristics such as age, race, genetic history, medical history, personal history (e.g ., smoking, alcohol, drugs, carcinogenic agents, diet, obesity, physical activity, sun exposure, radiation exposure, exposure to infectious agents such as viruses, and/or occupational hazard).
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or a population of subjects determined to currently be or have been a smoker (e.g. cigarettes, cigars, pipe, and/or hookah).
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or a population of subjects determined to have worked in conditions known to expose the subject(s) to inhalable carcinogens, including but not limited to: copper ore smelting, lead ore smelting, zinc ore smelting, manufacture of insecticides, arsenic mining, asbestos mining, asbestos textile production, brake lining work, cement production, construction work, insulation work, shipyard work, ceramic manufacture, electronic and aerospace equipment manufacture, chemical manufacturing, chromate production, chromium electroplating, leather tanning, pigment production, nickel mining, nickel refining, nickel electroplating, production of stainless and heat-resistant steel, polycyclic aromatic production, aluminum production, hydrocarbon compound production, coke production, ferrochromium alloy production, nickel-containing ore smelting, roofing, radon mining, ceramics and glass production, granite working, metal ore smelting, silica mining and quarrying stone.
• inhalable carcinogens including
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or a population of subjects determined to have one or more germline mutations in lung cancer-associated genes (e.g, genes associated with DNA repair pathways such as ATM or BRCA, and/or germline mutations in the potential oncogene epidermal growth factor receptor (EGFR)), and combinations thereof.
• lung cancer-associated genes e.g, genes associated with DNA repair pathways such as ATM or BRCA, and/or germline mutations in the potential oncogene epidermal growth factor receptor (EGFR)
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or a population of subjects diagnosed with an imaging-confirmed thoracic mass or pulmonary mass.
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or a population of subjects at hereditary risk or life-history associated risk before undergoing a risk-reducing pulmonary biopsy.
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or population of subjects determined to have COPD or pulmonary fibrosis.
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or population of subjects with a history of chronic bronchitis, tuberculosis, and/or pneumonia.
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or population of subjects determined to have HIV and/or AIDS.
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or population of subjects with high current or historical alcohol consumption.
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or population of subjects determined to have hereditary mutations in EGFR, cytochrome p450 enzymes, and/or DNA repair genes.
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or population of subjects exposed to radiation therapy and/or chemotherapy.
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or a population of subjects with one or more non-specific symptoms of lung cancer.
• exemplary non-specific symptoms of lung cancer may include symptoms similar to those of chronic obstructive pulmonary disease, and/or symptoms such as bloody sputum, persistent cough, shortness of breath, repeated and/or chronic respiratory infection, thoracic pain, unexplained weight loss, hemoptysis, airway obstruction, and/or fatigue.
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or a population of subjects of diverse descents such as Asians, African Americans, Caucasians, Native Hawaiians or other Pacific Islanders, Hispanics or Latinos, American Indians or Alaska natives, non-Hispanic blacks, or non-Hispanic whites.
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or a population of subjects of diverse descents such as Asian Pacific Islanders, Hispanics, American Indian/ Alaska natives, non-Hispanic black, or non-Hispanic white.
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be a subject or a population of subjects of any race and/or any ethnicity.
• a subject or population of subjects that are amenable to provided technologies for detection of lung cancer may be determined to have normal X-ray imaging, sputum testing, low-dose CT scanning, and/or molecular tests based on cell-free nucleic acids, serum proteins (e.g ., CEA, CYFRA 21-1, NSE, ProGRP, and/or SCCA).
• serum proteins e.g ., CEA, CYFRA 21-1, NSE, ProGRP, and/or SCCA.
• such subjects may have received a negative indication of lung cancer from such diagnostic tests.
• such subjects may have received a positive indication of lung cancer from such diagnostic tests.
• technologies provided herein can be used in combination with other diagnostics assays including e.g., but not limited to: (i) physicals, general practitioner visits, cholesterol/lipid blood tests, diabetes (type 2) screening, colonoscopies, blood pressure screening, thyroid function tests, prostate cancer screening, mammograms, HPV/Pap smears, and/or vaccinations; (ii) chest X-ray imaging, sputum testing, chest low-dose CT scanning, and/ or molecular tests based on cell-free nucleic acids, serum proteins (e.g, CEA, CYFRA 21-1, NSE, ProGRP, and/or SCCA); (iii) a genetic assay to screen blood plasma for genetic mutations in circulating tumor DNA and/or protein biomarkers linked to cancer; (iv) an assay involving immunofluorescence staining to identify cell phenotype and marker expression, followed by amplification and analysis by next-generation sequencing; and/or (v) EGFR, KRAS, A
• cancer therapy e.g., lung cancer therapy
• provided technologies can be used for selecting an appropriate treatment for a cancer patient (e.g, a patient suffering from or susceptible to lung cancer).
• a cancer patient e.g, a patient suffering from or susceptible to lung cancer.
• some embodiments provided herein relate to a companion diagnostic assay for classification of patients for cancer therapy (e.g, lung cancer and/or adjunct treatment) which comprises assessment in a patient sample (e.g, a blood or blood-derived sample from a lung cancer patient) of a selected combination of provided biomarkers using technologies provided herein.
• a cancer therapy e.g ., a lung cancer therapy and/or an adjunct therapy, including, e.g ., Abraxane, Afatinib Dimaleate, Alectinib, Atezolizumab, Bevacizumab, Brigatinib, Capmatinib Hydrochloride, Carboplatin, Ceritinib, Crizotinib, Dabrafenib Mesylate,
• a cancer therapy e.g a lung cancer therapy and/or an adjunct therapy, including, e.g ., Abraxane, Afatinib Dimaleate, Alectinib, Atezolizumab, Bevacizumab, Brigatinib, Capmatinib Hydrochloride, Carboplatin, Ceritinib, Crizotinib, Dabrafenib Mesylate,
• treatment efficacy e.g., cancer treatment efficacy
• technologies provided herein can be used for monitoring and/or evaluating efficacy of an anti-cancer therapy administered to a cancer patient (e.g, lung cancer patient).
• a blood or blood-derived sample can be collected from an lung cancer patient prior to or receiving an anti-cancer therapy (e.g, Abraxane, Afatinib Dimaleate,
• a second blood or blood-derived sample can be collected from the same lung cancer patient to detect changes in tumor burdens, e.g, by detecting absence or reduction in amount of extracellular vesicles comprising a selected combination of biomarkers that is specific to detection of lung cancer.
• appropriate course of action e.g, increasing or decreasing the dose of a therapeutic agent, and/or administering a different therapeutic agent, can be taken.
• kits that find use in practicing technologies as described above.
• a kit comprises a plurality of detection probes (e.g ., as described and/or utilized herein).
• a provided kit comprises two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) detection probes.
• individual detection probes may be directed at different targets.
• two or more individual detection probes may be directed to the same target.
• a provided kit comprises two or more different detection probes directed at different targets, and optionally may include at least one additional detection probe also directed at a target to which another detection probe is directed.
• a provided kit comprises a plurality of subsets of detection probes, each of which comprises two or more detection probes directed at the same target.
• a plurality of detection probes may be provided as a mixture in a container.
• multiple subsets of detection probes may be provided as individual mixtures in separate containers.
• each detection probe is provided individually in a separate container.
• a kit for detection of lung cancer comprises: (a) a capture agent comprising a target-capture moiety directed to an extracellular vesicle-associated membrane- bound polypeptide; and (b) a set of detection probes, which set comprises at least two detection probes each directed to a target biomarker of a target biomarker signature for lung cancer, wherein the detection probes each comprise:(i) a target binding moiety directed the target biomarker of the target biomarker signature for lung cancer; and (ii) an oligonucleotide domain coupled to the target binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single-stranded overhang portion extended from one end of the oligonucleotide domain, wherein the single-stranded overhang portions of the at least two detection probes are characterized in that they can hybridize to each other when the at least two detection probes are bound to the same extracellular vesicle.
• such a target biomarker signature for lung cancer comprises: at least one extracellular vesicle-associated membrane-bound polypeptide and at least one target biomarker selected from the group consisting of: surface protein biomarkers, intravesicular protein biomarkers, and intravesicular RNA biomarkers, wherein: • the surface protein biomarkers are selected from: ABCC3, ALCAM, ARSL, B3GNT3, CDCP1, CDH1, CDH3, CD55, CD274 (PD-L1) CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3, CLDN4, CLDN7, CLIC6, DMBT1, DSG2, EGFR, EpCAM, EPHX3, EVA1A, FOLR1, GJB1, GJB2, GPC4, HS6ST2, IG1FR, KDELR3, KRTCAP3, LAMB 3, LFNG, LSR, MANEAL, MET, MSLN, MUC1, MUC21, PIGT, PODXL2, PRRG4, ROS1, SDC
• the intravesicular protein biomarkers are selected from: AOC1, C12orf45, CRABP2, CST1, ETV4, FAM83A, FOXA2, HMGB3, LGALS3BP, MIF, NAPSA, PPP1R14D, S100A14, SBK1, SCGB3A2, SFTA2, SFTPA1, SFTPA2, SFTPB, SPINK1, TGFA, ZC3H11 A, and combinations thereof;
• the intravesicular RNA biomarkers are selected from ABCC3, AOC1, ARSL, B3GNT3, C12orf45, CDCP1, CDH1, CDH3, CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3, CLDN4, CLDN7, CLIC6, CRABP2, CST1, DMBTl, DSG2, EPCAM, EPHX3, ETV4, EVA1A, FAM83A, FOLR1, FOXA2, GJB1, GJB2, GPC4, HMGB3, HS6ST2, KDELR3, KRTCAP3, LAMB 3, LFNG, LGALS3BP, LSR, MANEAL, MIF, MSLN, MUC1, MUC21, NAPSA, PIGT, PODXL2, PPP1R14D, PRRG4, ROS1, S100A14, SBK1, SCGB3A2, SDC1, SERINC2, SEZ6L2, SFTA2, SFTPA1, SFTPA2, SFTPB, SLC34A2, SLC44A4, SLC
• a kit for detection of lung cancer comprises: (a) a capture agent comprising a target-capture moiety directed to an extracellular vesicle-associated membrane- bound polypeptide; and (b) a set of detection probes, which set comprises at least two detection probes each directed to a target biomarker of a target biomarker signature for lung cancer, wherein the detection probes each comprise:(i) a target binding moiety directed the target biomarker of the target biomarker signature for lung cancer; and (ii) an oligonucleotide domain coupled to the target binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single-stranded overhang portion extended from one end of the oligonucleotide domain, wherein the single-stranded overhang portions of the at least two detection probes are characterized in that they can hybridize to each other when the at least two detection probes are bound to the same extracellular vesicle.
• such a target biomarker signature for lung cancer comprises at least one extracellular vesicle-associated membrane-bound polypeptide ( e.g ., as described herein) and at least one target biomarker selected from the group consisting of: surface protein biomarkers (e.g., as described herein), intravesicular protein biomarkers (e.g, as described herein), and intravesicular RNA biomarkers (e.g, as described herein).
• one or more surface protein biomarkers utilized in a provided kit are selected from: ABC A3, ABCC1, ABCC3, ACBD3, ACSL5, AGER, ALCAM, API M2, APHIA, APOO, ATP11A, ATP11B, ATP1B1, ATP6AP2, B4GALT4, BCAP31, BSPRY,
• CD 109 CD55, CD9, CDC42, CDH1, CDH3, CDKALl, CEACAM5, CEACAM6, CELSR1, CIP2A, CISD2, CKAP4, CLCA2, CLDN1, CLIC6, CLPTM1L, CLSTN1, CNTN1, CPD, CYP2S1, CYP4F11, CYP4F3, DPY19L1, DSC2, DSC3, DSG2, DSG3, EGFR, EPCAM, EPHB3, FAT2, FBX045, FERMT1, FOLR1, FZD6, GALNTl, GALNT3, GALNT5, GALNT6, GGCX, GOLM1, GOLPH3L, GRHL2, HACD3, IER3IP1, IGSF3, IL1RAP, ITGA2, ITGB6, KLRG2, KPNA2, KRTCAP3, LAD1, LAMB 3, LAMC2, LAMP3, LAMTOR2, LCLAT1, LPCAT1, LSR, MAGT1, MARCKSLl, MET,
• one or more intravesicular protein biomarkers utilized in a provided kit are selected from: ABRACL, ACP5, ADH7, AGR2, AIF1, AKR1C1, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, AOC1, API M2,
• one or more intravesicular RNA biomarkers utilized in a provided kit are selected from: ABC A3, ABCCl, ABCC3, ABRACL, ACP5, ADAM23, ADH7, AGR2, AIF1, AKR1C1, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, ANTXRl, AOC1, API M2, APOBEC3B, APOBEC3C, AQP3, AREG, ARNTL2, ARSL, ASF IB, ATP8B1, AURKB, B3GNT3, B3GNT5, BAIAP2L1, BCAM, BIK, BIRC5, C12orf45, C15orf48, C19orf33, CIS, C8orf4, CA12, CA9, CALML3, CAPNS2, CBLC, CCL19, CCL5, CCNB2, CD109, CD24, CD53, CD74, CD9, CDC20, CDC42EP
• the selected surface protein biomarker(s) and the at least one extracellular vesicle-associated membrane-bound polypeptide are different. In some embodiments, when at least one target biomarker is selected from one or more of the provided surface protein biomarkers, the selected surface protein biomarker(s) and the at least one extracellular vesicle-associated membrane-bound polypeptide are the same (with the same or different epitopes).
• a capture agent provided in a kit comprises a target-capture moiety directed to an extracellular vesicle-associated membrane-bound polypeptide biomarker, which is or comprises one or more of: ALCAM, B3GNT3, CDCP1, CDH1, CDH3, CD55, CD274 (PD-L1), CEACAM5, CEACAM6, CLDN3, CLDN4, DSG2, EGFR, EPCAM, FOLR1, GJB1, GJB2, IG1FR, LAMB 3, MET, MSLN, MUC1, PIGT, PODXL2, ROS1, SDC1,
• a capture agent provided in a kit comprises a target-capture moiety directed to an extracellular vesicle-associated membrane-bound polypeptide, which is or comprises one or more polypeptides selected from SLC34A2, CEACAM5, CEACAM6, EpCAM, and/or combinations thereof.
• a target binding moiety of at least two detection probes provided in a kit is each directed to the same target biomarker of a target biomarker signature.
• such the same target biomarker is or comprises CEACAM6.
• an oligonucleotide domain of such at least two detection probes are different.
• a target binding moiety of at least two detection probes provided in a kit is each directed to a distinct target biomarker of a target biomarker signature.
• a kit comprises at least two detection probes directed to CEACAM6 and EpCAM, respectively.
• a kit comprises at least two detection probes directed to CEACAM6 and SLC34A2, respectively.
• a target binding moiety of a detection probe is or comprises an antibody (e.g ., a monoclonal antibody).
• a kit is directed to detection of a target biomarker signature for lung cancer as described herein (including, e.g., but not limited to ones depicted in Tables 4-5).
• a kit is directed to detection of a target biomarker signature for lung cancer comprising at least a surface protein biomarker as described herein (including, e.g, but not limited to ones provided in Table 3, as an extracellular vesicle-associated membrane-bound polypeptide) and at least a surface protein biomarker as described herein (including, e.g, but not limited to ones provided in Table 3, as a target surface protein biomarker).
• a kit is directed to a target biomarker signature for lung cancer comprising at least a SLC34A2 polypeptide (as an extracellular vesicle-associated membrane- bound polypeptide) and a CEACAM6 polypeptide (as a target surface protein biomarker).
• a kit is directed to a target biomarker signature for lung cancer comprising at least a SLC34A2 polypeptide (as an extracellular vesicle-associated membrane- bound polypeptide) and an EpCAM polypeptide (as a target surface protein biomarker).
• a kit is directed to a target biomarker signature for lung cancer comprising at least a CEACAM5 polypeptide (as an extracellular vesicle-associated membrane- bound polypeptide) and a CEACAM6 polypeptide (as a target surface protein biomarker).
• a kit is directed to a target biomarker signature for lung cancer comprising at least a CEACAM5 polypeptide (as an extracellular vesicle-associated membrane- bound polypeptide) and a SLC34A2 polypeptide (as a target surface protein biomarker).
• a kit is directed to a target biomarker signature for lung cancer comprising at least a CEACAM5 polypeptide (as an extracellular vesicle-associated membrane- bound polypeptide); and a CEACAM6 polypeptide and a SLC34A2 polypeptide (as two distinct target surface protein biomarkers).
• a kit is directed to a target biomarker signature for lung cancer comprising at least a SLC34A2 polypeptide (as an extracellular vesicle-associated membrane- bound polypeptide); and a CEACAM6 polypeptide and an EpCAM polypeptide (as two distinct target surface protein biomarkers).
• a kit comprises at least one chemical reagent such as a fixation agent, a permeabilization agent, and/or a blocking agent.
• a kit comprises one or more nucleic acid ligation reagents (e.g ., a nucleic acid ligase such as a DNA ligase and/or a buffer solution).
• nucleic acid ligation reagents e.g ., a nucleic acid ligase such as a DNA ligase and/or a buffer solution.
• a kit comprises at least one or more amplification reagents such as PCR amplification reagents.
• a kit comprises one or more nucleic acid polymerases (e.g., DNA polymerases), one or more pairs of primers, nucleotides, and/or a buffered solution.
• a kit comprises a solid substrate for capturing an entity (e.g, biological entity) of interest.
• a solid substrate may be or comprise a bead (e.g, a magnetic bead).
• a solid substrate may be or comprise a surface.
• a surface may be or comprise a capture surface (e.g, an entity capture surface) of an assay chamber, such as, e.g, a filter, a matrix, a membrane, a plate, a tube, a well (e.g, but not limited to a microwell), etc.
• a surface (e.g, a capture surface) of a solid substrate can be coated with a capture agent (e.g ., polypeptide or antibody agent) for an entity (e.g., biological entity) of interest.
• a capture agent e.g ., polypeptide or antibody agent
• a set of detection probes provided in a kit may be selected for diagnosis of lung cancer.
• a kit comprises a plurality of sets of detection probes, wherein each set of detection probes is directed for detection of a specific cancer and comprises at least 2 or more detection probes.
• a kit can be used to screen a subject for various cancers, one of which is lung cancer while other cancers may be selected from skin cancer, ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, brain cancer, and liver cancer in a single assay.
• kits provided herein may include instructions for practicing methods described herein. These instructions may be present in kits in a variety of forms, one or more of which may be present in the kits.
• One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g, a piece or pieces of paper on which the information is printed, in the packaging of kits, in a package insert, etc.
• Yet another means may be a computer readable medium, e.g, diskette, CD, USB drive, etc., on which instructional information has been recorded.
• Yet another means that may be present is a website address which may be used via the internet to access instructional information. Any convenient means may be present in the kits.
• kits can include instructions for identifying patients that are likely to respond to a therapeutic agent (e.g, identification of biomarkers that are indicative of patient responsiveness to the therapeutic agent).
• a therapeutic agent e.g., identification of biomarkers that are indicative of patient responsiveness to the therapeutic agent.
• kits can comprise a therapeutic agent for use in tandem with the companion diagnostic test.
• Example 1 Detection of an exemplary target biomarker signature in individual extracellular vesicles associated with lung adenocarcinoma
• the present Example describes synthesis of detection probes for targets (e.g ., target biomarker(s)) each comprising a target-binding moiety and an oligonucleotide domain (comprising a double-stranded portion and a single stranded overhang) coupled to the targetbinding moiety.
• targets e.g ., target biomarker(s)
• oligonucleotide domain comprising a double-stranded portion and a single stranded overhang
• a detection probe can comprise a double-stranded oligonucleotide with an antibody agent specific to a target cancer biomarker at one end and a single stranded overhang at another end.
• the single-stranded overhangs of the detection probes are in close proximity such that they can hybridize to each other to form a double-stranded complex, which can be subsequently ligated and amplified for detection.
• This study employed at least two detection probes in a set.
• such at least two detection probes are directed to the same target biomarker.
• such at least two detection probes directed to the same target are directed to different epitopes of the same target or to the same epitopes of the same target.
• such at least two detection probes are directed to distinct targets.
• two detection probes can be directed to different target biomarkers, or that three or more detection probes, each directed towards a distinct target protein, may be used.
• compositions and methods described in this Example can be extended to applications in different biological samples (e.g, comprising extracellular vesicles).
• the present Example shows experimental data from certain experiments demonstrating technologies provided herein are capable of detecting lung cancer (e.g, lung adenocarcinoma and/or lung squamous cell carcinoma) in patient samples using an exemplary biomarker combinations as described herein (e.g, SLC34A2, CEACAM5, CEACAM6, and EpCAM, e.g, in some embodiments, using SLC34A2 capture with CEACAM6 + CEACAM6 extracellular vesicles in PBS using a duplex system assay described herein. See, for example, Figure 1 and 2.
• lung cancer e.g, lung adenocarcinoma and/or lung squamous cell carcinoma
• an exemplary biomarker combinations as described herein e.g, SLC34A2, CEACAM5, CEACAM6, and EpCAM, e.g, in some embodiments, using SLC34A2 capture with CEACAM6 + CEACAM6 extracellular vesicles in PBS using a duplex system assay described here
• a target entity detection system described herein is a duplex system.
• such a duplex system e.g. , as illustrated in Figure 2 utilizes two antibodies that each recognize a different epitope. Paired double-stranded template DNAs are also utilized in qPCR, each of which has specific four-base 5' overhangs complementary to the 5' overhang on its partner. Each antibody is conjugated with one of the two double-stranded DNA templates. When the antibodies bind their target epitopes, the sticky ends of the respective templates can hybridize. These sticky ends are then ligated together by T7 ligase, prior to PCR amplification.
• the two antibodies For hybridization between the two DNA templates to occur, the two antibodies need to be bound close enough to each other (within 50 to 60 nm, the length of the DNA linker and antibody). Any templates that bind but remain unligated will not produce PCR product, as shown in Figure 2.
• Plasma samples from healthy controls and Lung Adenocarcinoma (LUAD) patients were processed to obtain purified extracellular vesicles, which were interrogated using an exemplary assay as described below.
• EVs were captured using magnetic beads covalently conjugated with anti- SLC34A2 , or anti-CEACAM5 antibodies.
• the EVs captured by the beads were profiled using a set of two detection probes, each comprising an antibody directed to a target biomarker (e.g, CEACAM6, EpCAM, or SLC34A2) and a distinct oligonucleotide domain (e.g, ones as described herein).
• a target biomarker e.g, CEACAM6, EpCAM, or SLC34A2
• oligonucleotide domain e.g, ones as described herein.
• biomarker combinations were carefully selected to minimize cross-reactivity with heal thy -tissue-derived extracellular vesicles, which are (i) SLC34A2 and CEACAM6, (ii) SLC34A2, CEACAM6, and EPCAM, and (iii) CEACAM5, CEACAM6, and SLC34A2.
• the cross-reactivity of such a biomarker combination with healthy tissues was bioinformatically predicted, in part, by using a heatmap of differentially expressed mRNAs in lung adenocarcinoma.
• different combinations of markers can be predicted to be much more abundant on the surface of lung cancer extracellular vesicles than on the surface of extracellular vesicles from healthy tissues.
• such a biomarker combination may be selected from: (i) SLC34A2 capture probe and CEACAM6 + CEACAM6 detection probes, (ii) SLC34A2 capture probe and CEACAM6 + EPCAM detection probes, and (iii) CEACAM5 capture probe and CEACAM6 + SLC34A2 detection probes.
• Table 1 represents transcript expression scores of indicated biomarkers, as expressed in a lung cancer cell line vs. negative control cell line (e.g., non-lung cancer cell line).
• oligonucleotides can have the following sequence structure and modifications. It is noted that the strand numbers below correspond to the numerical values associated with strands shown in Figure 2.
• /5AzideN/ refers to an azide group linked to the 5' oligonucleotide terminus via a NHS ester linker, or
• /5AmMC12/CAGTCTGACACAGCAGTCGTTAATCGTCGCTGCTACCCTTGACATCCGTGACTGG CTAGACAGAGGTGT where /5AmMC12/ refers to an amine group (e.g, a primary amino group) linked to the 5' oligonucleotide terminus via a 12-carbon spacer, or
• /5AzideN/ refers to an azide group linked to the 5' oligonucleotide terminus via a NHS ester linker, or
• /5AmMC12/GACCTGACCTACAGTGACCATAGCCTTGCCTGATTAGCCACTGTCCAGTTTGGCT CCTGGTCTCACTAG where /5AmMCl/ refers to an amine group (e.g, a primary amino group) linked to the 5' oligonucleotide terminus via a 12-carbon spacer, or
• /5Phos/GAGTACACCTCTGTCTAGCCAGTCACGGATGTCAAGGGTAGCAGCGACGATTAACGA CTGCTGTGTCAGACTG wherein /5Phos/ refers to a phosphate group linked to the 5' oligonucleotide terminus Strand 4 vl:
• /5Phos/ACTCCTAGTGAGACCAGGAGCCAAACTGGACAGTGGCTAATCAGGCAAGGCTATGGT CACTGTAGGTCAGGTC wherein /5Phos/ refers to a phosphate group linked to the 5' oligonucleotide terminus Strand 5 vl:
• /56- FAM/ refers to a fluorescein (e.g, 6-FAM) at the 5' oligonucleotide terminus
• /3IABkFQ/ refers to a fluorescein quencher at the 3' oligonucleotide terminus
• oligonucleotides can have the following sequence structure and modifications. It is noted that the strand numbers below correspond to the numerical values associated with strands shown in Figure 2.
• Strand 1 v2 /5AzideN/CAGTCTGACTCACCACTCGTTAATCGTCGCTGCTACCCTTGACATCCGTGACTGG CTAGACAGAGGTGT, where /5AzideN/ refers to an azide group linked to the 5' oligonucleotide terminus via a NHS ester linker, or
• /5AmMC12/CAGTCTGACTCACCACTCGTTAATCGTCGCTGCTACCCTTGACATCCGTGACTGG CTAGACAGAGGTGT where /5AmMC12/ refers to an amine group (e.g, a primary amino group) linked to the 5' oligonucleotide terminus via a 12-carbon spacer, or
• /5AzideN/CACCAGACCTACGAAGTCCATAGCCTTGCCTGATTAGCCACTGTCCAGTTTGGCT CCTGGTCTCACTAG refers to an azide group linked to the 5' oligonucleotide terminus via a NHS ester linker, or
• /5AmMC12/CACCAGACCTACGAAGTCCATAGCCTTGCCTGATTAGCCACTGTCCAGTTTGGCT CCTGGTCTCACTAG where /5AmMCl/ refers to an amine group (e.g, a primary amino group) linked to the 5' oligonucleotide terminus via a 12-carbon spacer, or
• /5Phos/GAGTACACCTCTGTCTAGCCAGTCACGGATGTCAAGGGTAGCAGCGACGATTAACGA GTGGTGAGTCAGACTG wherein /5Phos/ refers to a phosphate group linked to the 5' oligonucleotide terminus Strand 4 v2:
• /5Phos/ACTCCTAGTGAGACCAGGAGCCAAACTGGACAGTGGCTAATCAGGCAAGGCTATGGA CTTCGTAGGTCTGGTG wherein /5Phos/ refers to a phosphate group linked to the 5' oligonucleotide terminus Strand 5 v2:
• /56- FAM/ refers to a fluorescein (e.g, 6-FAM) at the 5' oligonucleotide terminus; and /3IABkFQ/ refers to a fluorescein quencher at the 3' oligonucleotide terminus
• oligonucleotides can have the following sequence structure and modifications. It is noted that the strand numbers below correspond to the numerical values associated with strands shown in Figure 2.
• /5ThiolMC6/ refers to a thiol linked to the 5' oligonucleotide terminus via a 6-carbon spacer.
• /5AzideN/ refers to an azide group linked to the 5' oligonucleotide terminus via a NHS ester linker
• /5 AmMC 1 / refers to an amine group (e.g, a primary amino group) linked to the 5' oligonucleotide terminus via a 12-carbon spacer, or
• /5ThiolMC6/ refers to a thiol linked to the 5' oligonucleotide terminus via a 6-carbon spacer
• /56- FAM/ refers to a fluorescein (e.g, 6-FAM) at the 5' oligonucleotide terminus
• /3IABkFQ/ refers to a fluorescein quencher at the 3' oligonucleotide terminus.
• Antibody-oligonucleotide (e.g., antibody-DNA) conjugation e.g., antibody-DNA conjugation:
• Antibody aliquots ranging from 25-100 pg were conjugated with oligonucleotide strands, for example, 60 pg aliquots of antibody was conjugated with hybridized strands 1+3 and 2+4, for example, using copper-free click chemistry.
• the first step was to prepare DBCO-functionalized antibodies to participate in the conjugation reaction with azide-modified oligonucleotide domain (e.g, DNA domain). This began with reacting the antibodies with the DBCO-PEG5-NHS heterobifunctional cross linker. The reaction between the NHS ester and available lysine groups was allowed to take place at room temperature for 2 hours, after which unreacted crosslinker was removed using centrifugal ultrafiltration. To complete the conjugation, azide-modified oligonucleotide domains (e.g, DNA domain) and the DBCO-functionalized antibodies were allowed to react overnight at room temperature. The concentration of conjugated antibody was measured using the Qubit protein assay.
• Negative control cells e.g, non-lung cancer cells such as melanoma cells or healthy cells
• EMEM Eagle’s Minimum Essential Medium
• Lung adenocarcinoma cells were grown in Roswell Park Memorial Institute (RPMI 1640) with 10% exosome-free FBS and 50 units of penicillin/streptomycin per mL.
• Exemplary lung cancer cell lines that may be useful to develop an assay for detection of lung cancer (e.g, ones as described herein) include, but are not limited to, HCC4006, PC9, L068, LUDLU-1, COR-L105, SKLU1, SKMES1, NCI-H727, LC-2/AD, NCIH358, ChaGo-K-1, MOR/CPR, MOR/0.4R, MOR/0.2R, NCIH-322 and cells lines described and discussed in Gazdar et al, “Lung Cancer Cell Lines as Tools for Biomedical Discovery and Research” Journal of the National Cancer Institute: 2010 September 8; 102(17): 1310-1321, which is incorporated herein by reference for the purpose described herein. All cell lines were maintained at 5% CO2 and 37°C and the passage number was below 20.
• lung cancer cells and negative control cells were grown in their respective media until they reached -80% confluence.
• the cell culture medium was collected and spun at 300 x ref for 5 minutes at room temperature (RT) to removes cells and debris. The supernatant was then collected and frozen at -80 °C.
• media was clarified as described in the MACS Miltenyi exosome retrieval kit instructions for cell media.
• the clarified cell culture medium was concentrated ( e.g ., to -500 pL) using a size-exclusion purification column (e.g., a single 15 mL Amicon filter was used to filter three additions of cell-line media; e.g. , with room temperature centrifugation at 2500 to 3000 x ref for 10 minutes for each addition). Nanoparticles having a size range of about 65 nm to about 1000 nm were collected for each sample. In some embodiments, a smaller particle range may be desirable.
• a size-exclusion purification column e.g., a single 15 mL Amicon filter was used to filter three additions of cell-line media; e.g. , with room temperature centrifugation at 2500 to 3000 x ref for 10 minutes for each addition.
• Nanoparticles having a size range of about 65 nm to about 1000 nm were collected for each sample. In some embodiments, a smaller particle range may be desirable.
• Particle counts were obtained, e.g. , using a SpectroDyne particle counting instrument using the TS400 chips, to measure nanoparticle range between 65 and 1000 nm. In some embodiments, a smaller particle range may be desirable.
• pooled patient plasma pools were utilized. In brief, 1 mL aliquots of patient plasma were thawed at room temperature for at least 30 minutes. The tubes were vortexed briefly and spun down to consolidate plasma to the bottom of each tube. Plasma samples from a given patient cohort were combined in an appropriately sized container and mixed thoroughly by end-over-end mixing. Each plasma pool was split into 1 mL aliquots in Protein Lo-bind 1.5 mL Eppendorf tubes and refrozen at -80°C.
• samples were blinded by personnel who would not participate in sample-handling.
• the patient-identification information was only revealed after the experiment was completed to enable data analysis. 1 mL aliquots of whole plasma were removed from storage at -80°C and subjected to three clarification spins to remove cells, platelets, and debris.
• Each clarified plasma sample (individual samples or pooled samples) was run through a single-use, size-exclusion purification column to isolate the EVs. Nanoparticles having a size range of about 65 nm to about 1000 nm were collected for each sample. In some embodiments, smaller particle range may be desirable.
• Antibodies were conjugated to magnetic beads (e.g. , epoxy-functionalized DynabeadsTM). Briefly, beads were weighed in a sterile environment and resuspended in buffer. Antibodies, at approximately 8 ⁇ g of Ab per mg of bead, were mixed with the functionalized beads and the conjugation reaction took place overnight at 37°C with end-over-end mixing. The beads were washed several times using the wash buffer provided by the conjugation kit and were stored at 4°C in the provided storage buffer, or at -20°C in a glycerol-based storage buffer.
• magnetic beads e.g. , epoxy-functionalized DynabeadsTM.
• purified plasma EVs were directly captured from clarified plasma samples.
• a diluted sample of purified plasma EVs were incubated with magnetic beads conjugated with respective antibodies for an appropriate time period, e.g. , at room temperature. Binding of antibody-oligonucleotide conjugates to EVs bound on magnetic capture beads:
• Antibody-oligonucleotide conjugates e.g. , anti-SLC34A2, CEACAM6, or EPCAM antibody-oligonucleotide conjugates; “antibody probes”
• Antibody probes were diluted in an appropriate buffer at their optimal concentrations. Antibody probes were allowed to interact with a sample comprising EVs bound on magnetic capture beads.
• samples were washed, e.g. , multiple times, in an appropriate buffer.
• the beads with bound extracellular vesicles and bound antibody-oligonucleotide conjugates were contacted with a ligation mix. The mixtures were incubated for 20 minutes at RT.
• PCR was performed in a 96-well plate, e.g. , on the Quant Studio 3, with the following exemplary PCR protocol: hold at 95 °C for 1 minute, perform 50 cycles of 95 °C for 5 seconds and 62 °C for 15 seconds. The rate of temperature change was chosen to be standard (2 °C per second). A single qPCR reaction was perform for each experimental replicate and ROX was used as the passive reference to normalize the qPCR signals. Data was then downloaded from the Quant Studio 3 machine and analyzed and plotted in Python 3.7.
• a binary classification system can be used for data analysis.
• signals from a detection assay may be normalized based on a reference signal. For example, in some embodiments, normalized signals for a single antibody duplex were calculated by choosing a reference sample. In some embodiments, the equations used to calculate the normalized signal for an arbitrary sample i are given below, where Signalmax is the signal from the highest concentration cell-line EVs standard.
• biomarker combinations as described herein e.g ., combinations of SLC34A2 + CEACAM6, or SLC34A2 + CEACAM6 + EPCAM, or CEACAM5+CEACAM6 + SLC34A2
• a biomarker combination includes SLC34A2 capture and CEACAM6 + CEACAM6 detection probes.
• a biomarker combination includes SLC34A2 capture and CEACAM6 + EpCAM detection probes.
• a biomarker combination includes CEACAM5 capture and CEACAM6 + SLC34A2 detection probes.
• use of two or more biomarker combinations in an assay may increase the sensitivity of the assay.
• a dendron which can add up to 16 strands of oligonucleotide domain (e.g, DNA) per antibody, can be used instead of one or two strands of DNA per antibody, for example, to enhance signal-to-noise.
• oligonucleotide domain e.g, DNA
• Example 2 Embodiments of biomarker signatures for lung cancer detection
• a biomarker combination including SLC34A2, CEACAM5, CEACAM6, and/or EPCAM e.g., in some embodiments, SLC34A2 capture and CEACAM6 + CEACAM6 detection probes, or SLC34A2 capture and CEACAM6 + EPCAM detection probes, or CEACAM5 capture and CEACAM6 + SLC34A2 detection probes
• SLC34A2 capture and CEACAM6 + CEACAM6 detection probes e.g, following an assay as described in Example 1
• subject populations including, e.g., healthy controls, Stage I LUAD; Stage II LUAD; Stage III LUAD; and Stage IV LUAD.
• two or more biomarker combinations described herein may be used together for detection of lung cancer, e.g, to increase sensitivity of an assay.
• an assay for lung cancer detection may involve at least two biomarker combinations, wherein such at least two biomarker combinations each may comprise a different biomarker combination described herein (e.g, but not limited to ones included in Tables 4-5).
• three or more biomarker combinations described herein may be used together for detection of lung cancer, e.g, to increase sensitivity of an assay.
• an assay for lung cancer detection may involve at least three biomarker combinations, wherein such at least three biomarker combinations each may comprise a different biomarker combination described herein ( e.g ., but not limited to ones included in Tables 4-5).
• Example 3 Assessment of extracellular vesicle (EV) surface proteins as lung cancer biomarkers
• lung cancer detection includes detection of at least EV surface protein(s) following immunoaffmity capture of extracellular vesicles.
• capture proteins can be used for immunoaffmity capture of lung cancer-associated extracellular vesicles.
• capture proteins may include, but are not limited to ALCAM, B3GNT3, CDCP1, CDH1, CDH3, CD55, CD274 (PD- Ll), CEACAM5, CEACAM6, CLDN3, CLDN4, DSG2, EGFR, EPCAM, FOLR1, IG1FR, GJB1, GJB2, LAMB 3, MET, MSLN, MUC1, PIGT, PODXL2, ROS1, SDC1, SLC34A2, SMPDL3B, ST14, sTn antigen, Tn antigen, T antigen, TACSTD2, TMPRSS4, TSPAN8, TNFRSF10B, and/or combinations thereof.
• capture proteins may include but are not limited to ABCA3, ABCC1, ABCC3, ACBD3, ACSL5, AGER, ALCAM, API M2, APHIA, APOO, ATP11A, ATP11B, ATP1B1, ATP6AP2, B4GALT4, BCAP31, BSPRY, CD 109, CD55, CD9, CDC42, CDH1, CDH3, CDKALl, CEACAM5, CEACAM6,
• EV immunoassay methodology e.g ., ones described herein such as in Example 1
• biomarker-validation process e.g., ones described herein such as in Example 1
• an antibody directed to a capture protein is conjugated to magnetic beads and evaluated, optionally first on cell-line EVs then on patient samples, for its ability to bind the specific target protein.
• the antibody-coated bead is assessed for its ability to capture lung cancer-associated EVs and the EVs captured by the antibody-coated bead are read out using a target entity detection system (e.g, a duplex system as described herein involving a set of two detection probes each directed to a target marker (e.g, as described herein).
• a target entity detection system e.g, a duplex system as described herein involving a set of two detection probes each directed to a target marker (e.g, as described herein).
• captured EVs can be read out using at least one or more (e.g, 1, 2, 3, or more) of the following surface protein biomarkers: ABCC3, ALCAM, ARSL, B3GNT3, CDCP1, CDH1, CDH3, CD55, CD274 (PD-L1), CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3, CLDN4, CLDN7, CLIC6, DMBT1, DSG2, EGFR, EPCAM, EPHX3, EVA1A,
• surface protein biomarkers ABCC3, ALCAM, ARSL, B3GNT3, CDCP1, CDH1, CDH3, CD55, CD274 (PD-L1), CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3, CLDN4, CLDN7, CLIC6, DMBT1, DSG2, EGFR, EPCAM, EPHX3, EVA1A,
• captured EVs can be read out using a set of detection probes (e.g, as utilized and/or described herein), at least two of which are directed to one or more (e.g, 1, 2, 3, or more) of the following surface protein biomarkers: ABCC3, ALCAM, ARSL, B3GNT3, CDCP1, CDH1, CDH3, CD55, CD274 (PD-L1), CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3, CLDN4, CLDN7, CLIC6, DMBT1, DSG2, EGFR, EPCAM, EPHX3, EVA1A, FOLR1, GJB1, GJB2, GPC4, HS6ST2, IG1FR, KDELR3, KRTCAP3, LAMB 3, LFNG, LSR, MANEAL, MET, MSLN, MUC1, MUC21, PIGT, PODXL2, PRRG4, ROS1, SDC1, SERINC2, SEZ6L2, SLC34A2, SLC44A4, SLC6A
• captured EVs can be read out using at least one or more (e.g ., 1, 2, 3, or more) of the following surface protein biomarkers: ABCA3, ABCCl, ABCC3, ACBD3, ACSL5, AGER, ALCAM, API M2, APHIA, APOO, ATP11A, ATP11B, ATP1B1, ATP6AP2, B4GALT4, BCAP31, BSPRY, CD109, CD55, CD9, CDC42, CDH1, CDH3, CDKALl, CEACAM5, CEACAM6, CELSR1, CIP2A, CISD2, CKAP4, CLCA2, CLDN1, CLIC6, CLPTM1L, CLSTN1, CNTN1, CPD, CYP2S1, CYP4F11, CYP4F3, DPY19L1, DSC2, DSC3, DSG2, DSG3, EGFR, EPCAM, EPHB3, FAT2, FBX045, FERMT1, FOLR1, FZD6,
• MSLN MUC1, MUC4, NCSTN, NECTINl, NECTIN4, NRAS, NT5E, NUP210, PARL, PEX13, PIGN, PIGT, PLA2G4A, PLCH1, PLEC, PSMD2, PTDSS1, PTGFRN, PTPRF,
• TTC33 UGT1A6, UPK1B, VAMP8, VMA21, VRK2, VWA1, XPR1, XXYLTl, ADAM28, AXL, BSG, CD274, CD47, CLU, DKK1, ERBB3, FLT4, GM3, HGF, IGF1R, IL6, KDR,
• captured EVs can be read out using a set of detection probes (e.g., as utilized and/or described herein), at least two of which are directed to one or more (e.g, 1, 2, 3, or more) of the following surface protein biomarkers: ABC A3, ABCCl, ABCC3, ACBD3, ACSL5, AGER, ALCAM, API M2, APHIA, APOO, ATP11A, ATP11B, ATP1B1, ATP6AP2, B4GALT4, BCAP31, BSPRY, CD109, CD55, CD9, CDC42, CDH1, CDH3, CDKALl, CEACAM5, CEACAM6, CELSR1, CIP2A, CISD2, CKAP4, CLCA2, CLDN1, CLIC6, CLPTM1L, CLSTN1, CNTN1, CPD, CYP2S1, CYP4F11, CYP4F3, DPY19L1, DSC2, DSC3, DSG2, DSG3, EGFR, EPCAM, EPHB
• Example 4 Assessment of mRNA in extracellular vesicles (intravesicular mRNA) as lung cancer biomarkers
• lung cancer detection includes detection of at least intravesicular mRNA(s) following immunoaffmity capture of extracellular vesicles.
• capture proteins can be used for immunoaffmity capture of lung cancer-associated extracellular vesicles.
• capture protein biomarkers may include, but are not limited to ALCAM, B3GNT3, CDCP1, CDH1, CDH3, CD55, CD274 (PD-L1), CEACAM5, CEACAM6, CLDN3, CLDN4, DSG2, EGFR, EPCAM, FOLR1, GJB1, GJB2, IG1FR, LAMB 3, MET, MSLN, MUC1, PIGT, PODXL2, ROS1, SDC1, SLC34A2, SMPDL3B, ST14, sTn antigen, Tn antigen, T antigen, TACSTD2, TMPRSS4, TSPAN8, TNFRSF10B, and combinations thereof.
• EV nucleic acid detection assay e.g ., reverse transcription PCR using primer-probe sets
• biomarker-validation process e.g., ones described herein such as in Example 1
• an antibody directed to a capture protein e.g, a surface protein present in lung cancer-associated EVs
• magnetic beads e.g., a surface protein present in lung cancer-associated EVs
• the antibody- coated bead is assessed for its ability to capture lung cancer-associated EVs and the captured EVs by the antibody-coated bead is profiled for their mRNA contents, for example, using one- step quantitative reverse transcription PCR (RT-qPCR) master mix.
• RT-qPCR quantitative reverse transcription PCR
• captured EVs can be read out by detection of at least one or more (e.g., 1, 2, 3, or more) of the following mRNAs: ABCC3, AOC1, ARSL, B3GNT3, C12orf45, CDCP1, CDH1, CDH3, CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3, CLDN4, CLDN7, CLIC6, CRABP2, CST1, DMBTl, DSG2, EPCAM, EPHX3, ETV4, EVA1A, FAM83A, FOLR1, FOXA2, GJB1, GJB2, GPC4, HMGB3, HS6ST2, KDELR3, KRTCAP3, LAMB3, LFNG, LGALS3BP, LSR, MANEAL, MIF, MSLN, MUC1, MUC21, NAPSA, PIGT, PODXL2, PPP1R14D, PRRG4, ROS1, S100A14, SBK1, SCGB3A2, SDC1, SERINC2,
• captured EVs can be read out by detection of at least one or more (e.g, 1, 2, 3, or more) of the following mRNAs using RT-qPCR: ABC A3, ABCCl, ABRACL, ACP5, ADAM23, ADH7, AGR2, AIFl, AKR1C1, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, ANTXR1, API M2, APOBEC3B, APOBEC3C, AQP3, AREG, ARNTL2, ASF1B, ATP8B1, AURKB, B3GNT5, BAIAP2L1, BCAM, BIK, BIRC5, C15orf48, C19orf33, CIS, C8orf4,
• captured EVs can be read out by detection of at least one or more (e.g ., 1, 2, 3, or more) of the following mRNAs: ABCC3, AOC1, ARSL, B3GNT3, C12orf45, CDCP1, CDH1, CDH3, CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3, CLDN4, CLDN7, CLIC6, CRABP2, CST1, DMBTl, DSG2, EPCAM, EPHX3, ETV4, EVA1A, FAM83A, FOLR1, FOXA2, GJB1, GJB2, GPC4, HMGB3, HS6ST2, KDELR3, KRTCAP3, LAMB3, LFNG, LGALS3BP, LSR, MANEAL, MIF, MSLN, MUC1, MUC21, NAPSA, PIGT, PODXL2, PPP1R14D, PRRG4, ROS1, S100A14, SBK1, SCGB3A2, SDC1, SERINC2,
• captured EVs can be read out by detection of at least one or more (e.g., 1, 2, 3, or more) of the following mRNAs using RT-qPCR: ABCA3, ABCCl, ABRACL, ACP5,
• captured EVs can be read out (i) by detection of one or more (e.g, 1, 2, 3, or more) of the following mRNAs using RT-qPCR: ABCC3, AOC1, ARSL, B3GNT3, C12orf45, CDCP1, CDH1, CDH3, CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3, CLDN4, CLDN7, CLIC6, CRABP2, CST1, DMBT1, DSG2, EPCAM, EPHX3, ETV4, EVA1A, FAM83A, FOLR1, FOXA2, GJB1, GJB2, GPC4, HMGB3, HS6ST2, KDELR3, KRTCAP3, LAMB3, LFNG, LGALS3BP, LSR, MANEAL, MIF, MSLN, MUC1, MUC21, NAPSA, PIGT, PODXL2, PPP1R14D, PRRG4, ROS1, S100A14, SBK1, SCGB3A2, SDC1,
• captured EVs can be read out (i) by detection of one or more (e.g, 1, 2, 3, or more) of the following mRNAs using RT-qPCR: ABCA3, ABCCl, ABRACL, ACP5, ADAM23, ADH7, AGR2, AIF1, AKR1C1, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, ANTXRl, API M2, APOBEC3B, APOBEC3C, AQP3, AREG, ARNTL2, ASF IB, ATP8B1, AURKB, B3GNT5, BAIAP2L1, BCAM, BIK, BIRC5, C15orf48, C19orf33, CIS, C8orf4, CA12, CA9, CALML3, CAPNS2, CBLC, CCL19, CCL5, CCNB2, CD109, CD24, CD53, CD74, CD9, CDC20, CDC42
• a set of detection probes comprises at least one detection probe directed to an EV surface protein. In some such embodiments, a set of detection probes comprises at least two detection probes directed to the same EV surface protein (with the same or different epitopes). In some such embodiments, a set of detection probes comprises at least two detection probes directed to distinct EV surface proteins.
• a sample comprising an EV surface protein and intravesicular mRNA can be contacted with an anti-EV surface protein antibody (e.g ., an antibody directed to EV surface protein as described in Example 3) conjugated to a single-stranded oligonucleotide (e.g., DNA) that serves as one of two primers in a pair for an intravesicular mRNA biomarker (e.g, described in Example 4) such that the anti-EV surface protein antibody is bound to the EV surface protein while the conjugated single-stranded oligonucleotide is hybridized with the intravesicular mRNA biomarker present in the same sample.
• a second primer of the pair and an RT-qPCR probe are then added to perform an RT-qPCR for detection of the presence of an intravesicular mRNA and an EV surface protein in a single sample.
• captured EVs can be read out by detection of at least one or more (e.g., 1, 2, 3, or more) of the following mRNAs: ABCC3, AOC1, ARSL, B3GNT3, C12orf45, CDCP1, CDH1, CDH3, CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3, CLDN4, CLDN7, CLIC6, CRABP2, CST1, DMBT1, DSG2, EPCAM, EPHX3, ETV4, EVA1A, FAM83A, FOLR1, FOXA2, GJB1, GJB2, GPC4, HMGB3, HS6ST2, KDELR3, KRTCAP3, LAMB3, LFNG, LGALS3BP, LSR, MANEAL, MIF, MSLN, MUC1, MUC21, NAPSA, PIGT, PODXL2, PPP1R14D, PRRG4, ROS1, S100A14, SBK1, SCGB3A2, SDC1, SERINC2,
• captured EVs can be read out (i) by detection of one or more (e.g, 1, 2, 3, or more) of the following mRNAs using RT-qPCR: ABCC3, AOC1, ARSL, B3GNT3, C12orf45, CDCP1, CDH1, CDH3, CEACAM5, CEACAM6, CELSR1, CLDN18, CLDN3, CLDN4, CLDN7, CLIC6, CRABP2, CST1, DMBT1, DSG2, EPCAM, EPHX3, ETV4, EVA1A, FAM83A, FOLR1, FOXA2, GJB1, GJB2, GPC4, HMGB3, HS6ST2, KDELR3, KRTCAP3, LAMB3, LFNG, LGALS3BP, LSR, MANEAL, MIF, MSLN, MUC1, MUC21, NAPSA, PIGT, PODXL2, PPP1R14D, PRRG4, ROS1, S100A14, SBK1, SCGB3A2, SDC1, SERINC2,
• a set of detection probes e.g ., as utilized and/or described herein
• captured EVs can be read out by detection of at least one or more (e.g, 1, 2, 3, or more) of the following mRNAs: ABC A3, ABCC1, ABRACL, ACP5, ADAM23, ADH7, AGR2, AIF1, AKR1C1, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, ANTXR1, API M2, APOBEC3B, APOBEC3C, AQP3, AREG, ARNTL2, ASF IB, ATP8B1, AURKB, B3GNT5, BAIAP2L1, BCAM, BIK, BIRC5, C15orf48, C19orf33, CIS, C8orf4, CA12, CA9, CALML3, CAPNS2, CBLC, CCL19, CCL5, CCNB2, CD109, CD24, CD53, CD74, CD9, CDC20, CDC42EP1, CDC45,
• mRNAs e
• captured EVs can be read out (i) by detection of one or more (e.g, 1, 2, 3, or more) of the following mRNAs using RT-qPCR: ABC A3, ABCCl, ABRACL, ACP5, ADAM23, ADH7, AGR2, AIF1, AKR1C1, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, ANTXR1,
• a set of detection probes comprises at least one detection probe directed to an intravesicular protein (e.g, as described herein). In some embodiments, a set of detection probes comprises at least two detection probes each directed to the same intravesicular protein (e.g, with the same epitope or different epitopes). In some embodiments, a set of detection probes comprises at least two detection probes each directed to a distinct intravesicular protein (e.g, as described herein).
• a sample comprising EV intravesicular protein and intravesicular mRNA can be contacted with an anti-EV intravesicular protein antibody (e.g, an antibody directed to EV intravesicular protein as described in Example 5) conjugated to a single-stranded oligonucleotide (e.g, DNA) that serves as one of two primers in a pair for an intravesicular mRNA biomarker (e.g, described in Example 4) such that the anti- EV intravesicular protein antibody is bound to the EV intravesicular protein while the conjugated single-stranded oligonucleotide is hybridized with the intravesicular mRNA biomarker present in the same sample.
• a second primer of the pair and an RT-qPCR probe are then added to perform an RT-qPCR for detection of the presence of an intravesicular mRNA and an intravesicular protein in a single sample.
• lung cancer detection includes detection of at least intravesicular protein(s) following immunoaffmity capture of extracellular vesicles.
• one or more surface proteins or extracellular membrane proteins that are present on extracellular vesicles (“capture proteins”) can be used for immunoaffmity capture of lung cancer-associated extracellular vesicles.
• capture protein biomarkers may include, but are not limited to ALCAM, B3GNT3, CDCP1, CDH1, CDH3, CD55, CD274 (PD-L1), CEACAM5, CEACAM6, CLDN3, CLDN4, DSG2, EGFR, EPCAM, FOLR1, GJB1, GJB2, IG1FR, LAMB 3, MET, MSLN, MUC1, PIGT, PODXL2, ROS1, SDC1, SLC34A2, SMPDL3B, ST14, sTn antigen, Tn antigen, T antigen, TACSTD2, TMPRSS4, TSPAN8, TNFRSF10B, and combinations thereof.
• EV immunoassay methodology e.g ., ones described herein such as in Example 1
• biomarker-validation process e.g., ones described herein such as in Example 1
• an antibody directed to a capture protein e.g, a surface protein present in lung cancer-associated EVs
• a capture protein e.g, a surface protein present in lung cancer-associated EVs
• the antibody-coated bead is assessed for its ability to capture lung cancer-associated EVs and the captured EVs by the antibody-coated beads are fixed and/or permeabilized prior to being profiled for their intravesicular proteins using a target entity detection system (e.g, a duplex system as described herein involving a set of two detection probes, each directed to a target marker that is distinct from the capture protein).
• a target entity detection system e.g, a duplex system as described herein involving a set of two detection probes, each directed to a target marker that is distinct from the capture protein.
• captured EVs after fixation and/or permeabilization can be read out using at least one or more (e.g, 1, 2, 3, or more) of the following intravesicular proteins: AOC1, C12orf45, CRABP2, CST1, ETV4, FAM83A, FOXA2, HMGB3, LGALS3BP, MIF, NAPSA, PPP1R14D, S100A14, SBK1, SCGB3A2, SFTA2, SFTPA1, SFTPA2, SFTPB,
• intravesicular proteins e.g, 1, 2, 3, or more of the following intravesicular proteins: AOC1, C12orf45, CRABP2, CST1, ETV4, FAM83A, FOXA2, HMGB3, LGALS3BP, MIF, NAPSA, PPP1R14D, S100A14, SBK1, SCGB3A2, SFTA2, SFTPA1, SFTPA2, SFTPB,
• captured EVs after fixation and/or permeabilization can be read out using a set of detection probes (e.g, as utilized and/or described herein), at least two of which are directed to one or more (e.g, 1, 2, 3, or more) of the following intravesicular proteins: AOC1, C12orf45, CRABP2, CST1, ETV4, FAM83A, FOXA2, HMGB3, LGALS3BP, MIF, NAPSA, PPP1R14D, S100A14, SBK1, SCGB3A2, SFTA2, SFTPA1, SFTPA2, SFTPB, SPINK1, TGFA, ZC3H11A, and combinations thereof.
• a set of detection probes comprises two detection probes each directed to the same intravesicular protein.
• a set of detection probes comprises two detection probes each directed to a distinct intravesicular protein.
• captured EVs after fixation and/or permeabilization can be read out using at least one or more (e.g ., 1, 2, 3, or more) of the following intravesicular proteins: ABRACL, ACP5, ADH7, AGR2, AIF1, AKR1C1, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, API M2, APOBEC3B, APOBEC3C, ARNTL2, ASF1B, AURKB, BAIAP2L1, BIRC5, C15orf48, C19orf33, CIS, C8orf4, CA9, CALML3, CAPNS2, CBLC, CCL19, CCNB2, CDC20, CDC45, CDCA4, CDCA5, CDK1, CDKN2A, CDKN2B, CENPW, CEP55, CES1, CHMP4C, CNN2, CP A3, CRABP2, CSTA, CTSC, CT
• captured EVs after fixation and/or permeabilization can be read out using a set of detection probes (e.g., as utilized and/or described herein), at least two of which are directed to one or more (e.g, 1, 2, 3, or more) of the following intravesicular proteins: ABRACL, ACP5, ADH7, AGR2, AIF1, AKR1C1, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, API M2, APOBEC3B, APOBEC3C, ARNTL2, ASF IB, AURKB, BAIAP2L1, BIRC5, C15orf48, C19orf33, CIS, C8orf4, CA9, CALML3, CAPNS2, CBLC, CCL19, CCNB2, CDC20, CDC45, CDCA4, CDCA5, CDK1, CDKN2A, CDKN2B, CENPW, CEP55, CES1, CHMP
• captured EVs after fixation and/or permeabilization can be read out using (i) at least one or more (e.g ., 1, 2, 3, or more) of the following intravesicular proteins: AOC1, C12orf45, CRABP2, CST1, ETV4, FAM83A, FOXA2, HMGB3, LGALS3BP, MIF, NAPSA, PPP1R14D, S100A14, SBK1, SCGB3A2, SFTA2, SFTPA1, SFTPA2, SFTPB,
• intravesicular proteins e.g ., 1, 2, 3, or more of the following intravesicular proteins: AOC1, C12orf45, CRABP2, CST1, ETV4, FAM83A, FOXA2, HMGB3, LGALS3BP, MIF, NAPSA, PPP1R14D, S100A14, SBK1, SCGB3A2, SFTA2, SFTPA1, SFTPA2, SFTPB,
• captured EVs after fixation and/or permeabilization can be read out using a set of detection probes (e.g, as utilized and/or described herein), which comprises (i) a first detection probe directed to one or more (e.g, 1, 2, 3, or more) of the following intravesicular proteins: AOC1, C12orf45, CRABP2, CST1, ETV4, FAM83A, FOXA2, HMGB3, LGALS3BP, MIF, NAPSA, PPP1R14D, S100A14, SBK1, SCGB3A2, SFTA2, SFTPA1, SFTPA2, SFTPB, SPINK1, TGFA, ZC3H11A, and combinations thereof; and (ii) a second detection probe directed to one or more (e.g, 1, 2, 3, or more) of
• captured EVs after fixation and/or permeabilization can be read out using (i) at least one or more (e.g, 1, 2, 3, or more) of the following intravesicular proteins: ABRACL, ACP5, ADH7, AGR2, AIF1, AKR1C1, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, API M2, APOBEC3B, APOBEC3C, ARNTL2, ASF1B, AURKB, BAIAP2L1, BIRC5, C15orf48, C19orf33, CIS, C8orf4, CA9, CALML3, CAPNS2, CBLC, CCL19, CCNB2, CDC20, CDC45, CDCA4, CDCA5, CDK1, CDKN2A, CDKN2B, CENPW, CEP55, CES1, CHMP4C, CNN2, CP A3, CRABP2, CSTA, CTSC, CT
• captured EVs after fixation and/or permeabilization can be read out using a set of detection probes (e.g., as utilized and/or described herein), which comprises (i) a first detection probe directed to one or more (e.g, 1, 2, 3, or more) of the following intravesicular proteins: ABRACL, ACP5, ADH7, AGR2, AIFl, AKRICI, AKR1C2, AKR1C3, ALDHIAI, ALDH3A1, ALDH3B2, ALGIL, API M2, APOBEC3B, APOBEC3C, ARNTL2, ASF IB, AURKB, BAIAP2L1, BIRC5, C15orf48, C19orf33, CIS, C8orf4, CA9, CALML3, CAPNS2, CBLC, CCL19, CCNB2, CDC20, CDC45, CDCA4,
• a first detection probe directed to one or more (e.g, 1, 2, 3, or more) of the following intravesicular proteins
• captured EVs after fixation and/or permeabilization can be read out by detecting an EV intravesicular protein and an EV intravesicular mRNA together in a single sample as described in Example 4 above.
• the present Example describes development of a lung cancer liquid biopsy assay, for example, for screening hereditary-, environmental-, and average-risk individuals.
• LDCT low-dose chest tomography
• USPSTF United States Preventative Services Task Force
• a lung cancer screening test may exhibit two features to provide clinical utility: (1) ultrahigh specificity (>99.5%) to minimize the number of false positives, and (2) high sensitivity (>40%) for stage I and II lung cancer when prognosis is most favorable.
• ultrahigh specificity >99.5%
• high sensitivity >40%) for stage I and II lung cancer when prognosis is most favorable.
• ctDNA circulating tumor DNA
• CTCs circulating tumor cells
• EVs extracellular vesicles
• EVs are particularly promising due to their abundance and stability in the bloodstream relative to ctDNA and CTCs, suggesting improved sensitivity for early-stage cancers.
• EVs contain cargo (e.g., proteins, RNA, metabolites) that originated from the same cell, providing superior specificity over bulk protein measurements. While the diagnostic utility EVs has been studied, much of this work has pertained to bulk EV measurements or low-throughput single-EV analyses.
• This present Example describes one aspect of an exemplary approach for early stage lung cancer detection through the profiling of individual extracellular vesicles (EVs) in human plasma.
• EVs including exosomes and microvesicles, contain co-localized proteins, RNAs, metabolites, and other compounds representative of their cell of origin (Kosaka etal ., 2019; which is incorporated herein by reference for the purpose described herein).
• the detection of strategically chosen co-localized markers within a single EV can enable the identification of cell type with ultrahigh specificity, including the ability to distinguish cancer cells from normal tissues.
• EVs are released at a high rate by functioning cells.
• Single cells have been shown to release as many as 10,000 EVs per day in vitro (Balaj et al ., 2011; which is incorporated herein by reference for the purpose described herein).
• cancer cells release EVs at a higher rate than healthy cells (Bebelman et al. 2018; which is incorporated herein by reference for the purpose described herein).
• the present disclosure provides insights and technologies involving identification of genes that are upregulated in lung cancer versus healthy tissues using Applicant’s proprietary bioinformatic biomarker discovery process. From a list of upregulated biomarkers, biomarker combinations that are predicted to exhibit high sensitivity and specificity for lung cancer are designed. Using an exemplary individual EV assay (see, e.g ., illustrated in Figure 1 or 2 and/or described herein), co-localization of such biomarkers on an individual vesicle is detected, indicating that the grouping of biomarkers originated from the same cell. This provides superior specificity to bulk biomarker measurements, including bulk EV assays, given that many upregulated cancer biomarkers may be expressed by one or more healthy tissues.
• the present disclosure provides technologies with high or ultrahigh specificity that is particularly helpful as a lung cancer screening test for which a positive-predictive value of at least comparable to that of a chest CT, which is the gold standard for screening high risk smokers, is sought. See, e.g. , National Lung Screening Trial Research Team (2013) “Results of initial low-dose computed tomographic screening for lung cancer” New England Journal of Medicine , 368(21): 1980-1991.
• the present disclosure provides technologies useful for lung cancer screening with a positive predictive value of at least 3.8 % or higher (including, e.g, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25% or higher) in a high-risk population.
• a biomarker discovery process leverages bioinformatic analysis of large databases and an understanding of the biology of lung cancer and extracellular vesicles.
• the detection of tumor-derived EVs in the blood requires an assay that has sufficient selectivity and sensitivity to detect relatively few tumor-derived EVs per milliliter of plasma in a background of 10 billion EVs from a diverse range of healthy tissues.
• the present disclosure provides technologies that address this challenge. For example, in some embodiments, an assay for individual extracellular vesicle analysis is illustrated in Figure 1, which is performed in three key steps as outlined below:
• EVs are purified from patient plasma using size-exclusion chromatography (SEC), which removes greater than 99% of soluble proteins and other interfering compounds.
• SEC size-exclusion chromatography
• Tumor-specific EVs are captured using antibody-functionalized magnetic beads specific to a membrane-bound protein.
• a modified version of proximity -ligation-immuno quantitative polymerase chain reaction (pliq-PCR) is performed to determine the co-localization of additional protein biomarkers contained on or within the captured EVs.
• two or more different antibody-oligonucleotide conjugates are added to the EVs captured by the antibody- functionalized magnetic bead and the antibodies subsequently bind to their protein targets.
• the oligonucleotides are composed of dsDNA with single-stranded overhangs that are complementary, and thus, capable of hybridizing when in close proximity ( i.e ., when the corresponding protein targets are located on the same EV). After washing away unbound antibody-oligonucleotide species, adjacently bound antibody-oligonucleotide species are ligated using a standard DNA ligase reaction.
• two to twenty distinct antibody-oligonucleotide probes can be incorporated into such an assay, e.g ., as described in U.S. Application No. 16/805,637, and International Application PCT/US2020/020529, both filed February 28, 2020 and entitled “Systems, Compositions, and Methods for Target Entity Detection”; which are both incorporated herein by reference in their entirety for any purpose.
• pliq-PCR has numerous advantages over other technologies to profile EVs.
• pliq-PCR has a sensitivity three orders of magnitude greater than other standard immunoassays, such as ELIS As (Darmanis et al ., 2010; which is incorporated herein by reference for the purpose described herein).
• ELIS As Error et al ., 2010; which is incorporated herein by reference for the purpose described herein.
• the ultra-low LOD of a well-optimized pliq-PCR reaction enables detection of trace levels of tumor-derived EVs, down to a thousand EVs per mL.
• nPLEX Nanoplasmic Exosome
• iMEX Integrated Magnetic-Electrochemical Exosome
• EV biomarkers include mRNA and intravesicular proteins (in addition to EV surface proteins) can be identified and included in an assay.
• biomarker candidates are validated to be present in EVs and capable of being detected by commercially available antibodies or mRNA primer-probe sets.
• one or more cell lines expressing (positive control) and not expressing the biomarker of interest (negative control) can be cultured to harvest their EVs through concentrating their cell culture media and performing purification to isolate nanoparticles having a size range of interest (e.g, using SEC).
• extracellular vesicles may range from 30 nm to several micrometers in diameter. See, e.g, Chuo et at.
• EV extracellular vesicle
• migrasomes 0.5-3 pm
• microvesicles 0.1-1 pm
• oncosomes (1-10 pm)
• exomeres ⁇ 50 nm
• small exosomes 60-80 nm
• large exosomes 90-120 nm.
• nanoparticles having a size range of about 30 nm to 1000 nm may be isolated for detection assay.
• specific EV subtype(s) may be isolated for detection assay.
• an assay configuration involving SLC34A2 immunoaffmity capture and two distinct antibody-oligonucleotide probes specific to CEACAM6 was developed.
• an assay configuration involving SLC34A2 immunoaffmity capture and two distinct antibody-oligonucleotide probes specific to CEACAM6 and EPCAM was developed.
• an assay configuration involving CEACAM5 immunoaffmity capture and two distinct antibody-oligonucleotide probes specific to CEACAM6 and SLC34A2 was developed.
• an assay configuration involving SLC34A2 immunoaffmity capture and two distinct antibody-oligonucleotide probes specific to CEACAM6 and EPCAM sensitivities of 20% for stage II LUAD, 50% for stage III LUAD, and 75% for stage IV LUAD were achieved.
• an assay configuration involving CEACAM5 immunoaffmity capture and two distinct antibody-oligonucleotide probes specific to CEACAM6 and SLC34A2 sensitivities of 16.7% for stage I LUAD, 20% for stage II LUAD, 50% for stage III LUAD, and 75% for stage IV LUAD were achieved.
• Figure 8 is a graphical representation of the correlation between exemplary lung adenocarcinoma diagnostic assays as described herein. Signal from SLC34A2 antibody based capture with CEACAM6 + CEACAM6 detection probes is depicted along the x-axis, while signal from SLC34A2 antibody based capture with CEACAM6 + EpCAM detection probes is depicted along the y-axis. Correlations were determined using the Pearson product-moment correlation coefficient. Strong correlations were observed, especially for stage III and stage IV LUAD samples.
• Figure 9 is a graphical representation of the correlation between exemplary lung adenocarcinoma diagnostic assays as described herein. Signal from SLC34A2 antibody based capture with CEACAM6 + CEACAM6 detection probes is depicted along the x-axis, while signal from CEACAM5 antibody based capture with CEACAM6 + SLC34A2 detection probes is depicted along the y-axis. Correlations were determined using the Pearson product-moment correlation coefficient. Strong correlations were observed, especially for stage III and stage IV LUAD samples.
• Figure 10 is a graphical representation of the correlation between exemplary lung adenocarcinoma diagnostic assays as described herein. Signal from SLC34A2 antibody based capture with CEACAM6 + EPCAM detection probes is depicted along the x-axis, while signal from CAECAM5 antibody based capture with CEACAM6 + SLC34A2 detection probes is depicted along the y-axis. Correlations were determined using the Pearson product-moment correlation coefficient. Strong correlations were observed, especially for stage III and stage IV LUAD samples.
• a specificity of 98%, 98.5%, 99%, 99.5%, 99.9%, or 100% can be used to evaluate the PPV for screening different LUAD at risk populations, e.g ., where prevalence is at ⁇ per 100,000 for smokers, or ⁇ per 100,000 for non-smokers.
• biomarker candidates including membrane-bound proteins and intravesicular mRNAs/proteins can be identified.
• mRNA detection using EVs can be performed by capturing EVs using capture probes (e.g, as described herein) and detecting a particular lung cancer mRNA biomarker. EVs that express both capture probe marker and lung cancer mRNA biomarker are selectively detected.
• the present Example illustrates an exemplary bioinformatically driven approach for identification of certain biomarkers and biomarker combinations that can be useful for lung cancer diagnosis.
• GTEx Genotype-Tissue Expression
• TCGA Cancer Genome Atlas
• UniProt filter was used. Biomarkers that have a valid UniProt entry, which includes some type of evidence that a biomarker protein was found to be associated with a membrane, were considered in the analysis (e.g, proteins with no evidence of being membrane associated were optionally filtered out). Such a filtering step may optionally distinguish between different membranes of interest or level of confidence of the provided evidence.
• Vesiclepedia filter was used. Vesiclepedia (a repository of extracellular vesicle publications) was used to filter the results. Vesiclepedia lists the number of EV related references published for each gene (e.g., Entrez). These references were used as a proxy for presence of a given biomarker in or on EVs. If no EV-related publications exist for a given biomarker, it is less likely to be an actual EV biomarker, and was thus filtered from the list of biomarkers for further consideration.
• Gene e.g., Entrez
• a minimum expression level of a biomarker is considered.
• Low biomarker expression may produce stochastic noise and make robust signal detection difficult and unreliable.
• one or more (including all of) of the following expression filters were applied.
• four expression filters were applied.
• TCGA cancer of interest e.g ., lung cancer
• TPM transcript per million
• positive control cell-lines were utilized for testing of antibodies directed towards bioinformatically-predicted biomarkers.
• the Cancer Cell Line Encyclopedia (CCLE) gene expression set which contains >1000 cell-line profiles, was utilized to reduce biomarker lists to those for which cell-lines expressing a biomarker of interest exist.
• the 90 th percentile of expression for each biomarker across relevant cell-lines was calculated, and in some embodiments, biomarkers with a TPM >15 were considered.
• assays described herein achieved superior specificity by requiring co-expression of at least two biomarkers, and in some embodiments, at least three biomarkers, on the same extracellular vesicle.
• Simple differential gene expression of normal tissues yielded too many false negative values.
• a biomarker signature comprises a combination of biomarkers that may include biomarkers that were highly expressed in multiple tissue types, but only when they were paired with other biomarkers that provided additional discriminatory power (e.g ., highly tissue specific and/or highly cancer specific).
• additional discriminatory power e.g ., highly tissue specific and/or highly cancer specific.
• such an analysis could capture housekeeping genes, such as GAPDH, which were ubiquitously expressed, and accordingly were not necessarily useful as discriminatory biomarkers.
• a Z-score comparing cancerous tissue (e.g., lung cancer) and every tissue type in GTEx for a given biomarker was calculated.
• a biomarker with a maximum z-score of 5 was selected (e.g, at least one normal tissue was clearly excluded by a biomarker candidate).
• Discriminatory power of a biomarker signature candidate or biomarker combination candidate comprising at least two or more (including, e.g, at least three or more) biomarkers can be determined by simulating and comparing expression of such a biomarker signature candidate in normal subjects (e.g, subjects who were determined not to have lung cancer) to that in cancer subjects.
• Combinations of at least 2 and at least 3 biomarkers were generated based on filtered biomarker sets.
• An EV score which estimated the number of EVs generated by a profiled tissue, was calculated for a given combination by multiplying TPM values of all markers in a given combination.
• a cohort of 5000 plasma samples from 5000 “healthy individuals” was created. Individual samples were created by randomly selecting a tissue sample from each of the 54 tissues in the GTEx database, combining these tissues into an individual, and multiplying the TPM values of expressed genes with the estimated weight in grams of each organ based on a healthy individual. EV scores were then summed for an individual across tissues. EV scores were then summed across tissues for a simulated individual.
• 5000 samples from “cancer individuals” were created by repeating the “healthy” pool generation technique, but with an added step of adding EV scores of randomly selected lung cancer (e.g, LUAD and/or LUSC) samples from TCGA, multiplying the sample by 1, 10, or 100, corresponding to a lg, a lOg, or a lOOg tumor.
• EV scores of randomly selected lung cancer e.g, LUAD and/or LUSC
• biomarker signature selection in some embodiments, 1 million combinations of three biomarkers were randomly sampled, and in some embodiments simulations were conducted using a lOOg tumor, and 1000 individuals in each of the cancer and the healthy pool. In some embodiments, biomarker combinations were then ranked based on their sensitivity value. IN some embodiments, single biomarkers were then ranked based on the top 0.5 percentile of their rank in the combination list.
• the present Example describes a gene set enrichment analysis for determination of overlap between certain bioinformatically-predicted biomarkers and published gene pathways.
• lists of single genes can be challenging to appropriately interpret.
• resources that provide functional lists of genes such as, for example, lists of genes that encode proteins that are components of the same biochemical pathway or phenomenon. Comparing a bioinformatically-identified list of biomarkers to known gene sets and biochemical pathways can impose structure on a list of biomarkers.
• Table 2 shows an enrichment analysis of certain bioinformatically-identified biomarkers when compared to all gene sets in the Molecular Signature Database Category 2 - Cannonical pathways (v.7.4.) from the Broad Institute.
• This database includes, among other resources, KEGG, Biocarta, and Reactome data.
• Each p-value is a result of a Chi-square test, comparing a particular gene set with a list of certain bioinformatically-identified biomarkers against the background of all genes in MSigDB C2-CP database. Biomarkers were ranked with the highest overlap first, and in some embodiments, overlaps with a nominal p-value of 0.05 were considered.
• Table 2 shows certain molecular pathways that are enriched in a list of bioinformatically identified biomarkers, following correction for multiple testing, several molecular pathways exhibited a false discovery rate (FDR) of less than 0.05. Such molecular pathways provide a biological theme for certain bioinformatically identified biomarkers.
• Table 2 - Enrichment analysis of certain bioinformatically identified biomarkers Example 9: Correlation of bioinformatically-identified biomarkers and biomarker combinations with clinical covariates and known mutational drivers.
• the present Example illustrates potential associations between known lung cancer clinical covariates and certain bioinformatically-predicted biomarkers; and potential associations between known lung cancer mutational drivers and certain bioinformatically-predicted biomarkers.
• a heatmap can be useful for such analyses. For example, a heatmap that shows the row-scaled gene expression of certain identified biomarker genes using The Cancer Genome Atlas (TCGA) LUSC samples was generated, where each row represented a biomarker candidate and each column represented gene expression in a LUSC sample. Pearson correlation metrics were used to cluster rows and Euclidean distance to cluster columns. Both dimensions were clustered using Ward’s algorithm, and an optimal leaf ordering algorithm was employed.
• TCGA Cancer Genome Atlas
• one or more clinical covariates were considered in addition to gene expression of certain bioinformatically-identified biomarkers. Such analysis can be useful to provide an indication on potential subgroups, including staging, lymph node involvement, microsatellite instability and others.
• clinical covariates included nodal involvement (e.g ., nO, nl, n2, n3, or n4), cancer stage, and/or smoking amount (e.g., pack years).
• cancer stage included stage I, stage II, stage III, or stage IV cancers.
• smoking amount was quantified in terms of pack years (e.g, as described herein, e.g, wherein 1 pack year is equal to 1 pack of cigarettes smoked per day for one year).
• one or more mutational drivers were considered in addition to gene expression of certain bioinformatically-identified biomarkers.
• certain major known mutational drivers of lung cancer include, but are not limited to mutations in TP53, RBI, PTEN, PIK3CA, NOTCH1, NFE2L2, KMT2D, KEAPl, or combinations thereof.
• cancer- associated mutations may include copy number alterations (CNAs; including, e.g, but not limited to amplification and/or deletion) and/or mutations (including, e.g., but not limited to inframe mutation, missense mutation, splice, and/or truncating mutation).
• CNAs copy number alterations
• mutations including, e.g., but not limited to inframe mutation, missense mutation, splice, and/or truncating mutation.
• a clustering analysis was performed to identify associations between bioinformatically-predicted biomarkers, biomarker combinations, and certain major mutational drivers of lung cancer.
• Example 10 Development of exemplary lung cancer liquid biopsy assays

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