WO2019232485A1 - Accurate blood test to predict cancer incidence, recurrence, guide and monitor treatment intervention - Google Patents

Accurate blood test to predict cancer incidence, recurrence, guide and monitor treatment intervention Download PDF

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WO2019232485A1
WO2019232485A1 PCT/US2019/035066 US2019035066W WO2019232485A1 WO 2019232485 A1 WO2019232485 A1 WO 2019232485A1 US 2019035066 W US2019035066 W US 2019035066W WO 2019232485 A1 WO2019232485 A1 WO 2019232485A1
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cancer
ctc
cell
biomarker
kit
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PCT/US2019/035066
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French (fr)
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Aihua Fu
Chungheng CHENG
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Nvigen, Inc.
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    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor 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
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour

Definitions

  • the subject is or has been treated with a hormone therapy, a chemotherapy, a targeted therapy or an immunotherapy.
  • the first nucleic acid biomarker is detected using an amplification assay, a hybridization assay, a sequencing assay or an array.
  • a single nanocomposition may comprise a single nanoparticle or a plurality or a cluster of mini-nanoparticles (A. Fu et al, J. Am. chem. Soc. 126, 10832-10833 (2004), J. Ge et al, Angew. Chem. Int. Ed. 46, 4342-4345 (2007), Zhenda Lu et al, Nano Letters 11, 3404-3412 (2011).).
  • the mini-nanoparticles can be homogeneous (e.g., made of the same composition/materials or having same size) or heterogeneous (e.g., made of different compositions/materials or having different sizes).
  • ionic bonding cation p-bond and salt bond
  • inter-molecular interaction such as hydrogen bond (e.g., Dihydrogen bond, Dihydrogen complex, Low-barrier hydrogen bond, Symmetric hydrogen bond) and non-covalent bonds (e.g., hydrophobic, hydrophilic, charge-charge, or p-stacking interactions, van der Waals force, London dispersion force, Mechanical bond, Halogen bond,
  • the number of large nanoparticles for a given core mass can be calculated using any suitable methods.
  • an individual large nanoparticle may be composed of a plurality of small nanoparticles which are visible under TEM.
  • the average size and volume of a small nanoparticle can be determined based on measurements under TEM, and the average mass of a small nanoparticle can be determined by multiplying the known density of the core material with the volume of the small particle.
  • the total number of small nanoparticles can be estimated.
  • the average number of small nanoparticles in it can be determined under TEM. Accordingly, the number of large nanoparticles for a given core mass can be estimated by dividing the total number of small nanoparticles with the average number of small nanoparticles in an individual large nanoparticle.
  • the porosity of the 3-D structure can be also evaluated by the capacity to load different molecules (see, e.g., Wang L. et al, Nano Research 1, 99-115 (2008)).
  • the 3-D structure provided herein has a low density and high porosity, it is envisaged that more payload can be associated with the 3-D structure than with other coatings.
  • organic fiuorophores such as Rhodamin
  • Rhodamin over 105 Rhodamin molecules can be loaded to 3-D structure of one nanoparticle.
  • the biomarker detected from CTC is the expression level, i.e., mRNA level, of a gene selected from CA15-3, CA27.29, CEA, ER, PR, HER2, uPA, PAI-l, TP53, Cathepsin D, Cyclin E, Cathepsin D, P53, Ki67, CK19, ALDH1, EGFR, Nestin, MSP-alpha, TIMP-4, PDGFR-alpha, OPG, AFP, 5- HIAA, CA19-9, CA27.29, CA72-4, hCGbeta, Calcitonin, CgA, ER, PR, Osteocalcin, Epithelial Membrane Antigen (EMA), AlphavBeta3 Integrin, AlphavBeta6 Integrin, Transferrin Receptor, Transthyretin, Alkaline Phosphatase, PSA, Lactate
  • Fig. 3 illustrates the CTC isolated from a whole blood sample using another exemplary nanocomposition of the present application

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
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  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
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Abstract

Provided is a blood test that can accurately predict cancer incidence or recurrence and guide and monitor the treatment intervention of the cancer. In one embodiment, the test includes obtaining a blood sample from a subject, capturing from the blood sample a circulating tumor DNA (ctDNA) using a first nanocomposition and a circulating tumor cell (CTC) using a second nanocomposition. The test further includes detecting at least a first nucleic acid biomarker from the ctDNA and detecting at least a CTC biomarker and at least a second nucleic acid biomarker from the CTC.

Description

ACCURATE BLOOD TEST TO PREDICT CANCER INCIDENCE, RECURRENCE, GUIDE AND MONITOR TREATMENT INTERVENTION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to ET.S. provisional patent application nos. 62/678,690, filed May 31, 2018, the disclosure of which is incorporated herein by reference.
FIELD OF THE DISCLOSURE
[0002] The present invention generally relates to cancer diagnosis and treatments. Specifically, the present invention relates to compositions and methods for predicting cancer incidence/recurrence and guide and monitor treatment intervention.
BACKGROUND OF THE DISCLOSURE
[0003] Cancer, which involves abnormal cell growth in a body, is the leading cause of human death in the world. Effective treatment of cancer depends on the accurate and timely diagnosis of the cancer, based on which a proper treatment intervention can be chosen. Frequently, cancer recurs because a small number of cancer cells can remain in the body after treatment. This recurrence of cancer is a major contribution to cancer death and raises an urgent need for early detection of cancer cell growth after treatment. The early diagnosis of recurrence, however, remains a challenge because most of the diagnostics currently available, such as symptom-based diagnosis, tissue biopsy, fine needle aspiration, MRI, PET and CT, can only detect cancer cell growth at a later stage.
[0004] Liquid biopsy, which detects cancer cells or pieces of DNA from cancer cells that circulate in the blood, has been used to help find cancer at an early stage. The accuracy of liquid biopsy depends on robust and reproducible detection of biomarkers from circulating proteins, circulating tumor cells and circulating tumor DNA. In particular, given the complexity and diversity of cancer types, in order to accurately predict the cancer incidence/recurrence, multiple biomarkers need to be detected in the same blood sample. However, due to the lack of standardized approaches, it remains a challenge to detect multiple biomarkers from circulating proteins, circulating tumor DNA and circulating cancer cells. [0005] Therefore, there is a continuing need to develop a blood test for predicting cancer incidence/recurrence and guiding and monitoring treatment intervention with high sensitivity and accuracy.
BRIEF SUMMARY OF THE DISCLOSURE
[0006] The present disclosure in one aspect provides a blood test that can accurately predict cancer incidence/recurrence and guide and monitor the treatment intervention of the cancer. In one embodiment, the test includes obtaining a blood sample from a subject, capturing from the blood sample a circulating tumor DNA (ctDNA) using a first nanocomposition and a circulating tumor cell (CTC) using a second nanocomposition. The test further includes detecting at least a first nucleic acid biomarker from the ctDNA and detecting at least a CTC biomarker and at least a second nucleic acid biomarker from the CTC. In certain embodiments, the test disclosed herein further includes the step of detecting the number of CTC or the amount of ctDNA.
[0007] In certain embodiments, the test disclosed herein further comprises the step of determining, based on information obtained in the detecting step, a likelihood of the subject having the cancer or a recurrence of the cancer.
[0008] In certain embodiments, the cancer is selected from the group consisting of a breast cancer, a lung cancer, a prostate cancer, a colorectal cancer, an ovarian cancer, a glioma, a soft-tissue sarcoma, a bladder cancer, a Melanoma, a renal cell carcinoma, an esophagogastric carcinoma, a germ cell tumor a biliary cancer, a thyroid cancer, an endometrial cancer, a head and neck cancer, a non-hodgkin lymphoma, a non-melanoma skin cancer, a gastrointestinal stromal tumor, a mesothelioma, a hepatocellular carcinoma, a salivary carcinoma, a uterine sarcoma, an appendiceal cancer, an embryonal tumor, a Gastrointestinal neuroendocrine tumor, a cervical cancer, an osteosarcoma, an Ewing sarcoma, a chondrosarcoma, a small- bowel cancer, an anal cancer, a meningothelial tumor, an adrenocortical carcinoma, am ampullary carcinoma, a histiocytosis, a thymic tumor, an ependymomal tumor, a nerve sheath tumor, a chordoma, a gestational trophoblastic disease, and a pancreatic cancer. In certain embodiments, wherein the cancer is a breast cancer. In certain embodiments, wherein the cancer is a lung cancer. In certain embodiments, wherein the cancer is a prostate cancer. In certain embodiments, wherein the cancer is an ovarian cancer. In certain embodiments, wherein the cancer is a colorectal cancer. In certain embodiments, wherein the cancer is a pancreatic cancer. In certain
embodiments, wherein the cancer is a glioma.
[0009] In certain embodiments, the subject is or has been treated with a hormone therapy, a chemotherapy, a targeted therapy or an immunotherapy.
[0010] In certain embodiments, the blood sample has been fractionated before the capturing step. In certain embodiments, the ctDNA is captured from plasma and the CTC is captured from buffy coat.
[0011] In certain embodiments, the first nucleic acid biomarker is a mutation, deletion, amplification, rearrangement or fusion of a gene selected from the group consisting of BRCA1, BRCA2, BIRC5, MMP9, MCM6, MELK, LIN9, UBE2C, UBE2T, EGFR, RPM2, PGR, ORC6L, ERBB2, ERBB3, KRAS, TERT, APC,
ARID 1 A, KMT2D, RB1, KMT2C, NF1, FAT1, BRAF, NOTCH1, KDM6A, KIT, CTNNB1, BAP1, IDH1, GNAS, VHL, ESR1, NTRK1, NTRK2, NTRK3, Trop-2, ROS1, MET, AA555029 RC, AKT1 , AKT2, AKT3, ABL1, ACVRIB, ALDH4A1, AP2B1, AYTL2, BBC3, Cl6orf6l, C20orf46, C9orf30, CCNE2, CDC42BPA, CDCA7, CENPA, COL4A2, COLAA2, DCK, DIAPH3, DTL, EBF4, ECT2, EGLN1, ESM1, EXT1, ALOX12B, AMER1, APC, AR, ARAF, ARFRP1, ARID 1 A, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, AZGP1, BAP1, BARD1, BCL2, BCL2L1, BCL2L2, BCL6, BIRC5, ALK, BCOR, BCORL1, BRD4, BRIP1, BTG1, BTG2, BTK, Cl lorf30, CALR, CALM2, CARD11, CASP8, CBFB, CBL, CCND1, CCND2, BRAF, CDH1, CHEK2, CDK4, CDK6, CDK8, CDKN1A, CDKN2A, CDKN2B, CDKN2C, CDK12, CEBPA, CHEK1, CUL3, CUL4A,
CXCR4, CYP17A1, DAXX, DDR1, DDR2, DIS3, DHCR7, DNMT3A, DOTIL,
EED, EP300, EPHA3, EPHB1, EPHB4, ERCC4, ERG, ERRFI1, ESR1, ESR2,
EZH2, FAM46C, FANCA, FANCC, FANCG, FANCL, FAS, FBXW7, FGF10, FGF12, FGF14, FGF18, FGF19, FGF23, FGF3, FGF4, FGFR1, FGFR2, FGFR3, FGFR4, FH, FLCN, FLT1, FLT3, FOXL2, FUBP1, GABRA6, GAT A3, GATA4, GATA6, GID4, GMPS, GNAZ, GRP126, GPR180, GSTM3, HRASLS, IGFBP5, JHDM1D, GNA11, GNA13, GNAQ, GNAS, GRM3, GSK3B, H3F3A, HBB, HDAC1, HGF, HNF1A, HRAS, HSD3B1, ID3, IDH1, IDH2, IGF1R, IKBKE, IKZF1, IL6ST, INPP4B, IRF2, IRF4, IRS2, JAK1, JAK2, JAK3, JUN, KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIT, KLHL6, KMT2A, KMT2D, KNTC2, LGP2, LIN9, LOC100288906, LOC730018, MCM6, MELK, MMP9, LTK, LYN, MAF, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K13, MAPK1, MCL1, MDM2, MDM4, MED 12, MEF2B, MEN1, MERTK, MET, MGP, MITF, MKNK1, MLH1, MPL, MRE11A, MS4A7, MSH2, MSH3, MSH6, MST1R, MTAP, MTH, NMU, MTOR, MUTYH, MYC, MYCL, MYCN, MYD88, NBN, NF1, NF2, NFE2L2, NFKB1A, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NPM1, NUSAP1, ORC6L, OXCT1, NRAS, NT5C2, OAZ1, P2RY8, PALB2, PALM2, PECI, PITRM1, PR1, QSCN6L1, PARK2, PARP1, PARP2, PARP3, PAXS, PBRM1, PDCD1, PDCD1LG2, PDGFRA, PDGFRB, PDK1, PIK3C2B, PIK3C2G, PIK3CB, PIK3RI, PIMI, PMS2, POLD1, POLE, PPARG, PPP2R1A, PPP2R2A, PRDM1, PPKAR1A, PPKC1, PTCH1, PTPN11, PTPRO, QK1, RAB6B, RASSF7, RECQL5, RFC4, RTN4RL1, RUNDC1, SCUBE2, RAC1, RAD21, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RAF1, RARA, RBBP8, RBM10, RPL37A, REL, RRET, RICTOR, RNF43, RPTOR, SDHA, SDHB, SDHC, SDHD, SERF 1 A, SLC3A3, STK32B, SETD2, SF3B1, SGK1, SMAD2, SMAD4, SMARCA4,
SMARCB1, SNCAIP, SOCS1, SOX2, SOX9, SPEN, SPOP, SRC, STAG2, STAT3, STC2, STK11, SUFU, SYK, TBX3, TEK, TET2, TGFBR2, TGFB3, TSPYL5,
TIP ARP, TNFAIP3, TNFRSF14, TSC1, TSC2, TYR03, U2AF1, VEGFA, VHL, WHSC1, WHSCIL1, WT1, XPOl, XRCC2, ZNF217, ZNF703, ALK, BCL@, BCR, CD74, ETV4, ETV5, ETV6, EWSR1, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MSH2, MYB, MYC, NUTM1, PDGFRA, RAF1, RARA, RET, RSP02, SDC4, SLC34A2, TERC, TERT, TMPRSS2, DDR2, FGFR2, FGFR3, HRAS, KIT,
MAP2K1, MAP2K2, MET, MTOR, NRAS, PDGFRA, RET, SMO, UBE2C,
UCHL5, WISP1, ZNF533, PALM2, RASSF7, PR1, OXCT1, MCM6, MS4A7, RPL37A, TP53, STC2, UBE2C, EST1, ATM, NBV, NF1, PALB2, PTEN, RAD50, AZGP1, BIRC5, PALB2, PIK3CA, CDC20, CD19, ATRX, Ki67, MUC-l, MUC-7, EX01, ESR1, CeP55, Cyclin Bl, GRB7, Stromelysin 3, Cathepsin L2, GRP-7, RB-l, Ki-67, STK15, Survivin, Cyclin Bl, MyBL2, ER, PR, Cathepsin L2, GRB7, BCL2, SCUBE2, GSTM1, CD68, ACTR3B, ANLN, BAG1, BCL2, BIRC5, BLVRA, CCNB1, CCNE1, CDC20, CDC6, CDH3, CENPF, CEP55, CXXC5, EX01, FGFR4, FOXA1, FOXC1, GPR160, GRB7, KIF2C, KRT14, KRT17, KRT5, MAPT, MDM2, MELK, MIA, MKI67, MLPH, MMPl l, MVBL2, MYC, NAT1, NDC80, NUF2, ORC6L, PGR, PHGDH, PTTG1, RRM2, SFRP1, SLC39A6, TMEM45B, TYMS, UBE2C, UBE2T, and a combination thereof. The analysis includes all molecular events including structural (CNVs, deletions, duplications, LOH regions, fusions, translocations) and/or molecular (SNVs, MNVs, insertions, deletions, and copy- number alterations (CNAs)).
[0012] In certain embodiments, the first nucleic acid biomarker is detected using an amplification assay, a hybridization assay, a sequencing assay or an array.
[0013] In certain embodiments, the surface marker is selected from the group consisting of Her2, CD19, CD20, BCMA, GD2, NY-ESO-l, EBV, Mesothelin,
CD33, CD22, CD30, CD123, PSMA, WT1, GPC3, CD38, EGFRvIII, MUC-l, PDL1, ALK, MAGE- A3, NKG2D, RoRl, SLAMF7, CD 138, CD171, PD-L1, Mena, EpCAM, EGFR, N-Cadherin, vimentin, CD44, CD 133, CD 146, Trop-2, ER, PR and a combination thereof.
[0014] In certain embodiments, the second nucleic acid biomarker is a mutation, deletion, amplification, rearrangement or fusion of a gene selected from the group consisting of BRCA1, BRCA2, BIRC5, MMP9, MCM6, MELK, LIN9,
UBE2C, UBE2T, EGFR, RPM2, PGR, ORC6L, ERBB2, ERBB3, KRAS, TERT, APC, ARID 1 A, KMT2D, RB1, KMT2C, NF1, FAT1, BRAF, NOTCH1, KDM6A, KIT, CTNNB1, BAP1, IDH1, GNAS, VHL, ESR1, NTRK1, NTRK2, NTRK3, Trop- 2, ROS1, MET, AA555029 RC, AKT1 , AKT2, AKT3, ABL1, ACVRIB,
ALDH4A1, AP2B1, AYTL2, BBC3, Cl6orf6l, C20orf46, C9orf30, CCNE2, CDC42BPA, CDCA7, CENPA, COL4A2, COLAA2, DCK, DIAPH3, DTL, EBF4, ECT2, EGLN1, ESM1, EXT1, ALOX12B, AMER1, APC, AR, ARAF, ARFRP1, ARID 1 A, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, AZGP1, BAP1, BARD1, BCL2, BCL2L1, BCL2L2, BCL6, BIRC5, ALK, BCOR, BCORL1, BRD4, BRIP1, BTG1, BTG2, BTK, Cl lorf30, CALR, CALM2, CARD11, CASP8, CBFB, CBL, CCND1, CCND2, BRAF, CDH1, CHEK2, CDK4, CDK6, CDK8, CDKN1A, CDKN2A, CDKN2B, CDKN2C, CDK12, CEBPA, CHEK1, CUL3, CUL4A, CXCR4, CYP17A1, DAXX, DDR1, DDR2, DIS3, DHCR7, DNMT3A, DOTIL, EED, EP300, EPHA3, EPHB1, EPHB4, ERCC4, ERG, ERRFI1, ESR1, EZH2, FAM46C, FANCA, FANCC, FANCG, FANCL, FAS, FBXW7, FGF10, FGF12, FGF14, FGF18, FGF19, FGF23, FGF3, FGF4, FGFR1, FGFR2, FGFR3, FGFR4, FH, FLCN, FLT1, FLT3, FOXL2, FUBP1, GABRA6, GAT A3, GATA4, GATA6, GID4, GMPS, GNAZ, GRP126, GPR180, GSTM3, HRASLS, IGFBP5, JHDM1D, GNA11, GNA13, GNAQ, GNAS, GRM3, GSK3B, H3F3A, HBB, HDAC1, HGF, HNF1A, HRAS, HSD3B1, ID3, IDH1, IDH2, IGF1R, IKBKE, IKZF1, IL6ST, INPP4B, IRF2, IRF4, IRS2, JAK1, JAK2, JAK3, JUN, KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIT, KLHL6, KMT2A, KMT2D, KNTC2, LGP2, LIN9, LOC100288906, LOC730018, MCM6, MELK, MMP9, LTK, LYN, MAF, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K13, MAPK1, MCL1, MDM2, MDM4, MED 12, MEF2B, MEN1, MERTK, MET, MGP, MITF, MKNK1, MLH1, MPL, MRE11A, MS4A7, MSH2, MSH3, MSH6, MST1R, MTAP, MTH, NMU, MTOR, MUTYH, MYC, MYCL, MYCN, MYD88, NBN, NF1, NF2, NFE2L2, NFKB1A, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NPM1, NUSAP1, ORC6L, OXCT1, NRAS, NT5C2, OAZ1, P2RY8, PALB2, PALM2, PECI, PITRM1, PR1, QSCN6L1, PARK2, PARP1, PARP2, PARP3, PAXS, PBRM1, PDCD1, PDCD1LG2, PDGFRA, PDGFRB, PDK1, PIK3C2B, PIK3C2G, PIK3CB, PIK3RI, PIMI, PMS2, POLD1, POLE, PPARG, PPP2R1A, PPP2R2A, PRDM1, PPKAR1A, PPKC1, PTCH1, PTPN11, PTPRO, QK1, RAB6B, RASSF7, RECQL5, RFC4, RTN4RL1, RUNDC1, SCUBE2, RAC1, RAD21, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RAF1, RARA, RBBP8, RBM10, RPL37A, REL, RRET, RICTOR, RNF43, RPTOR, SDHA, SDHB, SDHC, SDHD, SERF 1 A, SLC3A3, STK32B, SETD2, SF3B1, SGK1, SMAD2, SMAD4, SMARCA4,
SMARCB1, SNCAIP, SOCS1, SOX2, SOX9, SPEN, SPOP, SRC, STAG2, STAT3, STC2, STK11, SUFU, SYK, TBX3, TEK, TET2, TGFBR2, TGFB3, TSPYL5,
TIP ARP, TNFAIP3, TNFRSF14, TSC1, TSC2, TYR03, U2AF1, VEGFA, VHL, WHSC1, WHSCIL1, WT1, XPOl, XRCC2, ZNF217, ZNF703, ALK, BCL@, BCR, CD74, ETV4, ETV5, ETV6, EWSR1, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MSH2, MYB, MYC, NUTM1, PDGFRA, RAF1, RARA, RET, RSP02, SDC4, SLC34A2, TERC, TERT, TMPRSS2, DDR2, FGFR2, FGFR3, HRAS, KIT,
MAP2K1, MAP2K2, MET, MTOR, NRAS, PDGFRA, RET, SMO, UBE2C,
UCHL5, WISP1, ZNF533, PALM2, RASSF7, PR1, OXCT1, MCM6, MS4A7, RPL37A, TP53, STC2, UBE2C, EST1, ATM, NBV, NF1, PALB2, PTEN, RAD50, AZGP1, BIRC5, PALB2, PIK3CA, CDC20, CD19, ATRX, Ki67, MUC-l, MUC-7, EX01, ESR1, CeP55, Cyclin Bl, GRB7, Stromelysin 3, Cathepsin L2, GRP-7, RB-l, Ki-67, STK15, Survivin, Cyclin Bl, MyBL2, ER, PR, Cathepsin L2, GRB7, BCL2, SCUBE2, GSTM1, CD68, ACTR3B, ANLN, BAG1, BCL2, BIRC5, BLVRA, CCNB1, CCNE1, CDC20, CDC6, CDH3, CENPF, CEP55, CXXC5, EX01, FGFR4, FOXA1, FOXC1, GPR160, GRB7, KIF2C, KRT14, KRT17, KRT5, MAPT, MDM2, MELK, MIA, MKI67, MLPH, MMP11, MVBL2, MYC, NAT1, NDC80, NUF2, ORC6L, PGR, PHGDH, PTTG1, RRM2, SFRP1, SLC39A6, TMEM45B, TYMS, UBE2C, UBE2T, and a combination thereof.
[0015] In certain embodiments, the second nucleic acid biomarker is expression level of a gene selected from CA15-3, CA27.29, CEA, ER, PR, HER2, uPA, PAI-l, TP53, cathepsin D, cyclin E, nestin, MSP-alpha, TIMP-4, PDGFR-alpha, OPG, AFP, 5 -HI A A, CA19-9, CA27.29, CA72-4, hCGbeta, Calcitonin, CgA, ER,
PR, Osteocalcin, epithelial membrane antigen (EMA), AlphavBeta3 integrin,
AlphavBeta6 integrin, transferrin receptor, transthyretin, Alkaline phosphatase, PSA, Lactate dehydrogenase, Neuron specific enolase, Nuclear matrix protein 22,
Plasminogen activator inhibitor, SCC, Apolipoprotein Al, Beta-2 -microglobulin, Cyfra2l-l, HE4, Ferritin, Fibrinogen, Fibrin D-dimer, S100, TP A, Thyroglobulin, Aldehyde dehydrogenase, CD20, CD24, CD44, IL-2, IL-6, IL-10, IL-12, IL-15, interferon gamma, CA125, HE4, PD-l and PD-L1 and a combination thereof.
[0016] In certain embodiments, the second nucleic acid biomarker is detected using an amplification assay, a hybridization assay, a sequencing assay or an array.
[0017] In certain embodiments, the CTC biomarker is selected from the group consisting of Her2, CD19, CD20, CD22, PD-L1, Mena, EpCAM, EGFR, N-Cadherin, E-cadherin, vimentin, CD44, CD 133, CD 146, CD151, EGFR, Trop-2, ER, PR,
PSMA, AlphavBeta3 integrin, AlphavBeta6 integrin, CEA, Folate receptor-alpha, urokinase-type plasminogen activator receptor, Epithelial membrane antigen, and a combination thereof.
[0018] In certain embodiments, the CTC biomarker is a protein marker or nucleic acid marker.
[0019] In certain embodiments, the CTC biomarker is detected using a third nanocomposition comprising a fluorescent label that barcodes the CTC biomarker.
[0020] In certain embodiments, the CTC biomarker is detected using an immunohistochemistry (IHC) assay or a fluorescence in situ hybridization (FISH) assay.
[0021] In certain embodiments, the method further comprises detecting a circulating protein in the blood sample.
[0022] The method of claim 1, wherein the circulating protein is selected from the group consisting of CA15-3, CA27.29, CEA, ER, PR, Her-2, Cyclin E, Cathepsin D, Nestin, P53, CA125, HE4, IL-6, IL-10, Ki67, CK19, AIDH1, EGFR, PD-l, and PD-L1, uPA, PAI-l, MSP-alpha, TIMP-4, PDGFR-alpha, OPG, AFP, 5-HIAA, CA19-9, CA27.29, CA72-4, hCGbeta, Calcitonin, CgA, ER, PR, Osteocalcin, epithelial membrane antigen (EMA), AlphavBeta3 integrin, AlphavBeta6 integrin, transferrin receptor, transthyretin, Alkaline phosphatase, PSA, Lactate
dehydrogenase, Neuron specific enolase, Nuclear matrix protein 22, Plasminogen activator inhibitor, SCC, Apolipoprotein Al, Beta-2-microglobulin, Cyfra2l-l, HE4, Ferritin, Fibrinogen, Fibrin D-dimer, S100, TP A, Thyroglobulin, Aldehyde dehydrogenase, CD20, CD24, CD44, CD34, CD45, IL-2, IL-6, IL-10, IL-12, IL-15, interferon gamma, CA125, HE4, PD-l and PD-L1, and a combination thereof.
[0023] In certain embodiments, the first or the second nanocomposition comprising a superparamagnetic iron oxide (SPIO) nanoparticle. In certain embodiments, the nanocomposition comprises a silanization coating. In certain embodiments, the silanization coating forms a low density, porous 3-D structure.
[0024] In certain embodiments, the test disclosed herein further comprises the step of recommending, suggesting or selecting, based on information obtained in the detecting step, a treatment for the subject.
[0025] In some embodiments, the treatment is selected from the group consisting of endocrine therapy, fulvestrant, Tamoxifen, Toremifene, Aromatase inhibitors, Megestrol acetate, Her-2 targeted therapy such as trastuzumab, ado- trastuzumab-emtansine, and Pertuzumab, Everolimus, Larotrectinib, Everolimus, Fulvestrant, Robocliclib, palbociclib, ADC, Atezolizumab, Avetumab, Durvalumb, Olaparib, EGFR - targeted therapy, such as afatinib, gefitinib, ertotinib, and
Osimertinib, ALK- targeted therapy such as alectinib, crizotinib, and ceritinib, BRAF- targeted therapy such as dabrafenib in combination with trametinib, or vemurafenib, trametinib, cobimetinib in combination with Zelboraf, KRAS-targeted therapy such as cetuximab and panitumumab, BRCA1/2 - targeted therapy such as rucaparib, 5-Fu, MTX, Doxorubicin, Etoposide, Alkylating agents, Cisplatin Doxorubicin,
Bevacizumab, Ramucirumab, Erlotinib, Afatinib, Gefitinib, Osimertinib, Osimertinib, Necitumumab, Crizotinib, Ceritinib, Alectinib, Brigatinib, Dabrafenib, Trametinib.
DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 illustrates a blood test according to an exemplary embodiment of the invention. [0027] FIG. 2 illustrated a workflow to analyze comprehensive circulating markers from a blood sample according to an exemplary embodiment of the invention.
[0028] FIG. 3 illustrates the high capture yield and better sample quality of capturing ctDNA, CTC and protein using nanocompostions according to an exemplary embodiment of the invention.
[0029] FIG. 4 illustrates the high performance of detecting ctDNA using a nanocompostion according to an exemplary embodiment of the invention.
[0030] FIG. 5 illustrates the high sensitivity of detecting ctDNA using a nanocompostion according to an exemplary embodiment of the invention.
[0031] FIG. 6 illustrates the high accuracy and cleaner samples for targeted sequencing using a nanocompostion according to an exemplary embodiment of the invention.
[0032] FIG. 7 illustrates the huffy coat and plasma/cfDNA extraction from whole blood samples. Two on-market blood collection tubes were used. The huffy coat images show one sample of the collected huffy coat cells and a 50X diluted samples. Buffy coat layer contains cells that can be clearly imaged and used for downstream diagnostics. Bottom row shows separated plasma samples and extracting cfDNA elution step. cfDNA eluates can be quantified using optical method, and house keeping genes or cancer mutations can be measured using molecular detection approach for example qPCR.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
[0034] ETnless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. [0035] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
[0036] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Definitions
[0037] The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over the definition of the term as generally understood in the art.
[0038] As used herein, the singular forms“a”,“an” and“the” include plural references unless the context clearly dictates otherwise.
[0039] The term“amount” or“level” refers to the quantity of a polynucleotide of interest or a polypeptide of interest present in a sample. Such quantity may be expressed in the absolute terms, i.e., the total quantity of the polynucleotide or polypeptide in the sample, or in the relative terms, i.e., the concentration of the polynucleotide or polypeptide in the sample.
[0040] As used herein, the term“cancer” refers to any diseases involving an abnormal cell growth and include all stages and all forms of the disease that affects any tissue, organ or cell in the body. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre- and post-metastatic cancers. In general, cancers can be categorized according to the tissue or organ from which the cancer is located or originated and morphology of cancerous tissues and cells. As used herein, cancer types include, without limitation, acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, childhood cerebellar or cerebral, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, brain cancer, breast cancer, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, Burkitf s lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, emphysema, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, retinoblastoma, gastric (stomach) cancer, glioma, head and neck cancer, heart cancer, Hodgkin lymphoma, islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer, leukemia, liver cancer, lung cancer, neuroblastoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, Ewing family of tumors, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, vaginal cancer.
[0041] It is noted that in this disclosure, terms such as“comprises”,
“comprised”,“comprising”,“contains”,“containing” and the like have the meaning attributed in United States Patent law; they are inclusive or open-ended and do not exclude additional, un-recited elements or method steps. Terms such as“consisting essentially of’ and“consists essentially of’ have the meaning attributed in United States Patent law; they allow for the inclusion of additional ingredients or steps that do not materially affect the basic and novel characteristics of the claimed invention. The terms“consists of’ and“consisting of’ have the meaning ascribed to them in United States Patent law; namely that these terms are close ended.
[0042] A“cell”, as used herein, can be prokaryotic or eukaryotic. A prokaryotic cell includes, for example, bacteria. A eukaryotic cell includes, for example, a fungus, a plant cell, and an animal cell. The types of an animal cell (e.g., a mammalian cell or a human cell) includes, for example, a cell from
circulatory/immune system or organ (e.g., a B cell, a T cell (cytotoxic T cell, natural killer T cell, regulatory T cell, T helper cell), a natural killer cell, a granulocyte (e.g., basophil granulocyte, an eosinophil granulocyte, a neutrophil granulocyte and a hypersegmented neutrophil), a monocyte or macrophage, a red blood cell (e.g., reticulocyte), a mast cell, a thrombocyte or megakaryocyte, and a dendritic cell); a cell from an endocrine system or organ (e.g., a thyroid cell (e.g., thyroid epithelial cell, parafollicular cell), a parathyroid cell (e.g., parathyroid chief cell, oxyphil cell), an adrenal cell (e.g., chromaffin cell), and a pineal cell (e.g., pinealocyte)); a cell from a nervous system or organ (e.g., a glioblast (e.g., astrocyte and oligodendrocyte), a microglia, a magnocellular neurosecretory cell, a stellate cell, a boettcher cell, and a pituitary cell (e.g., gonadotrope, corticotrope, thyrotrope, somatotrope, and lactotroph)); a cell from a respiratory system or organ (e.g., a pneumocyte (a type I pneumocyte and a type II pneumocyte), a clara cell, a goblet cell, an alveolar macrophage); a cell from circular system or organ (e.g., myocardiocyte and pericyte); a cell from digestive system or organ (e.g., a gastric chief cell, a parietal cell, a goblet cell, a paneth cell, a G cell, a D cell, an ECL cell, an I cell, a K cell, an S cell, an enteroendocrine cell, an enterochromaffm cell, an APUD cell, a liver cell (e.g., a hepatocyte and Kupffer cell)); a cell from integumentary system or organ (e.g., a bone cell (e.g., an osteoblast, an osteocyte, and an osteoclast), a teeth cell (e.g., a cementoblast, and an ameloblast), a cartilage cell (e.g., a chondroblast and a chondrocyte), a skin/hair cell (e.g., a trichocyte, a keratinocyte, and a melanocyte (Nevus cell)), a muscle cell (e.g., myocyte), an adipocyte, a fibroblast, and a tendon cell), a cell from urinary system or organ (e.g., a podocyte, a juxtaglomerular cell, an intraglomerular mesangial cell, an extraglomerular mesangial cell, a kidney proximal tubule brush border cell, and a macula densa cell), and a cell from reproductive system or organ (e.g., a spermatozoon, a Sertoli cell, a leydig cell, an ovum, an oocyte). A cell can be normal, healthy cell; or a diseased or unhealthy cell (e.g., a cancer cell). A cell further includes a mammalian zygote or a stem cell which include an embryonic stem cell, a fetal stem cell, an induced pluripotent stem cell, and an adult stem cell. A stem cell is a cell that is capable of undergoing cycles of cell division while maintaining an undifferentiated state and differentiating into specialized cell types. A stem cell can be an omnipotent stem cell, a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell and a unipotent stem cell, any of which may be induced from a somatic cell. A stem cell may also include a cancer stem cell. A mammalian cell can be a rodent cell, e.g., a mouse, rat, hamster cell. A mammalian cell can be a lagomorpha cell, e.g., a rabbit cell. A mammalian cell can also be a primate cell, e.g., a human cell.
[0043] The term“complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non- traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%>, 70%>, 80%>, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
[0044] The terms“determining,”“assessing,”“assaying,”“measuring” and
“detecting” can be used interchangeably and refer to both quantitative and semi- quantitative determinations. Where either a quantitative and semi -quantitative determination is intended, the phrase“determining a level” of a polynucleotide or polypeptide of interest or“detecting” a polynucleotide or polypeptide of interest can be used.
[0045] The term“nucleic acid” and“polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either
deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.
[0046] In general, a“protein” is a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a functional portion thereof. Those of ordinary skill will further appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
[0047] The term“recommending” or“suggesting” in the context of a treatment of a disease, refers to making a suggestion or a recommendation for therapeutic intervention (e.g., drug therapy, adjunctive therapy, stopping using a therapy, using a different drug, etc.) and/or disease management which are specifically applicable to the patient.
[0048] As used herein, the term“subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term“subject” is used herein interchangeably with“individual” or“patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
[0049] The term“treatment,”“treat,” or“treating” refer to a method of reducing the effects of a cancer (e.g., breast cancer, lung cancer, ovarian cancer or the like) or symptom of cancer. Thus, in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a cancer or symptom of the cancer. For example, a method of treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any percent reduction between 10 and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
Blood Test
[0050] In one aspect, the present disclosure provides a blood test that can accurately provide comprehensive cancer profiling to predict cancer
incidence/recurrence or guide and monitor the treatment intervention of the cancer. In one embodiment, the test includes obtaining a blood sample from a subject, and capturing from the blood sample a circulating tumor DNA (ctDNA) using a first nanocomposition and a circulating tumor cell (CTC) using a second nanocomposition. The test further includes detecting at least a first nucleic acid biomarker from the ctDNA and detecting at least a CTC biomarker and at least a second nucleic acid biomarker from the CTC. In certain embodiments, the test further includes the step of determining, based on information obtained in the detecting step, a likelihood of the subject having the cancer or a recurrence of the cancer.
[0051] By testing protein, CTC, ctDNA comprehensive circulating markers from a blood sample, the multiple type of biomarker information can be
complementary and corelate with each other for more accurate diagnostics than testing using individual types of markers.
[0052] A. Nanocomposition
[0053] In some embodiments, the nanocomposition used in the test described herein has a diameter ranging from about 1 nm to about 50 pm (e.g. from 1 nm to 40 pm, from 1 nm to 30 pm, from 1 nm to 20 pm, from 1 nm to 10 pm, from 1 nm to 1 pm, 1-900 nm, 1-800 nm, 1-700 nm, 1-600 nm, 1-500 nm, 1-400 nm, 1-300 nm, 1 200 nm, 1-100 nm, 1-50 nm, 1-40 nm, 1-30 nm, 1-20 nm etc.). In certain
embodiments, the nanocomposition used in the test described herein comprises at least a nanoparticle or a cluster of nanoparticles. In certain embodiments, the nanocopositin further comprises an analyte-associating member operably linked to the nanoparticle or nanoparticles.
[0054] The diameter of the nanoparticles ranges from 1 nm to 900 nm
(preferably 1-800 nm, 1-700 nm, 1-600 nm, 1-500 nm, 1-400 nm, 1-300 nm, 1-200 nm, 1-100 nm, 1-50 nm, 2-40 nm, 5-20 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm). The size of nanoparticles can be controlled by selecting appropriate synthesis methods and/or systems. For example, to control the size of nanoparticles, synthesis of nanoparticles can be carried out in a polar solvent which provides ionic species that can adsorb on the surface of the nanoparticles, thereby providing electrostatic effect and particle-particle repulsive force to help stabilize the
nanoparticles and inhibit the growth of the nanoparticles. For another example, nanoparticles can be synthesized in a micro-heterogeneous system that allows compartmentalization of nanoparticles in constrained cavities or domains. Such a micro-heterogeneous system may include, liquid crystals, mono and multilayers, direct micelles, reversed micelles, microemulsions and vesicles. To obtain
nanoparticles within a desired size range, the synthesis conditions may be properly controlled or varied to provide for, e.g., a desired solution concentration or a desired cavity range (a detailed review can be found at, e.g., Vincenzo Liveri, Controlled synthesis of nanoparticles in microheterogeneous systems, Published by Springer, 2006).
[0055] The shape of the nanoparticles can be spherical, cubic, rod shaped (see, e.g., A. Fu et al, Nano Letters, 7, 179-182 (2007)), tetrapod-shaped (see, e.g., L.
Manna et al, Nature Materials, 2, 382-385 (2003)), pyramidal, multi-armed, nanotube, nanowire, nanofiber, nanoplate, or any other suitable shapes. Methods are known in the art to control the shape of the nanoparticles during the preparation (see, e.g., Waseda Y. et al., Morphology control of materials and nanoparticles: advanced materials processing and characterization, published by Springer, 2004). For example, when the nanoparticles are prepared by the bottom -up process (i.e. from molecule to nanoparticle), a shape controller which adsorbs strongly to a specific crystal plane may be added to control the growth rate of the particle.
[0056] A single nanocomposition may comprise a single nanoparticle or a plurality or a cluster of mini-nanoparticles (A. Fu et al, J. Am. chem. Soc. 126, 10832-10833 (2004), J. Ge et al, Angew. Chem. Int. Ed. 46, 4342-4345 (2007), Zhenda Lu et al, Nano Letters 11, 3404-3412 (2011).). The mini-nanoparticles can be homogeneous (e.g., made of the same composition/materials or having same size) or heterogeneous (e.g., made of different compositions/materials or having different sizes). A cluster of homogeneous mini-nanoparticles refers to a pool of particles having substantially the same features or characteristics or consisting of substantially the same materials. A cluster of heterogeneous mini -nanoparticles refers to a pool of particles having different features or characteristics or consisting of substantially different materials. For example, a heterogeneous mini-nanoparticle may comprise a quantum dot in the center and a discrete number of gold (Au) nanocrystals attached to the quantum dot. When the nanoparticles are associated with a coating (as described below), different nanoparticles in a heterogeneous nanoparticle pool do not need to associate with each other at first, but rather, they could be individually and separately associated with the coating. [0057] In some embodiments, the nanocomposition provided herein comprises a plurality of nanoparticles. For example, the nanocomposition comprises 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, lOOs or lOOOs nanoparticles.
[0058] In some embodiments, the nanocomposition provided herein comprises nanoparticle(s) which comprises a magnetic material. Suitable magnetic materials include, but are not limited to, ferrimagnetic or ferromagnetic materials (e.g., iron, nickel, cobalt, some alloys of rare earth metals, and some naturally occurring minerals such as lodestone), paramagnetic materials (such as platinum, aluminum), and superparamagnetic materials (e.g., superparamagnetic iron oxide (SPIO), or SPIO doped with other metal elements such as gold, silver, manganese, cobalt, magnesium, nickel.).
[0059] In some embodiments, the nanoparticle of the nanocomposition provided herein is a SPIO nanoparticle. The SPIO nanoparticle is an iron oxide nanoparticle, either maghemite (y-Fe 03) or magnetite (Fe304), or nanoparticles composed of both phases. The SPIO can be synthesized with a suitable method and dispersed as a colloidal solution in organic solvents or water. Methods to synthesize the SPIO nanoparticles are known in the art (see, for example, Morteza Mahmoudi et al, Superparamagnetic Iron Oxide Nanoparticles: Synthesis, Surface Engineering, Cytotoxicity and Biomedical Applications, published by Nova Science Pub Inc,
2011). In one embodiment, the SPIO nanoparticles can be made through wet chemical synthesis methods which involve co-precipitation of Fe and Fe salts in the presence of an alkaline medium. During the synthesis, nitrogen may be introduced to control oxidation, surfactants and suitable polymers may be added to inhibit agglomeration or control particle size, and/or emulsions (such as water-in-oil microemulsions) may be used to modulate the physical properties of the SPIO nanoparticle (see, for example, Jonathan W. Gunn, The preparation and
characterization of superparamagnetic nanoparticles for biomedical imaging and therapeutic application, published by ProQuest, 2008). In another embodiment, the SPIO nanoparticles can be generated by thermal decomposition of iron pentacarbonyl, alone or in combination with transition metal carbonyls, optionally in the presence of one or more surfactants (e.g., lauric acid and oleic acid) and/or oxidatants (e.g., trimethylamine-N-oxide), and in a suitable solvent (e.g., dioctyl ether or hexadecane) (see, for example, US patent application PG Pub 20060093555). In another embodiment, the SPIO nanoparticles can also be made through gas deposition methods, which involves laser vaporization of iron in a helium atmosphere containing different concentrations of oxygen (see, Miller J.S. et al., Magnetism: Nanosized magnetic materials, published by Wiley-VCH, 2002). In certain embodiments, the SPIO nanoparticles are those disclosed in US patent application PG Pub
20100008862.
[0060] In certain embodiments, the nanocomposition provided herein can further comprise non-SIPO nanoparticle(s). The non-SPIO nanoparticles include, for example, metallic nanoparticles (e.g., gold or silver nanoparticles (see, e.g., Hiroki Hiramatsu, F.E.O., Chemistry of Materials 16, 2509-2511 (2004)), semiconductor nanoparticles (e.g., quantum dots with individual or multiple components such as CdSe/ZnS (see, e.g., M. Bruchez, et al, science 281, 2013-2016 (1998))), doped heavy metal free quantum dots (see, e.g., Narayan Pradhan et al, J. Am. chem. Soc. 129, 3339-3347 (2007)) or other semiconductor quantum dots); polymeric nanoparticles (e.g., particles made of one or a combination of PLGA (poly(lactic-co-gly colic acid) (see, e.g., Minsoung Rhee et al, Adv. Mater. 23, H79-H83 (2011)), PCL
(polycaprolactone) (see, e.g., Marianne Labet et al, Chem. Soc. Rev. 38, 3484-3504 (2009)), PEG (poly ethylene glycol) or other polymers); siliceous nanoparticles; and non-SPIO magnetic nanoparticles (e.g., MnFe204 (see, e.g., Jae-Hyun Lee et al, Nature Medicine 13, 95-99 (2006)), synthetic antiferromagnetic nanoparticles (SAF) (see, e.g., A. Fu et al, Angew. Chem. Int. Ed. 48, 1620-1624 (2009)), and other types of magnetic nanoparticles). In certain embodiments, the non-SPIO nanoparticle is a colored nanoparticle, for example, a semiconductor nanoparticle such as a quantum dot.
[0061] The non-SPIO nanoparticles can be prepared or synthesized using suitable methods known in the art, such as for example, sol-gel synthesis method, water-in-oil micro-emulsion method, gas deposition method and so on. For example, gold nanoparticles can be made by reduction of chloroaurate solutions (e.g., HAuCU) by a reducing agent such as citrate, or acetone dicarboxulate. For another example, CdS semiconductor nanoparticle can be prepared from Cd(Cl04)2 and Na2S on the surface of silica particles. For another example, II- VI semiconductor nanoparticles can be synthesized based on pyrolysis of organometallic reagents such as dimethyl cadmium and trioctylphosphine selenide, after injection into a hot coordinating solvent (see, e.g., Gunter Schmid, Nanoparticles: From Theory to Application, published by John Wiley & Sons, 2011). Doped heavy metal free quantum dots, for example Mn-doped ZnSe quantum dots can be prepared using nucleation-doping strategy, in which small-sized MnSe nanoclusters are formed as the core and ZnSe layers are overcoated on the core under high temperatures. For another example, polymeric nanoparticles can be prepared by emulsifying a polymer in a two-phase solvent system, inducing nanosized polymer droplets by sonication or
homogenization, and evaporating the organic solvent to obtain the nanoparticles. For another example, siliceous nanoparticles can be prepared by sol-gel synthesis, in which silicon alkoxide precursors (e.g., TMOS or TEOS) are hydrolyzed in a mixture of water and ethanol in the presence of an acid or a base catalyst, the hydrolyzed monomers are condensed with vigorous stirring and the resulting silica nanoparticles can be collected. For another example, SAFs, a non- SPIO magnetic nanoparticle, can be prepared by depositing a ferromagenetic layer on each of the two sides of a nonmagnetic space layer (e.g., ruthenium metal), along with a chemical etchable copper release layer and protective tantalum surface layers, using ion-bean deposition in a high vacuum, and the SAF nanoparticle can be released after removing the protective layer and selective etching of copper.
[0062] In some embodiments, the magnetic material in the composition allows the nanocomposition to be pulled or attracted to a magnet or in a magnetic field. The magnetic nanocomposition can be attracted to or magnetically guided to an intended site when subject to an applied magnetic field, for example a magnetic field from high-filed and/or high-gradient magnets. For example, a magnet (e.g., magnetic grid) can be placed in the proximity of the beads so as to attract the magnetic beads.
[0063] In some embodiments, the nanocomposition further comprises a coating. The at least one nanoparticle can be embedded in or coated with the coating. Any suitable coatings known in the art can be used, for example, a polymer coating and a non-polymer coating. In certain embodiment, the nanocoposition described herein has a silanization coating. The coating may interact with the nanoparticle(s) through 1) intra-molecular interaction such as covalent bonds (e.g., Sigma bond, Pi bond, Delta bond, Double bond, Triple bond, Quadruple bond, Quintuple bond, Sextuple bond, 3c-2e, 3c-4e, 4c-2e, Agostic bond, Bent bond, Dipolar bond, Pi backbond, Conjugation, Hyperconjugation, Aromaticity, Hapticity, and Antibonding), metallic bonds (e.g., chelating interactions with the metal atom in the core
nanoparticle), or ionic bonding (cation p-bond and salt bond), and 2) inter-molecular interaction such as hydrogen bond (e.g., Dihydrogen bond, Dihydrogen complex, Low-barrier hydrogen bond, Symmetric hydrogen bond) and non-covalent bonds (e.g., hydrophobic, hydrophilic, charge-charge, or p-stacking interactions, van der Waals force, London dispersion force, Mechanical bond, Halogen bond,
Aurophilicity, Intercalation, Stacking, Entropic force, and chemical polarity).
[0064] In some embodiments, the coating includes a low density, porous 3-D structure, as disclosed in U.S. Patent No. 9711266, the disclosure of which is incorporated by reference.
[0065] As used herein, the low density, porous 3-D structure refers to a structure with density much lower (e.g., lOs times, 20s times, 30s times, 50s times,
70s times, lOOs times) than existing mesoporous nanoparticles (e.g., mesoporous nanoparticles having a pore size ranging from 2 nm to 50 nm). (A. Vincent, et. al., J. Phys. Chem. C, 2007, 111, 8291- 8298; J. E. Lee, et. al, J. Am. Chem. Soc, 2010, 132, 552-557; Y. -S. Lin, et. al, J. Am. Chem. Soc, 2011, 133, 20444-20457; Z. Lu,
Angew. Chem. Int. Ed., 2010, 49, 1862-1866.)
[0066] In certain embodiments, the low density, porous 3-D structure refers to a structure having a density of <1.0 g/cc (e.g., <l00mg/cc, <l0mg/cc, <5mg/cc, <lmg/cc, <0.5mg/cc, <0.4mg/cc, <0.3mg/cc, <0.2mg/cc, or <0. lmg/cc) (for example, from 0.01 mg/cc to 10 mg/cc, from 0.01 mg/cc to 8 mg/cc, from 0.01 mg/cc to 5 mg/cc, from 0.01 mg/cc to 3 mg/cc, from 0.01 mg/cc to 1 mg/cc, from 0.01 mg/cc to 1 mg/cc, from 0.01 mg/cc to 0.8 mg/cc, from 0.01 mg/cc to 0.5 mg/cc, from 0.01 mg/cc to 0.3 mg/cc, from 0.01 mg/cc to 1000 mg/cc, from 0.01 mg/cc to 915 mg/cc, from 0.01 mg/cc to 900 mg/cc, from 0.01 mg/cc to 800 mg/cc, from 0.01 mg/cc to 700 mg/cc, from 0.01 mg/cc to 600 mg/cc, from 0.01 mg/cc to 500 mg/cc, from 0.1 mg/cc to 800 mg/cc, from 0.1 mg/cc to 700 mg/cc, from 0.1 mg/cc to 1000 mg/cc, from 1 mg/cc to 1000 mg/cc, from 5 mg/cc to 1000 mg/cc, from 10 mg/cc to 1000 mg/cc, from 20 mg/cc to 1000 mg/cc, from 30 mg/cc to 1000 mg/cc, from 30 mg/cc to 1000 mg/cc, from 30 mg/cc to 900 mg/cc, from 30 mg/cc to 800 mg/cc, or from 30 mg/cc to 700 mg/cc).
[0067] The density of 3-D structure can be determined using various methods known in the art (see, e.g., Lowell, S. et al, Characterization of porous solids and powders: surface area, pore size and density, published by Springer, 2004).
Exemplary methods include, Brunauer Emmett Teller (BET) method and helium pycnometry (see, e.g., Varadan V. K. et al., Nanoscience and Nanotechnology in Engineering, published by World Scientific, 2010). Briefly, in BET method, dry powders of the testing 3-D structure is placed in a testing chamber to which helium and nitrogen gas are fed, and the change in temperature is recorded and the results are analyzed and extrapolated to calculate the density of the testing sample. In helium pycnometry method, dry powders of the testing 3-D structure are filled with helium, and the helium pressure produced by a variation of volume is studied to provide for the density. The measured density based on the dry power samples does not reflect the real density of the 3-D structure because of the ultralow density of the 3-D structure, the framework easily collapses during the drying process, hence providing much smaller numbers in the porosity measurement than when the 3-D structure is fully extended, for example, like when the 3-D structure is fully extended in a buffer solution. In certain embodiments, the density of the 3-D structure can be determined using the dry mass of the 3-D structure divided by the total volume of such 3-D structure in an aqueous solution. For example, dry mass of the core particles with and without the 3-D structure can be determined respectively, and the difference between the two would be the total mass of the 3-D structure. Similarly, the volume of a core particle with and without the 3-D structure in an aqueous solution can be determined respectively, and the difference between the two would be the volume of the 3-D structure on the core particle in an aqueous solution.
[0068] In certain embodiments, the nanocomposition can be dispersed as a plurality of large nanoparticles coated with the 3-D structure in an aqueous solution.
In such case, the total volume of the 3-D structure can be calculated as the average volume of the 3-D structure for an individual large nanoparticle multiplied with the number of the large nanoparticles. For each individual large nanoparticle, the size (e.g., radius) of the particle with 3-D structure can be determined with Dynamic Light Scattering (DLS) techniques, and the size (e.g., radius) of the particle core without the 3-D structure can be determined under Transmission Electron Microscope (TEM), as the 3-D structure is substantially invisible under TEM. Accordingly, the volume of the 3-D structure on an individual large nanoparticle can be obtained by subtracting the volume of the particle without 3-D structure from the volume of the particle with the 3-D structure.
[0069] The number of large nanoparticles for a given core mass can be calculated using any suitable methods. For example, an individual large nanoparticle may be composed of a plurality of small nanoparticles which are visible under TEM. In such case, the average size and volume of a small nanoparticle can be determined based on measurements under TEM, and the average mass of a small nanoparticle can be determined by multiplying the known density of the core material with the volume of the small particle. By dividing the core mass with the average mass of a small nanoparticle, the total number of small nanoparticles can be estimated. For an individual large nanoparticle, the average number of small nanoparticles in it can be determined under TEM. Accordingly, the number of large nanoparticles for a given core mass can be estimated by dividing the total number of small nanoparticles with the average number of small nanoparticles in an individual large nanoparticle.
[0070] In certain embodiments, the low density, porous 3-D structure is highly porous. For example, the low density, porous 3-D structure can be a structure having 40%-99.9% (preferably 50% to 99.9%) of empty space or pores in the structure, where 80% of the pores having size of 1 nm to 500 nm in pore radius.
[0071] The porosity of the 3-D structure can be characterized by the
Gas/Vapor adsorption method. In this technique, usually nitrogen, at its boiling point, is adsorbed on the solid sample. The amount of gas adsorbed at a particular partial pressure could be used to calculate the specific surface area of the material through the Brunauer, Emmit and Teller (BET) nitrogen adsorption/desorption equation. The pore sizes are calculated by the Kelvin equation or the modified Kelvin equation, the BJH equation (see, e.g., D. Niu et al, J. Am. chem. Soc. 132, 15144-15147 (2010)). The porosity of the 3-D structure can also be characterized by mercury porosimetry (see, e.g., Varadan V. K. et al, supra). Briefly, gas is evacuated from the 3-D structure, and then the structure is immersed in mercury. As mercury is non-wetting at room temperature, an external pressure is applied to gradually force mercury into the sample. By monitoring the incremental volume of mercury intruded for each applied pressure, the pore size can be calculated based on the Washburn equation.
[0072] In some embodiments, the low density, porous 3-D structure has a porous structure (except to the core nanoparticle or core nanoparticles) which could not be obviously observed or substantially transparent under TEM, for example, even when the feature size of the 3-D structure is in the lOs or lOOs nanometer range. The term "obviously observed" or "substantially transparent" as used herein means that, the thickness of the 3-D structure cannot be readily estimated or determined based on the image of the 3-D structure under TEM. The bead (e.g., nanoparticle(s) coated with or embedded in a low density, porous 3-D structure) can be observed or measured by ways known in the art. For example, the size (e.g., radius) of the bead with the 3-D structure can be measured using DLS methods, and the size (e.g., radius) of the core particle without the 3-D structure can be measured under TEM. In certain embodiments, the thickness of the 3-D structure is measured as lOs, lOOs, lOOOs nanometer range by DLS, but cannot be readily determined under TEM. For example, when the beads provided herein are observed under TEM, the nanoparticles can be identified, however, the low density, porous 3-D structure cannot be obviously observed, or is almost transparent. This distinguishes the low density, porous 3-D structures from those reported in the art that comprise nanoparticles coated with crosslinked and size tunable 3-D structure, including the mesoporous silica nanoparticles or coating (see, e.g., J. Kim, et. al, J. Am. Chem. Soc, 2006, 128, 688- 689; J. Kim, et. al, Angew. Chem. Int. Ed., 2008, 47, 8438-8441). This feature also indicates that the low density, porous 3-D structure has a much lower density and/or is highly porous in comparison to other coatings known in the art. The porosity of the 3-D structure can be also evaluated by the capacity to load different molecules (see, e.g., Wang L. et al, Nano Research 1, 99-115 (2008)). As the 3-D structure provided herein has a low density and high porosity, it is envisaged that more payload can be associated with the 3-D structure than with other coatings. For example, when 3-D structure is loaded with organic fiuorophores such as Rhodamin, over 105 Rhodamin molecules can be loaded to 3-D structure of one nanoparticle.
[0073] In some embodiments, the low density, porous 3-D structure is made of silane-containing or silane-like molecules (e.g., silanes, organosilanes,
alkoxysilanes, silicates and derivatives thereof).
[0074] In certain embodiments, the silane-containing molecule comprises an organosilane, which is also known as silane coupling agent. Organosilane has a general formula of RxSiY4-x, wherein R group is an alkyl, aryl or organic functional group. Y group is a methoxy, ethoxy or acetoxy group, x is 1, 2 or 3. The R group could render a specific function such as to associate the organosilane molecule with the surface of the core nanoparticle or other payloads through covalent or non- covalent interactions. The Y group is hydrolysable and capable of forming a siloxane bond to crosslink with another organosilane molecule. Exemplary R groups include, without limitation, disulphidealkyl, aminoalkyl, mercaptoalkyl, vinylalkyl, epoxyalkyl, and methacrylalkyl, carboxylalkyl groups. The alkyl group in an R group can be methylene, ethylene, propylene, and etc. Exemplary Y groups include, without limitation, alkoxyl such as OCH3, OC2H5, and OC2H4OCH3. For example, the organosilane can be amino-propyl-trimethoxysilane, mercapto-propyl- trimethoxysilane, carboxyl-propyl-trimethoxysilane, amino-propyl-triethoxysilane, mercapto-propyl-triethoxysilane, carboxyl-propyl-triethoxysilane, bis-[3- (triethoxysilyl) propyl]- tetrasulfide, bis-[3-(triethoxysilyl) propyl]- disulfide, aminopropyltriethoxysilane, N-2-(aminoethyl)-3 -amino propyltrimethoxysilane, vinyltrimethoxysilane, vinyl-tris(2-methoxyethoxy)silane, 3- methacryloxypropyltrimethoxy silane, 2-(3, 4-epoxy cy cl ohexy)-ethyl
trimethoxy silane, 3 -glycidoxy-propyltri ethoxy silane, 3- isocyanatopropyltriethoxysilane, and 3- cyanatopropyltriethoxy silane.
[0075] In certain embodiments, the silanization coating contains one or more functional groups within or on the surface of the coating. The functional groups may be introduced during the formation of the coating during the cross-linking process, for example, by adding silicon-containing compounds containing such functional groups during the cross-linking, in particular, during the ending stage of the cross-linking process. The functional groups may also be introduced after the formation of the cross-linking product, for example, by introducing functional groups to the surface of the cross-linking product by chemical modification. In certain embodiments, the functional groups are inherent in the coating.
[0076] The functional groups serve as linkage between the nanocomposition and the analyte, e.g., ctDNA to be captured. Examples of the functional groups include, but are not limited to amino, mercapto, carboxyl, phosphonate, biotin, streptavidin, avidin, hydroxyl, alkyl or other hydrophobic molecules, polyethylene glycol or other hydrophilic molecules, and photo cleavable, thermo cleavable or pH responsive linkers.
[0077] In certain embodiment, the silanization coating does not contain a carboxyl functional group. In certain embodiments, the nanocomposition comprising a silanization coating that does not contain a carboxyl group demonstrates a higher binding efficiency in capturing ctDNA.
[0078] In some embodiments, the nanocomposition provided herein can be colored or non-colored. "Colored" as used herein, means that the bead is capable of generating a color signal under a suitable condition. For example, the colored nanostructure may emit a fluorescent color signal upon excitation with a light of a certain wavelength. The nanostructures may alternatively be non-colored. A non- colored nanostructure does not emit a color signal when subject to a condition that would otherwise induce a color signal for a colored nanostructure.
[0079] In certain embodiments, the nanocomposition is bar-coded or associated with a detectable agent to show color. The term "bar-coding" or "bar- coded" means that the nanostructure is associated with a known code or a known label that allows identification of the nanostructure. "Code" as used herein, refers to a molecule capable of generating a detectable signal that distinguishes one bar-coded bead from another. For example, the colored bead may comprise a colored nanoparticle (e.g. a quantum dot) which emits a detectable color signal at a known wave length.
[0080] In certain embodiments, the characteristics or the identity of a bar- coded nanocomposition is based on multiplexed optical coding system as disclosed in Han et al, Nature Biotechnology, Vol. 19, pp: 631-635 (2001) or US Pat. Appl.
10/185, 226. Briefly, multicolor semiconductor quantum-dots (QDs) are embedded in the bead. For each QD, there is a given intensity (within the levels of, for example 0- 10) and a given color (wavelength). For each single-color coding, the
nanocomposition has different intensity of QDs depending on the number of QDs embedded therein. If QDs of multiple colors (n colors) and multiple intensity (m levels of intensity) are used, then the bead may have a total number of unique identities or codes, which is equal to m to the exponent of n less one (m11 1). In addition, since the porous structure can be associated with additional payloads (e.g., fluorescent organic molecules), if there are Y number of additional fluorescent colors available, the total number of code can be Yx (m11 1).
[0081] In certain embodiments, the nanocomposition (with or without bar coding) is colored by being operably linked to a detectable agent. A detectable agent can be a fluorescent molecule, a chemo-luminescent molecule, a bio-luminescent molecule, a radioisotope, a MRI contrast agent, a CT contrast agent, an enzyme- substrate label, and/or a coloring agent etc.
[0082] Examples of fluorescent molecules include, without limitation, fluorescent compounds (fluorophores) which can include, but are not limited to: 1,5 IAEDANS; l,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofiuorescein; 5- Carboxyfluorescein (5-FAM); 5 -Carboxynaptho fluorescein; 5- Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5 -Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X- rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6- JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7- Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin
(Photoprotein); AFPs— AutoFluorescent Protein— (Quantum Biotechnologies); Alexa® Fluor 350; Alexa® Fluor 405; Alexa® Fluor 500; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA- S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D;
Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine;
ATTO- TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G;
Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; Bimane; Bisbenzamide;
Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; Blancophor SV; BOBO™-l; BOBO™-3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy
576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy FI; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP;
Bodipy TR-X SE; BO-PRO™- 1; BO- PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green- 1 Ca 2+ Dye; Calcium Green-2 Ca 2+ ; Calcium Green-5N Ca 2+ ; Calcium Green-Cl8 Ca 2+ ; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5- ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer);
CFDA; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA;
Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3. l 8; Cy3.5™; Cy3™; Cy5. l 8; Cy5.5™; Cy5™; Cy7™; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3' DCFDA; DCFH (Dichlorodihydro fluorescein Diacetate); DDAO; DHR (Dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-l6- ASP); Dichlorodihydro fluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD (DiICl8(5)); DIDS; Dihydrorhodamine 123 (DHR); Dil (DiICl8(3)); Dinitrophenol; DiO (DiOCl8(3)); DiR; DiR (DiICl8(7)); DM-NERF (high pH); DNP; Dopamine; DTAF; DY-630-NHS; DY-635-NHS; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer- 1 (EthD-l); Euchrysin; EukoLight;
Europium (III) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4;
Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; FluoroGold
(Hydroxy stilbamidine); Fluor-Ruby; Fluor X; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF;
GeneBlazer (CCF2); Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxy coumarin;
Hydroxy stilbamidine (FluoroGold); Hydroxytryptamine; Indo-l, high calcium; Indo- 1, low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-l; JO-JO- 1; JO-PRO- 1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-l; LO-PRO-l; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue -White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-l; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin; Mitotracker Green FM; Mitotracker Orange;
Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr- GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine;Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE- TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL;
Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-l ; POPO-3; PO— PRO-l ; PO-PRO-3; Primuline; Procion Yellow; Propidium lodid (PI); PYMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed] ; Resorufm; RH 414; Rhod-2; Rhodamine; Rhodamine 1 10; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB;
RhodamineBG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phy cocyanine; R-phycoerythrin (PE); S65A; S65C; S65L; S65T; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-l ; SNAFL-2; SNARF calcein; SNARF 1 ; Sodium Green; SpectrumAqua;
Spectrum Green; Spectrum Orange; Spectrum Red; SPQ (6-m ethoxy -N-(3- sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine Extra; SYTO 1 1 ; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21 ; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41 ; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61 ; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81 ; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange;
Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thio flavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofiuor White); TMR; TO-PRO-l ; TO-PRO-3 ; TO-PRO-5; TOTO-l ; TOTO-3; Tricolor (PE- Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue;
TruRed; Ultralite; ETranine B; Uvitex SFC; WW 781 ; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; YO-PRO-l ; YO-PRO-3; YOYO-l ; YOYO-3, Sybr Green, Thiazole orange (interchelating dyes), fluorescent semiconductor
nanostructures, lanthanides or combinations thereof.
[0083] Examples of radioisotopes include, 123I, 124I, 125I, 1311, 35S, 3H, U 1ln,
112In, 14C, 64Cu, 67Cu, 86Y, 88Y, 90Y, 177Lu, 211At, 186Re, 188Re, 153Sm, 212Bi, 32P, 18F, 201Tl, 67Ga, 137Cs and other radioisotopes.
[0084] Examples of enzyme-substrate labels include, luciferases (e.g., firefly luciferase and bacterial luciferase), luciferin, 2,3-dihydrophthalazinedionesm, alate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
[0085] In some embodiments, the nanocomposition is surface modified with a functional group, an oligo, a nucleic acid, a peptide, a protein, a charge, a
hydrophobic group, a hydrophilic group, a polymer, a small organic molecule, a ligand, a receptor, a guest molecule, a host molecule, a sugar molecule, a lipid, a signaling molecule, a detergent, a guest molecule, a host molecule, a nanoparticle, a bead, and any combination thereof.
[0086] B. Analyte-associating member
[0087] The nanocomposition provided herein comprises an analyte- associating member operably linked to the nanoparticle.
[0088] In certain embodiments, the analyte-associating members are molecules capable of specifically associated with an analyte, such as ctDNA or CTC. The term“specifically associated with” as used herein, means that a non-random binding interaction between two molecules or non-random non-covalent association between two molecules. The specific binding can be characterized by binding affinity (Kd), which is calculated as the ratio of dissociation rate to association rate (k0ff/kon) when the binding between the two molecules reaches equilibrium. The dissociation rate (koe) measured at the binding equilibrium may also be used when measurement of kon is difficult to obtain, for example, due to aggregation of one molecule. The antigen-binding affinity (e.g. Kd or k0ff) can be appropriately determined using suitable methods known in the art, including, for example, Biacore (see, for example, Murphy, M. et al, Current protocols in protein science, Chapter 19, unit 19.14, 2006) and Kinexa techniques (see, for example, Darling, R. I, et al, Assay Drug Dev.
Technok, 2(6): 647-657 (2004)).
[0089] Examples of analyte-associating members include but not limited to
Protein A; Protein G, antigen-binding members (e.g., antibodies or fragments thereof), protein such as streptavidin or avidin, peptide, a nucleic acid (or a fragment of nuclei acid, an oligo nucleotide), an oligodeoxynucleotide, an antibody or fragments thereof, a polymer, a small organic molecule such as a drug, a charge, a functional group (e.g., hydroxyl, carboxyl, phosphonate, epoxyl, amino, methyl, ethyl), a ligand (e.g., a peptide, small molecule, hormone, a drug, toxin,
neurotransmitter), a receptor, a guest chemical (e.g., prostaglandin , itraconazole), a host chemical (e.g. cyclodextrins, calixarenes, cucurbiturils, porphyrins ,
metallacrowns, crown ethers, zeolites, cyclotriveratrylenes, cryptophanes and carcerands), an analyte-binding bead capable of binding to an analyte.
[0090] Protein A is an affinity ligand for an antibody having an
immunoglobulin Fc domain, and can be useful in purification of antibodies that are based on human gammal, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Similarly, protein G is recommended for specific binding to antibodies of all mouse isotypes and for antibodies based on
human .gamma.3 heavy chains (Guss et al., EMBO J. 5: 1567 1575 (1986)). Avidin (or streptavdin) and biotin can specifically bind to each other to form strong and specific non-covalent association. An antigen binding member can be an antibody, an antibody fragment or an antibody memetics, such as, for example, scFV, Fab, Fab’, Fv, single domain antibody, diabody, nanobody, domain antibody, dsFv, or canelized antibody. The antibodies or fragments can be polyclonal, monoclonal, of animal origin (e.g., murine, rabbit, camel), of human origin (e.g., fully human), chimeric, humanized, variable regions, CDRs, ScFv, bispecific, diabody, or other forms of antibodies with antigen-binding capabilities. In certain embodiments, the antibodies or antigen-binding fraction thereof specifically binds to a biomarker or biomarkers such as a specific antigen of a cancer cell of interest.
[0091] In certain embodiments, the analyte-associating member is an antibody specifically binding to a surface marker of the CTC. In some embodiments, the surface marker is selected from the group consisting of Her2, CD19, CD20, PD-L1, Mena, EpCAM, EGFR, N-Cadherin, vimentin, CD44, CD133, Trop-2, ER, PR and a combination thereof.
[0092] In certain embodiments, the analyte-associating member may be covalently linked or non-covalently linked to the nanoparticle, directly, or indirectly. In certain embodiments, the analyte-associating member is linked to the nanoparticle via a linker.
Biomarkers
[0093] One of the advantages of the blood test described herein is that a number of biomarkers from various sources, including proteins, ctDNA and CTC, can be detected in a single test, thus providing comprehensive and more accurate prediction of the cancer incidence/recurrence and guidance for cancer treatment. [0094] In certain embodiments, the biomarker detected from ctDNA is a mutation, deletion, amplification, rearrangement or fusion of a gene selected from the group consisting of BRCA1, BRCA2, BIRC5, MMP9, MCM6, MELK, LIN9, UBE2C, UBE2T, EGFR, RPM2, PGR, ORC6L, ERBB2, ERBB3, KRAS, TERT, APC, ARID 1 A, KMT2D, RB1, KMT2C, NF1, FAT1, BRAF, NOTCH1, KDM6A, KIT, CTNNB1, BAP1, IDH1, GNAS, VHL, ESR1, NTRK1, NTRK2, NTRK3, Trop- 2, ROS1, MET, AA555029 RC, AKT1 , AKT2, AKT3, ABL1, ACVRIB,
ALDH4A1, AP2B1, AYTL2, BBC3, Cl6orf6l, C20orf46, C9orf30, CCNE2, CDC42BPA, CDCA7, CENPA, COL4A2, COLAA2, DCK, DIAPH3, DTL, EBF4, ECT2, EGLN1, ESM1, EXT1, ALOX12B, AMER1, APC, AR, ARAF, ARFRP1, ARID 1 A, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, AZGP1, BAP1, BARD1, BCL2, BCL2L1, BCL2L2, BCL6, BIRC5, ALK, BCOR, BCORL1, BRD4, BRIP1, BTG1, BTG2, BTK, Cl lorf30, CALR, CALM2, CARD11, CASP8, CBFB, CBL, CCND1, CCND2, BRAF, CDH1, CHEK2, CDK4, CDK6, CDK8, CDKN1A, CDKN2A, CDKN2B, CDKN2C, CDK12, CEBPA, CHEK1, CUL3, CUL4A, CXCR4, CYP17A1, DAXX, DDR1, DDR2, DIS3, DHCR7, DNMT3A, DOTIL, EED, EP300, EPHA3, EPHB1, EPHB4, ERCC4, ERG, ERRFI1, ESR1, EZH2, FAM46C, FANCA, FANCC, FANCG, FANCL, FAS, FBXW7, FGF10, FGF12, FGF14, FGF18, FGF19, FGF23, FGF3, FGF4, FGFR1, FGFR2, FGFR3, FGFR4, FH, FLCN, FLT1, FLT3, FOXL2, FUBP1, GABRA6, GAT A3, GATA4, GATA6, GID4, GMPS, GNAZ, GRP126, GPR180, GSTM3, HRASLS, IGFBP5, JHDM1D, GNA11, GNA13, GNAQ, GNAS, GRM3, GSK3B, H3F3A, HBB, HDAC1, HGF, HNF1A, HRAS, HSD3B1, ID3, IDH1, IDH2, IGF1R, IKBKE, IKZF1, IL6ST, INPP4B, IRF2, IRF4, IRS2, JAK1, JAK2, JAK3, JUN, KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIT, KLHL6, KMT2A, KMT2D, KNTC2, LGP2, LIN9, LOC100288906, LOC730018, MCM6, MELK, MMP9, LTK, LYN, MAF, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K13, MAPK1, MCL1, MDM2, MDM4, MED 12, MEF2B, MEN1, MERTK, MET, MGP, MITF, MKNK1, MLH1, MPL, MRE11A, MS4A7, MSH2, MSH3, MSH6, MST1R, MTAP, MTH, NMU, MTOR, MUTYH, MYC, MYCL, MYCN, MYD88, NBN, NF1, NF2, NFE2L2, NFKB1A, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NPM1, NUSAP1, ORC6L, OXCT1, NRAS, NT5C2, OAZ1, P2RY8, PALB2, PALM2, PECI, PITRM1, PR1, QSCN6L1, PARK2, PARP1, PARP2, PARP3, PAXS, PBRM1, PDCD1, PDCD1LG2, PDGFRA, PDGFRB, PDK1, PIK3C2B, PIK3C2G, PIK3CB, PIK3RI, PIMI, PMS2, POLD1, POLE, PPARG, PPP2R1A, PPP2R2A, PRDM1, PPKAR1A, PPKC1, PTCH1, PTPN11, PTPRO, QK1, RAB6B, RASSF7, RECQL5, RFC4, RTN4RL1, RUNDC1, SCUBE2, RAC1, RAD21, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RAF1, RARA, RBBP8, RBM10, RPL37A, REL, RRET, RICTOR, RNF43, RPTOR, SDHA, SDHB, SDHC, SDHD, SERF 1 A, SLC3A3, STK32B, SETD2, SF3B1, SGK1, SMAD2, SMAD4, SMARCA4,
SMARCB1, SNCAIP, SOCS1, SOX2, SOX9, SPEN, SPOP, SRC, STAG2, STAT3, STC2, STK11, SUFU, SYK, TBX3, TEK, TET2, TGFBR2, TGFB3, TSPYL5,
TIP ARP, TNFAIP3, TNFRSF14, TSC1, TSC2, TYR03, U2AF1, VEGFA, VHL, WHSC1, WHSCIL1, WT1, XPOl, XRCC2, ZNF217, ZNF703, ALK, BCL@, BCR, CD74, ETV4, ETV5, ETV6, EWSR1, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MSH2, MYB, MYC, NUTM1, PDGFRA, RAF1, RARA, RET, RSP02, SDC4, SLC34A2, TERC, TERT, TMPRSS2, DDR2, FGFR2, FGFR3, HRAS, KIT,
MAP2K1, MAP2K2, MET, MTOR, NRAS, PDGFRA, RET, SMO, UBE2C,
UCHL5, WISP1, ZNF533, PALM2, RASSF7, PR1, OXCT1, MCM6, MS4A7, RPL37A, TP53, STC2, UBE2C, EST1, ATM, NBV, NF1, PALB2, PTEN, RAD50, AZGP1, BIRC5, PALB2, PIK3CA, CDC20, CD19, ATRX, Ki67, MUC-l, MUC-7, EX01, ESR1, CeP55, Cyclin Bl, GRB7, Stromelysin 3, Cathepsin L2, GRP-7, RB-l, Ki-67, STK15, Survivin, Cyclin Bl, MyBL2, ER, PR, Cathepsin L2, GRB7, BCL2, SCUBE2, GSTM1, CD68, ACTR3B, ANLN, BAG1, BCL2, BIRC5, BLVRA, CCNB1, CCNE1, CDC20, CDC6, CDH3, CENPF, CEP55, CXXC5, EX01, FGFR4, FOXA1, FOXC1, GPR160, GRB7, KIF2C, KRT14, KRT17, KRT5, MAPT, MDM2, MELK, MIA, MKI67, MLPH, MMPl l, MVBL2, MYC, NAT1, NDC80, NUF2, ORC6L, PGR, PHGDH, PTTG1, RRM2, SFRP1, SLC39A6, TMEM45B, TYMS, UBE2C, UBE2T, and a combination thereof.
[0095] In certain embodiments, the biomarker from ctDNA can be detected using an amplification assay, a hybridization assay, a sequencing assay or an array.
[0096] In certain embodiments, the biomarker detected from CTC is a mutation, deletion, amplification, rearrangement or fusion of a gene selected from the group consisting of BRCA1, BRCA2, BIRC5, MMP9, MCM6, MELK, LIN9, UBE2C, UBE2T, EGFR, RPM2, PGR, ORC6L, ERBB2, ERBB3, KRAS, TERT, APC, ARID 1 A, KMT2D, RB1, KMT2C, NF1, FAT1, BRAF, NOTCH1, KDM6A, KIT, CTNNB1, BAP1, IDH1, GNAS, VHL, ESR1, NTRK1, NTRK2, NTRK3, Trop- 2, ROS1, MET, AA555029 RC, AKT1 , AKT2, AKT3, ABL1, ACVRIB,
ALDH4A1, AP2B1, AYTL2, BBC3, Cl6orf6l, C20orf46, C9orf30, CCNE2, CDC42BPA, CDCA7, CENPA, COL4A2, COLAA2, DCK, DIAPH3, DTL, EBF4, ECT2, EGLN1, ESM1, EXT1, ALOX12B, AMER1, APC, AR, ARAF, ARFRP1, ARID 1 A, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, AZGP1, BAP1, BARD1, BCL2, BCL2L1, BCL2L2, BCL6, BIRC5, ALK, BCOR, BCORL1, BRD4, BRIP1, BTG1, BTG2, BTK, Cl lorf30, CALR, CALM2, CARD11, CASP8, CBFB, CBL, CCND1, CCND2, BRAF, CDH1, CHEK2, CDK4, CDK6, CDK8, CDKN1A, CDKN2A, CDKN2B, CDKN2C, CDK12, CEBPA, CHEK1, CUL3, CUL4A, CXCR4, CYP17A1, DAXX, DDR1, DDR2, DIS3, DHCR7, DNMT3A, DOTIL, EED, EP300, EPHA3, EPHB1, EPHB4, ERCC4, ERG, ERRFI1, ESR1, ESR2, EZH2, FAM46C, FANCA, FANCC, FANCG, FANCL, FAS, FBXW7, FGF10, FGF12, FGF14, FGF18, FGF19, FGF23, FGF3, FGF4, FGFR1, FGFR2, FGFR3, FGFR4, FH, FLCN, FLT1, FLT3, FOXL2, FUBP1, GABRA6, GAT A3, GATA4, GATA6, GID4, GMPS, GNAZ, GRP126, GPR180, GSTM3, HRASLS, IGFBP5, JHDM1D, GNA11, GNA13, GNAQ, GNAS, GRM3, GSK3B, H3F3A, HBB, HDAC1, HGF, HNF1A, HRAS, HSD3B1, ID3, IDH1, IDH2, IGF1R, IKBKE, IKZF1, IL6ST, INPP4B, IRF2, IRF4, IRS2, JAK1, JAK2, JAK3, JUN, KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIT, KLHL6, KMT2A, KMT2D, KNTC2, LGP2, LIN9, LOC100288906, LOC730018, MCM6, MELK, MMP9, LTK, LYN, MAF, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K13, MAPK1, MCL1, MDM2, MDM4, MED 12, MEF2B, MEN1, MERTK, MET, MGP, MITF, MKNK1, MLH1, MPL, MRE11A, MS4A7, MSH2, MSH3, MSH6, MST1R, MTAP, MTH, NMU, MTOR, MUTYH, MYC, MYCL, MYCN, MYD88, NBN, NF1, NF2, NFE2L2, NFKB1A, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NPM1, NUSAP1, ORC6L, OXCT1, NRAS, NT5C2, OAZ1, P2RY8, PALB2, PALM2, PECI, PITRM1, PR1, QSCN6L1, PARK2, PARP1, PARP2, PARP3, PAXS, PBRM1, PDCD1, PDCD1LG2, PDGFRA, PDGFRB, PDK1, PIK3C2B, PIK3C2G, PIK3CB, PIK3RI, PIMI, PMS2, POLD1, POLE, PPARG, PPP2R1A, PPP2R2A, PRDM1, PPKAR1A, PPKC1, PTCH1, PTPN11, PTPRO, QK1, RAB6B, RASSF7, RECQL5, RFC4, RTN4RL1, RUNDC1, SCUBE2, RAC1, RAD21, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RAF1, RARA, RBBP8, RBM10, RPL37A, REL, RRET, RICTOR, RNF43, RPTOR, SDHA, SDHB, SDHC, SDHD, SERF 1 A, SLC3A3, STK32B, SETD2, SF3B1, SGK1, SMAD2, SMAD4, SMARCA4,
SMARCB1, SNCAIP, SOCS1, SOX2, SOX9, SPEN, SPOP, SRC, STAG2, STAT3, STC2, STK11, SUFU, SYK, TBX3, TEK, TET2, TGFBR2, TGFB3, TSPYL5,
TIP ARP, TNFAIP3, TNFRSF14, TSC1, TSC2, TYR03, U2AF1, VEGFA, VHL, WHSC1, WHSCIL1, WT1, XPOl, XRCC2, ZNF217, ZNF703, ALK, BCL@, BCR, CD74, ETV4, ETV5, ETV6, EWSR1, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MSH2, MYB, MYC, NUTM1, PDGFRA, RAF1, RARA, RET, RSP02, SDC4, SLC34A2, TERC, TERT, TMPRSS2, DDR2, FGFR2, FGFR3, HRAS, KIT,
MAP2K1, MAP2K2, MET, MTOR, NRAS, PDGFRA, RET, SMO, UBE2C,
UCHL5, WISP1, ZNF533, PALM2, RASSF7, PR1, OXCT1, MCM6, MS4A7, RPL37A, TP53, STC2, UBE2C, EST1, ATM, NBV, NF1, PALB2, PTEN, RAD50, AZGP1, BIRC5, PALB2, PIK3CA, CDC20, CD19, ATRX, Ki67, MUC-l, MUC-7, EX01, ESR1, CeP55, Cyclin Bl, GRB7, Stromelysin 3, Cathepsin L2, GRP-7, RB-l, Ki-67, STK15, Survivin, Cyclin Bl, MyBL2, Stromelysin 3, ER, PR, Cathepsin L2, GRB7, BCL2, SCUBE2, GSTM1, CD68, ACTR3B, ANLN, BAG1, BCL2, BIRC5, BLVRA, CCNB1, CCNE1, CDC20, CDC6, CDH3, CENPF, CEP55, CXXC5, EX01, FGFR4, FOXA1, FOXC1, GPR160, GRB7, KIF2C, KRT14, KRT17, KRT5, MAPT, MDM2, MELK, MIA, MKI67, MLPH, MMPl l, MVBL2, MYC, NAT1, NDC80, NUF2, ORC6L, PGR, PHGDH, PTTG1, RRM2, SFRP1, SLC39A6, TMEM45B, TYMS, UBE2C, UBE2T, and a combination thereof.
[0097] In certain embodiments, the biomarker detected from CTC is the expression level, i.e., mRNA level, of a gene selected from CA15-3, CA27.29, CEA, ER, PR, HER2, uPA, PAI-l, TP53, Cathepsin D, Cyclin E, Cathepsin D, P53, Ki67, CK19, ALDH1, EGFR, Nestin, MSP-alpha, TIMP-4, PDGFR-alpha, OPG, AFP, 5- HIAA, CA19-9, CA27.29, CA72-4, hCGbeta, Calcitonin, CgA, ER, PR, Osteocalcin, Epithelial Membrane Antigen (EMA), AlphavBeta3 Integrin, AlphavBeta6 Integrin, Transferrin Receptor, Transthyretin, Alkaline Phosphatase, PSA, Lactate
dehydrogenase, Neuron Specific Enolase, Nuclear matrix protein 22, Plasminogen Activator Inhibitor, SCC, Apolipoprotein Al, Beta-2-microglobulin, Cyfra2l-l, HE4, Ferritin, Fibrinogen, Fibrin D-dimer, S100, TP A, Thyroglobulin, Aldehyde dehydrogenase, CD20, CD24, CD44, CD278, IL-2, IL-6, IL-10, IL-12, IL-15, Interferon Gamma, CA125, PD-l and PD-L1, Folate Receptor and a combination thereof. [0098] In certain embodiments, the gene expression level is detected using an amplification assay, a hybridization assay, a sequencing assay or an array.
[0099] In certain embodiments, the biomarker detected from CTC is the protein level or mRNA level of a gene selected from the group consisting of CA15-3, CA27.29, CEA, ER, PR, HER2, uPA, PAI-l, TP53, Cathepsin D, Cyclin E, Cathepsin D, P53, Ki67, CK19, ALDH1, EGFR, Nestin, MSP-alpha, TIMP-4, PDGFR-alpha, OPG, AFP, 5 -HI A A, CA19-9, CA27.29, CA72-4, hCGbeta, Calcitonin, CgA, ER,
PR, Osteocalcin, Epithelial Membrane Antigen (EMA), AlphavBeta3 Integrin, AlphavBeta6 Integrin, Transferrin Receptor, Transthyretin, Alkaline Phosphatase, PSA, Lactate dehydrogenase, Neuron Specific Enolase, Nuclear matrix protein 22, Plasminogen Activator Inhibitor, SCC, Apolipoprotein Al, Beta-2 -microglobulin, Cyfra2l-l, HE4, Ferritin, Fibrinogen, Fibrin D-dimer, S100, TP A, Thyroglobulin, Aldehyde dehydrogenase, CD20, CD24, CD44, CD278, IL-2, IL-6, IL-10, IL-12, IL- 15, Interferon Gamma, CA125, PD-l and PD-L1, Folate Receptor and a combination thereof.
[00100] In certain embodiments, the protein level is detected using a nanocomposition having a fluorescent label that barcodes the CTC biomarker. In certain embodiments, the mRNA level is detected using an immunohistochemistry (IHC) assay or a fluorescence in situ hybridization (FISH) assay.
Prediction and Machine-Learning Method
[00101] After the information of the biomarkers is collected, the test disclosed herein, in some embodiments, includes determining a likelihood of the patient having the cancer or having recurrence of the cancer. In certain embodiments, the likelihood can be determined based on models using machine learning techniques.
[00102] As used herein,“machine learning” refers to a computer-implemented technique that gives computer systems the ability to progressively improve performance on a specific task with data, i.e., to learn from the data, without being explicitly programmed. Machine learning technique adopts algorithms that can learn from and make prediction on data through building a model, i.e., a description of a system using mathematical concepts, from sample inputs. A core objective of machine learning is to generalize from the experience, i.e., to perform accurately on new data after having experienced a learning data set. In the context of biomedical diagnosis or prognosis, machine learning techniques generally involves supervised learning process, in which the computer is presented with example inputs (e.g., signature of gene expression) and their desired outputs (e.g., responsiveness) to learn a general rule that maps inputs to outputs. Different models, i.e., hypothesis, can be employed in the generalization process. For the best performance in the
generalization, the complexity of the hypothesis should match the complexity of the function underlying the data.
[00103] As used in the test disclosed herein, the machine learning technique can build a model based on the information of a panel of biomarkers and the cancer incidence/recurrence or responsiveness to a treatment that has been observed or confirmed, for example, information based on doctors’ experience and diagnostic results and patient outcome, such that the model can accurately predict the cancer incident/recurrence or the responsiveness to a treatment for future data.
[00104] In some embodiments, the machine learning technique used in the test includes, without limitations, partial least square (Wold S et al., PLS for Multivariate Linear Modeling, In H van de Waterbeemd (ed.), Chemometric Methods in Molecular Design, pp. 195-218. VCH, Weinheim), elastic net (Zou H et al., Regularization and Variable Selection via the Elastic Net, Journal of the Royal Statistical Society, Series B (2005) 67(2): 301-320), support vector machine (Vapnik V), random forest (Breiman L), neural net (Bishop C, Neural Networks for Pattern Recognition (1995) Oxford University Press, Oxford) and gradient boosting machine (Friedman J, Greedy Function Approximation: A Gradient Boosting Machine, Annals of Statistics (2001) 29(5), 1189-1232).
[00105] In some embodiments, the data of biomarkers information, prediction of cancer incidence/recurrence and recommendation of treatment is uploaded to a cloud data server for storage and for matching with patient outcome, and ultimately be used to predict patient survival and treatment selection through machine learning algorithm.
[00106] As a result, the test described herein may be totally or partially performed with a computer system including one or more processors, which can be configured to perform the steps. Thus, embodiments are directed to computer systems configured to perform the steps of any of the test described herein, potentially with different components performing a respective step or a respective group of steps. Although presented as numbered steps, steps of test herein can be performed at a same time or in a different order. Additionally, portions of these steps may be used with portions of other steps from other test or methods. Also, all or portions of a step may be optional. Any of the steps of any of the tests can be performed with modules, circuits, or other means for performing these steps.
[00107] Any of the computer systems mentioned herein may utilize any suitable number of subsystems. In some embodiments, a computer system includes a single computer apparatus, where the subsystems can be the components of the computer apparatus. In other embodiments, a computer system can include multiple computer apparatuses, each being a subsystem, with internal components. The subsystems can be interconnected via a system bus. Additional subsystems include, for examples, a printer, keyboard, storage device(s), monitor, which is coupled to display adapter, and others. Peripherals and input/output (I/O) devices, which couple to I/O controller, can be connected to the computer system by any number of means known in the art, such as serial port. For example, serial port or external interface (e.g. Ethernet, Wi-Fi, etc.) can be used to connect computer system to a wide area network such as the Internet, a mouse input device, or a scanner. The interconnection via system bus allows the central processor to communicate with each subsystem and to control the execution of instructions from system memory or the storage device(s) (e.g., a fixed disk, such as a hard drive or optical disk), as well as the exchange of information between subsystems. The system memory and/or the storage device(s) may embody a computer readable medium. Any of the data mentioned herein can be output from one component to another component and can be output to the user.
[00108] A computer system can include a plurality of the same components or subsystems, e.g., connected together by external interface or by an internal interface. In some embodiments, computer systems, subsystem, or apparatuses can
communicate over a network. In such instances, one computer can be considered a client and another computer a server, where each can be part of a same computer system. A client and a server can each include multiple systems, subsystems, or components.
[00109] It should be understood that any of the embodiments of the present disclosure can be implemented in the form of control logic using hardware (e.g., an application specific integrated circuit or field programmable gate array) and/or using computer software with a generally programmable processor in a modular or integrated manner. As used herein, a processor includes a multi-core processor on a same integrated chip, or multiple processing units on a single circuit board or networked. Based on the disclosure and teachings provided herein, a person of ordinary skill in the art will know and appreciate other ways and/or methods to implement embodiments of the present disclosure using hardware and a combination of hardware and software.
[00110] Any of the software components or functions described in this application may be implemented as software code to be executed by a processor using any suitable computer language such as, for example, Java, Python, C++ or Perl using, for example, conventional or object- oriented techniques. The software code may be stored as a series of instructions or commands on a computer readable medium for storage and/or transmission, suitable media include random access memory (RAM), a read only memory (ROM), a magnetic medium such as a hard- drive or a floppy disk, or an optical medium such as a compact disk (CD) or DVD (digital versatile disk), flash memory, a cloud based storage server, and the like. The computer readable medium may be any combination of such storage or transmission devices.
[00111] Such programs may also be encoded and transmitted using carrier signals adapted for transmission via wired, optical, and/or wireless networks conforming to a variety of protocols, including the Internet and cloud server. As such, a computer readable medium according to an embodiment of the present invention may be created using a data signal encoded with such programs. Computer readable media encoded with the program code may be packaged with a compatible device or provided separately from other devices (e.g., via Internet download). Any such computer readable medium may reside on or within a single computer product (e.g. a hard drive, a CD, or an entire computer system), and may be present on or within different computer products within a system or network. A computer system may include a monitor, printer, or other suitable display for providing any of the results mentioned herein to a user.
[00112] In some embodiments, the computer-implemented technique can improve the prediction of cancer incidence/recurrence or the guidance of treatment intervention from a specific testing data of a cancer patient using all the previously measured data from the testing technology and any other data upload onto the Data Server for machine learning purpose;
[00113] In some embodiments, the machine learning technique can build a model based on the information of a panel of biomarkers (cancer profiling) and the cancer incidence/recurrence or responsiveness to a treatment that has been observed or confirmed, for example, information based on doctors’ experience and diagnostic results and patient outcome, such that the model can accurately predict the cancer incident/recurrence or the responsiveness to a treatment for future data.
[00114] In some embodiments, the cancer profiling information obtained from the method described herein can predict at the earliest time possible for cancer recurrence and guide the most effective personalized therapy to improve patient outcome compared to other technologies on market.
[00115] In some embodiments, the method described herein can change traditional cancer treatment scheme by turning fatal cancers into manageable diseases because of the earliest possible timing in predict cancer incidence/recurrence and the most effective personalized treatment intervention guided by the testing technology.
Examples
[00116] Example 1
[00117] An exemplary workflow of the method in an example of the present application is illustrated in Fig. 2.
[00118] The blood sample was centrifuged to separate plasma. The plasma was further centrifuged. The bottom layer was kept for further step. The upper layer plasma was treated with lysis buffer. The nanocomposition of the present application such as Mag Vigen™ or MyQu Vigen™ beads (generated based on low-density, highly porous nanostructure) was added to the treated plasma in a reaction tube and incubated at 65°C. The reaction tube was put on a magnetic rack to pellet the beads until the solution is clear. The supernatant was slowly removed. The beads were washed with wash buffer for several times and the supernatants during the washing were removed. A desired amount of elution buffer was added to the beads in the tube to re-disperse the beads completely. The tube was put on a magnetic stand for 2-3 minutes. The supernatant was then carefully collected from the bottom of the tube without disturbing the beads. The supernatant contains the extracted cell free DNA.
[00119] The bottom layer obtained by centrifugation of the blood sample was added with Ficoll® and then the mixture was centrifuged to separate plasma and huffy coat, and red cell layers. The huffy coat layer was transferred to another new tube, and RBC lysis buffer was added to lyse RBC. After washing, cell pellet was re- suspended in PBS with 0.2% BSA and 4mM EDTA, followed by addition of Mag Vigen™ or MyQuVigen™-Anti-EpCAM beads in buffer and incubation for 1 hr. After incubation, the beads with captured cells were separated from the solution by a magnet, and then washed. After washing, the captured cells were redispersed in PBS buffer and a little drop was counted under a fluorescence microscope. CTC nucleic acids such as genomic DNA or RNA expression profile could also be analyzed.
[00120] Table 1 shows the results of cfDNA extraction in EDTA or Streck tubes using the same plasma sample but different methods, i.e., the method of the present application, Qiagen method and MagMax method. The methods were compared in terms of Picogreen Reading, Truseq Nano library construction, Kapa library qPCR quantification, NGS WGS reads, genomic sequencing coverage and GC content.
[00121] Table 1
Figure imgf000041_0001
[00122] From Table 1, it can be seen that the extraction method of the present application outperforms Qiagen method and MagMax method.
[00123] Fig. 3 shows the CTC capture using the method of the present application as stated above and other products on market. The data show 100 spiking- in H1650 cell capture from whole blood using antibody conjugated Streptavidin (SA) beads (Mag Vigen™ SA Bead of the present application and other SA beads). It is clear that the beads of the present application show much higher capture yield than other beads on market.
[00124] The insert of Fig. 3 illustrates the CTC isolated from a whole blood sample using another exemplary nanocomposition of the present application
MyQuVigen™-SA fluorescent magnetic nanoparticles (emission: 635 nm). It can be seen that the tumor cell surface markers were stained with these fluorescent magnetic nanoparticles through binding with antibodies conjugated to the nanoparticle surfaces. The exemplary fluorescent magnetic nanoparticles enable simultaneous CTC capture and phenotyping. Such workflow could dramatically save assay time, and gain multiplexed CTC surface marker information to correlate with further CTC nucleic acid analysis for more accurate diagnostic information.
[00125] Figure 4 shows lOOx better detection sensitivity of ctDNA using the exemplary nanoparticles for cfDNA extraction combined with MagVigen-streptavidin beads for target specific nucleic acid capture and enrichment compared to when the target enrichment are performed with other on market magnetic beads-streptvidin.
The MagVigen-streptavidin beads target capture enabled low copy number gene mutation detection at 10 copies while other on-market magnetic beads only presented a real signal at 1000 copy experiments not able to achieve rare mutation detection.
[00126] Figure 5 shows that using the exemplary nanoparticle for combination of cfDNA extraction and target enrichment enabled a much better differentiation of patient vs. normal people samples, with the Ct difference about 9 reflecting 5l2x difference of mutant copy number; while enrichment with other on market magnetic beads only gave a Ct difference of less than 2 or less than 4x mutant copy number difference. This also manifested an over lOOx difference in mutant copies to differentiate patient vs. normal people samples. Such better differentiating power in a cancer diagnostics with our technology allows less amplification cycles so less chance for false positive signal due to more PCR amplification cycles for more accurate cancer molecular diagnostics.
[00127] The blood test described herein allows a number of biomarkers from various sources, such as proteins, ctDNAs and CTCs, to be detected in a single test. This provides comprehensive and more accurate prediction of the cancer
incidence/recurrence and guidance for cancer treatment.
[00128] The foregoing description is considered as illustrative only of the principles of the present disclosure. Further, since numerous modifications and changes will be readily apparent to those skilled in the art, it is not desired to limit the invention to the exact construction and process shown as described above.
Accordingly, all suitable modifications and equivalents may be considered to fall within the scope of the invention as defined by the claims that follow.

Claims

WHAT IS CLAIMED IS:
1. A method comprising:
(1) obtaining a blood sample from a subject having or suspect of having a cancer;
(2) capturing from the blood sample
(2a) a circulating tumor DNA (ctDNA) using a first
nanocomposition, and
(2b) a circulating tumor cell (CTC) using a second
nanocomposition, wherein the second nanocomposition comprises an antibody/protein/peptide/or other binding ligands specifically binding to a surface marker of the CTC; and
(3) detecting
(3a) from the ctDNA, at least a first nucleic acid biomarker,
(3b) from the CTC, at least a CTC biomarker,
(3c) from the CTC, at least a second nucleic acid biomarker.
2. The method of claim 1, further comprising the step of detecting the number of
CTC or the quantity of ctDNA.
3. The method of claim 1, further comprising the step of
(4) determining, based on information obtained in the detecting step, a likelihood of the subject having a cancer or a recurrence of a cancer.
4. The method of claim 1, wherein the cancer is selected from the group consisting of a breast cancer, a lung cancer, a prostate cancer, a colorectal cancer, an ovarian cancer, a liver cancer, a glioma, a melanoma, and a pancreatic cancer.
5. The method of claim 1, wherein the subject is or has been treated with a hormone therapy, a chemotherapy, a targeted therapy or an immunotherapy.
6. The claim of claim 1, wherein the blood sample has been fractionated before the capturing step.
7. The claim of claim 7, wherein the ctDNA is captured from plasma and the CTC is captured from huffy coat.
8. The method of claim 1, wherein the first nucleic acid biomarker is a mutation, insertion, deletion, amplification, rearrangement or fusion of a gene selected from the group consisting of BRCA1, BRCA2, BIRC5, MMP9, MCM6, MELK, LIN9, UBE2C, UBE2T, EGFR, RPM2, PGR, ORC6L, ERBB2, ERBB3, KRAS, TERT, APC, ARID 1 A, KMT2D, RB1, KMT2C, NF1, FAT1, BRAF, NOTCH1, KDM6A, KIT, CTNNB1, BAP1, IDH1, GNAS, VHL, ESR1, NTRK1, NTRK2, NTRK3, Trop- 2, ROS1, MET, AA555029 RC, AKT1 , AKT2, AKT3, ABL1, ACVRIB,
ALDH4A1, AP2B1, AYTL2, BBC3, Cl6orf6l, C20orf46, C9orf30, CCNE2, CDC42BPA, CDCA7, CENPA, COL4A2, COLAA2, DCK, DIAPH3, DTL, EBF4, ECT2, EGLN1, ESM1, EXT1, ALOX12B, AMER1, APC, AR, ARAF, ARFRP1, ARID 1 A, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, AZGP1, BAP1, BARD1, BCL2, BCL2L1, BCL2L2, BCL6, BIRC5, ALK, BCOR, BCORL1, BRD4, BRIP1, BTG1, BTG2, BTK, Cl lorfiO, CALR, CALM2, CARD11, CASP8, CBFB, CBL, CCND1, CCND2, BRAF, CDH1, CHEK2, CDK4, CDK6, CDK8, CDKN1A, CDKN2A, CDKN2B, CDKN2C, CDK12, CEBPA, CHEK1, CUL3, CUL4A, CXCR4, CYP17A1, DAXX, DDR1, DDR2, DIS3, DHCR7, DNMT3A, DOTIL, EED, EP300, EPHA3, EPHB1, EPHB4, ERCC4, ERG, ERRFI1, ESR1, ESR2, EZH2, FAM46C, FANCA, FANCC, FANCG, FANCL, FAS, FBXW7, FGF10, FGF12, FGF14, FGF18, FGF19, FGF23, FGF3, FGF4, FGFR1, FGFR2, FGFR3, FGFR4, FH, FLCN, FLT1, FLT3, FOXL2, FUBP1, GABRA6, GAT A3, GATA4, GATA6, GID4, GMPS, GNAZ, GRP126, GPR180, GSTM3, HRASLS, IGFBP5, JHDM1D, GNA11, GNA13, GNAQ, GNAS, GRM3, GSK3B, H3F3A, HBB, HDAC1, HGF, HNF1A, HRAS, HSD3B1, ID3, IDH1, IDH2, IGF1R, IKBKE, IKZF1, IL6ST, INPP4B, IRF2, IRF4, IRS2, JAK1, JAK2, JAK3, JUN, KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIT, KLHL6, KMT2A, KMT2D, KNTC2, LGP2, LIN9, LOC100288906, LOC730018, MCM6, MELK, MMP9, LTK, LYN, MAF, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K13, MAPK1, MCL1, MDM2, MDM4, MED 12, MEF2B, MEN1, MERTK, MET, MGP, MITF, MKNK1, MLH1, MPL, MRE11A, MS4A7, MSH2, MSH3, MSH6, MST1R, MTAP, MTH, NMU, MTOR, MUTYH, MYC, MYCL, MYCN, MYD88, NBN, NF1, NF2, NFE2L2, NFKB1A, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NPM1, NUSAP1, ORC6L, OXCT1, NRAS, NT5C2, OAZ1, P2RY8, PALB2, PALM2, PECI, PITRM1, PR1, QSCN6L1, PARK2, PARP1, PARP2, PARP3, PAXS, PBRM1, PDCD1, PDCD1LG2, PDGFRA, PDGFRB, PDK1, PIK3C2B, PIK3C2G, PIK3CB, PIK3RI, PIMI, PMS2, POLD1, POLE, PPARG, PPP2R1A, PPP2R2A, PRDM1, PPKAR1A, PPKC1, PTCH1, PTPN11, PTPRO, QK1, RAB6B, RASSF7, RECQL5, RFC4, RTN4RL1, RUNDC1, SCUBE2, RAC1, RAD21, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RAF1, RARA, RBBP8, RBM10, RPL37A, REL, RRET, RICTOR, RNF43, RPTOR, SDHA, SDHB, SDHC, SDHD, SERF 1 A, SLC3A3, STK32B, SETD2, SF3B1, SGK1, SMAD2, SMAD4, SMARCA4,
SMARCB1, SNCAIP, SOCS1, SOX2, SOX9, SPEN, SPOP, SRC, STAG2, STAT3, STC2, STK11, SUFU, SYK, TBX3, TEK, TET2, TGFBR2, TGFB3, TSPYL5,
TIP ARP, TNFAIP3, TNFRSF14, TSC1, TSC2, TYR03, U2AF1, VEGFA, VHL, WHSC1, WHSCIL1, WT1, XPOl, XRCC2, ZNF217, ZNF703, ALK, BCL@, BCR, CD74, ETV4, ETV5, ETV6, EWSR1, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MSH2, MYB, MYC, NUTM1, PDGFRA, RAF1, RARA, RET, RSP02, SDC4, SLC34A2, TERC, TERT, TMPRSS2, DDR2, FGFR2, FGFR3, HRAS, KIT,
MAP2K1, MAP2K2, MET, MTOR, NRAS, PDGFRA, RET, SMO, UBE2C,
UCHL5, WISP1, ZNF533, PALM2, RASSF7, PR1, OXCT1, MCM6, MS4A7, RPL37A, TP53, STC2, UBE2C, EST1, ATM, NBV, NF1, PALB2, PTEN, RAD50, AZGP1, BIRC5, PALB2, PIK3CA, CDC20, CD19, ATRX, Ki67, MUC-l, MUC-7, EX01, ESR1, CeP55, Cyclin Bl, GRB7, Stromelysin 3, Cathepsin L2, GRP-7, RB-l, Ki-67, STK15, Survivin, Cyclin Bl, MyBL2, Stromelysin 3, ER, PR, Cathepsin L2, GRB7, BCL2, SCUBE2, GSTM1, CD68, ACTR3B, ANLN, BAG1, BCL2, BIRC5, BLVRA, CCNB1, CCNE1, CDC20, CDC6, CDH3, CENPF, CEP55, CXXC5, EX01, FGFR4, FOXA1, FOXC1, GPR160, GRB7, KIF2C, KRT14, KRT17, KRT5, MAPT, MDM2, MELK, MIA, MKI67, MLPH, MMPl l, MVBL2, MYC, NAT1, NDC80, NUF2, ORC6L, PGR, PHGDH, PTTG1, RRM2, SFRP1, SLC39A6, TMEM45B, TYMS, UBE2C, UBE2T, and a combination thereof.
9. The claim of claim 1, wherein the first nucleic acid biomarker is detected using an amplification assay, a hybridization assay, a sequencing assay or an array.
10. The claim of claim 1, wherein the surface marker is selected from the group consisting of Her2, CD19, CD20, CD22, BCMA, GD2, NY-ESO-l, EBV,
Mesothelin, CD33, CD30, CD123, PSMA, WT1, GPC3, CD38, EGFRvIII, MUC-l, PDL1, ALK, MAGE- A3, NKG2D, RoRl, SLAMF7, CD 138, CD171, Mena, EpCAM, EGFR, N-Cadherin, E-Cadherin, Vimentin, CD44, CD 133, CD 146, CD278, Trop-2 and a combination thereof.
11. The claim of claim 1, wherein the second nucleic acid biomarker is a mutation, deletion, amplification, rearrangement or fusion of a gene selected from the group consisting of BRCA1, BRCA2, BIRC5, MMP9, MCM6, MELK, LIN9, UBE2C, UBE2T, EGFR, RPM2, PGR, ORC6L, ERBB2, ERBB3, KRAS, TERT, APC, ARID 1 A, KMT2D, RB1, KMT2C, NF1, FAT1, BRAF, NOTCH1, KDM6A, KIT, CTNNB1, BAP1, IDH1, GNAS, VHL, ESR1, NTRK1, NTRK2, NTRK3, Trop- 2, ROS1, MET, AA555029 RC, AKT1 , AKT2, AKT3, ABL1, ACVRIB,
ALDH4A1, AP2B1, AYTL2, BBC3, Cl6orf6l, C20orf46, C9orf30, CCNE2, CDC42BPA, CDCA7, CENPA, COL4A2, COLAA2, DCK, DIAPH3, DTL, EBF4, ECT2, EGLN1, ESM1, EXT1, ALOX12B, AMER1, APC, AR, ARAF, ARFRP1, ARID 1 A, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, AZGP1, BAP1, BARD1, BCL2, BCL2L1, BCL2L2, BCL6, BIRC5, ALK, BCOR, BCORL1, BRD4, BRIP1, BTG1, BTG2, BTK, Cl lorfiO, CALR, CALM2, CARD11, CASP8, CBFB, CBL, CCND1, CCND2, BRAF, CDH1, CHEK2, CDK4, CDK6, CDK8, CDKN1A, CDKN2A, CDKN2B, CDKN2C, CDK12, CEBPA, CHEK1, CUL3, CUL4A, CXCR4, CYP17A1, DAXX, DDR1, DDR2, DIS3, DHCR7, DNMT3A, DOTIL, EED, EP300, EPHA3, EPHB1, EPHB4, ERCC4, ERG, ERRFI1, ESR1, ESR2, EZH2, FAM46C, FANCA, FANCC, FANCG, FANCL, FAS, FBXW7, FGF10, FGF12, FGF14, FGF18, FGF19, FGF23, FGF3, FGF4, FGFR1, FGFR2, FGFR3, FGFR4, FH, FLCN, FLT1, FLT3, FOXL2, FUBP1, GABRA6, GAT A3, GATA4, GATA6, GID4, GMPS, GNAZ, GRP126, GPR180, GSTM3, HRASLS, IGFBP5, JHDM1D, GNA11, GNA13, GNAQ, GNAS, GRM3, GSK3B, H3F3A, HBB, HDAC1, HGF, HNF1A, HRAS, HSD3B1, ID3, IDH1, IDH2, IGF1R, IKBKE, IKZF1, IL6ST, INPP4B, IRF2, IRF4, IRS2, JAK1, JAK2, JAK3, JUN, KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIT, KLHL6, KMT2A, KMT2D, KNTC2, LGP2, LIN9, LOC100288906, LOC730018, MCM6, MELK, MMP9, LTK, LYN, MAF, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K13, MAPK1, MCL1, MDM2, MDM4, MED 12, MEF2B, MEN1, MERTK, MET, MGP, MITF, MKNK1, MLH1, MPL, MRE11A, MS4A7, MSH2, MSH3, MSH6, MST1R, MTAP, MTH, NMU, MTOR, MUTYH, MYC, MYCL, MYCN, MYD88, NBN, NF1, NF2,
NFE2L2, NFKB1A, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NPM1, NUSAP1, ORC6L, OXCT1, NRAS, NT5C2, OAZ1, P2RY8, PALB2, PALM2, PECI, PITRM1, PR1, QSCN6L1, PARK2, PARP1, PARP2, PARP3, PAXS, PBRM1, PDCD1, PDCD1LG2, PDGFRA, PDGFRB, PDK1, PIK3C2B, PIK3C2G, PIK3CB, PIK3RI, PI3K-delta, PI3K-gamma, PIMI, PMS2, POLD1, POLE, PPARG, PPP2R1A, PPP2R2A, PRDM1, PPKAR1A, PPKC1, PTCH1, PTPN11, PTPRO, QK1, RAB6B, RASSF7, RECQL5, RFC4, RTN4RL1, RUNDC1, SCUBE2, RAC1, RAD21,
RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RAF1, RARA, RBBP8, RBM10, RPL37A, REL, RRET, RICTOR, RNF43, RPTOR, SDHA, SDHB, SDHC, SDHD, SERF 1 A, SLC3A3, STK32B, SETD2, SF3B1, SGK1, SMAD2, SMAD4, SMARCA4, SMARCB1, SNCAIP, SOCS1, SOX2, SOX9, SPEN, SPOP, SRC, STAG2, STAT3, STC2, STK11, SUFU, SYK, TBX3, TEK, TET2, TGFBR2, TGFB3, TSPYL5, TIP ARP, TNFAIP3, TNFRSF14, TSC1, TSC2, TYR03, U2AF1, VEGFA, VHL, WHSC1, WHSCIL1, WT1, XPOl, XRCC2, ZNF217, ZNF703, ALK, BCL@, BCR, CD74, ETV4, ETV5, ETV6, EWSR1, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MSH2, MYB, MYC, NUTM1, PDGFRA, RAF1, RARA, RET, RSP02, SDC4, SLC34A2, TERC, TERT, TMPRSS2, DDR2, FGFR2, FGFR3, HRAS, KIT, MAP2K1, MAP2K2, MET, MTOR, NRAS, PDGFRA, RET, SMO, UBE2C, UCHL5, WISP1, ZNF533, PALM2, RASSF7, PR1, OXCT1, MCM6, MS4A7, RPL37A, TP53, STC2, UBE2C, EST1, ATM, NBV, NF1, PALB2, PTEN, RAD50, AZGP1, BIRC5, PALB2, PIK3CA, CDC20, CD19, ATRX, Ki67, MUC-l, MUC-7, EX01, ESR1, CeP55, Cyclin Bl, GRB7, Stromelysin 3, Cathepsin L2, GRP- 7, RB-l, Ki-67, STK15, Survivin, Cyclin Bl, MyBL2, Stromelysin 3, ER, PR, Cathepsin L2, GRB7, BCL2, SCUBE2, GSTM1, CD68, ACTR3B, ANLN, BAG1, BCL2, BIRC5, BLVRA, CCNB1, CCNE1, CDC20, CDC6, CDH3, CENPF, CEP55, CXXC5, EX01, FGFR4, FOXA1, FOXC1, GPR160, GRB7, KIF2C, KRT14, KRT17, KRT5, MAPT, MDM2, MELK, MIA, MKI67, MLPH, MMPl l, MVBL2, MYC, NAT1, NDC80, NUF2, ORC6L, PGR, PHGDH, PTTG1, RRM2, SFRP1, SLC39A6, TMEM45B, TYMS, UBE2C, UBE2T, and a combination thereof,
12. The method of claim 1, wherein the second nucleic acid biomarker is expression level of a gene selected from CA15-3, CA27.29, CEA, ER, PR, HER2, uPA, PAI-l, TP53, Cathepsin D, Cyclin E, Cathepsin D, P53, Ki67, CK19, ALDH1, EGFR, Nestin, MSP-alpha, TIMP-4, PDGFR-alpha, OPG, AFP, 5-HIAA, CA19-9, CA27.29, CA72-4, hCGbeta, Calcitonin, CgA, ER, PR, Osteocalcin, Epithelial Membrane Antigen (EMA), AlphavBeta3 Integrin, ASGPR, HSA, snail, twist, slug, ZEB1, ZEB2, OV6, OV1, CK19, c-Kit, Thy-l, CD90, CD44, ALDH, CD13, ABCG2, eNOS, vWF, AlphavBeta6 Integrin, Transferrin Receptor, Transthyretin, Alkaline Phosphatase, PSA, Lactate dehydrogenase, Neuron Specific Enolase, Nuclear matrix protein 22, Plasminogen Activator Inhibitor, SCC, Apolipoprotein Al, Beta-2 - microglobulin, Cyfra2l-l, HE4, Ferritin, Fibrinogen, Fibrin D-dimer, S100, TP A, Thyroglobulin, Aldehyde dehydrogenase, CD20, CD24, CD44, CD278, IL-2, IL-6, IL-10, IL-12, IL-15, Interferon Gamma, CA125, PD-l and PD-L1, Folate Receptor and a combination thereof.
13. The method of claim 1, wherein the second nucleic acid biomarker is detected using an amplification assay, a hybridization assay, a sequencing assay or an array.
14. The method of claim 1, wherein the CTC biomarker is selected from the group consisting of Her2, CD19, CD20, BCMA, GD2, NY-ESO-l, EBV, CD278,
Mesothelin, CD33, CD22, CD30, CD123, CD278, PSMA, WT1, GPC3, CD38, EGFRvIII, MUC-l, ALK, MAGE- A3, NKG2D, RoRl, SLAMF7, CD 138, CD171, PD-L1, Mena, EpCAM, EGFR, N-Cadherin, E-Cadherin, Vimentin, CD44, CD 133, CD 146, Trop-2, ER, PR, HE4, CK19, AIDF11, ASGPR, HSA, snail, twist, slug,
ZEB1, ZEB2, OV6, OV1, CK19, c-Kit, Thy-l, CD90, CD44, ALDH, CD13, ABCG2, eNOS, vWF, PAI-l, uPA, CA27.29, CEA, Cyclin E, Cathepsin D, Nestin, P53, Folate receptor and a combination thereof.
15. The method of claim 1, wherein the CTC biomarker is a protein marker or nucleic acid marker.
16. The method of claim 1, wherein the CTC biomarker is detected using a third nanocomposition comprising a fluorescent label that barcodes the CTC biomarker.
17. The method of claim 1, wherein the CTC biomarker is detected using an immunohistochemistry (IHC) assay, a fluorescence in situ hybridization (FISH) assay or a sequence-based assay.
18. The method of claim 1, further comprising detecting a circulating protein in the blood sample.
19. The method of claim 1, wherein the circulating protein is selected from the group consisting of CA15-3, CA27.29, CEA, ER, PR, HER2, uPA, PAI-l, TP53, Cathepsin D, Cyclin E, Nestin, MSP-alpha, TIMP-4, PDGFR-alpha, OPG, AFP, 5- HIAA, CA19-9, CA27.29, CA72-4, hCGbeta, ASGPR, HSA, snail, twist, slug, ZEB1, ZEB2, OV6, OV1, CK19, c-Kit, Thy-l, CD90, CD44, ALDH, CD13, ABCG2, eNOS, vWF, Calcitonin, CgA, ER, PR, Osteocalcin, Epithelial Membrane Antigen (EMA), AlphavBeta3 Integrin, AlphavBeta6 Integrin, Transferrin Receptor, Transthyretin, Alkaline Phosphatase, PSA, Lactate dehydrogenase, Neuron Specific Enolase,
Nuclear matrix protein 22, Plasminogen Activator Inhibitor, SCC, Apolipoprotein Al, Beta-2 -microglobulin, Cyfra2l-l, HE4, Ferritin, Fibrinogen, Fibrin D-dimer, S100,
TP A, Thyroglobulin, Aldehyde dehydrogenase, CD20, CD24, CD44, CD278, folate receptor, IL-2, IL-6, IL-10, IL-12, IL-15, Interferon Gamma, CA125, HE4, PD-l and PD-L1 and a combination thereof.
20. The method of claim 1, wherein the first or the second nanocomposition comprising a superparamagnetic iron oxide (SPIO) nanoprticle.
21. The method of claim 1, wherein the nanocomposition comprises a silanization coating.
22. The method of claim 21, wherein the silanization coating forms a low density, porous 3-D structure.
23. The method of claim 1, further comprising the step of
(5) recommending, based on information obtained in the detecting step, a treatment for the subject.
24. The method of claim 23, wherein the treatment is selected from the group consisting of endocrine therapy, fulvestrant, Tamoxifen, Toremifene, Aromatase inhibitors, Megestrol acetate, Her-2 targeted therapy such as trastuzumab, ado- trastuzumab-emtansine, Pertuzumab, Lepatinib, and neratinib, Everolimus,
Larotrectinib, Everolimus, Fulvestrant, Robocliclib, palbociclib, ADC, Atezolizumab, Avetumab, Durvalumb, Olaparib, EGFR- targeted therapy, such as afatinib, gefitinib, ertotinib, and Osimertinib, ALK- targeted therapy such as alectinib, crizotinib, and ceritinib, BRAF -targeted therapy such as dabrafenib in combination with trametinib, or vemurafenib, trametinib, cobimetinib in combination with Zelboraf, KRAS- targeted therapy such as cetuximab and panitumumab, BRCA1/2 - targeted therapy such as rucaparib, 5-Fu, MTX, Doxorubicin, Etoposide, Alkylating agents, Cisplatin Doxorubicin, Bevacizumab, Ramucirumab, Erlotinib, Afatinib, Gefitinib,
Osimertinib, Osimertinib, Necitumumab, Crizotinib, Ceritinib, Alectinib, Brigatinib, Dabrafenib, Trametinib.
25. The method of claim 1, further comprising the step of
(6) monitoring, based on information obtained in the detecting step, the treatment response for the subject.
26. The method of claim 25, wherein the monitoring step further provides prognosis or prediction information, and further guide treatment intervention.
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