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US20140031250A1 - Biomarkers of Cancer - Google Patents

Biomarkers of Cancer Download PDF

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US20140031250A1
US20140031250A1 US13/877,373 US201113877373A US2014031250A1 US 20140031250 A1 US20140031250 A1 US 20140031250A1 US 201113877373 A US201113877373 A US 201113877373A US 2014031250 A1 US2014031250 A1 US 2014031250A1
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cancer
satellite
level
value
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David Tsai Ting
Daniel A. Haber
Shyamala Maheswaran
Doron Lipson
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General Hospital Corp
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Assigned to THE GENERAL HOSPITAL CORPORATION reassignment THE GENERAL HOSPITAL CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HABER, DANIEL A.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • This invention relates to methods of diagnosing cancer, based on detecting the presence of increased levels of expression of satellite repeats and/or Line-1.
  • heterochromatin is comprised of centric (minor) and pericentric (major) satellite repeats that are required for formation of the mitotic spindle complex and faithful chromosome segregation (M. Guenatri, D. Bailly, C. Maison, G. Almouzni, J Cell Biol 166, 493 (Aug.
  • Kanellopoulou et al. Genes Dev 19, 489 (Feb. 15, 2005); T. Fukagawa et al., Nat Cell Biol 6, 784 (August, 2004)) and from DNA demethylation, heat shock, or the induction of apoptosis (H. Bouzinba-Segard, A. Guais, C. Francastel, Proc Natl Acad Sci USA 103, 8709 (Jun. 6, 2006); R. Valgardsdottir et al., Nucleic Acids Res 36, 423 (February, 2008)).
  • the present invention is based, at least in part, on the identification of massive expression of satellite repeats in tumor cells, and of increased levels of Line-1, e.g., in tumor cells including circulating tumor cells (CTCs).
  • CTCs circulating tumor cells
  • Described herein are methods for diagnosing cancer, e.g., solid malignancies of epithelial origin such as pancreatic, lung, breast, prostate, renal, ovarian or colon cancer, based on the presence of increased levels of expression of satellite repeats and/or Line-1.
  • the invention provides methods, e.g., in vitro methods, for detecting the presence of cancer in a subject, including determining a level of LINE-1 in a sample from the subject to obtain a test value; and comparing the test value to a reference value, wherein a test value compared to the reference value indicates whether the subject has cancer.
  • the reference value represents a threshold level of LINE-1, wherein the presence of a level of LINE-1 in the subject that is above the reference value indicates that the subject has cancer, and the presence of a level of LINE-1 in the subject that is below the reference value indicates that the subject is unlikely to have cancer.
  • the invention provides methods, e.g., in vitro methods, for detecting the presence of cancer in a subject, including determining a level of satellite transcripts in a sample from the subject to obtain a test value; and comparing the test value to a reference value, wherein a test value compared to the reference value indicates whether the subject has cancer.
  • the satellite transcripts comprise one or more of ALR, HSATII, GSATII, TAR1, and SST1.
  • the satellite transcript is ALR and/or HSATII, and the presence of a level of ALR and/or HSATII satellite transcripts above the reference level indicates that the subject has a tumor.
  • the satellite transcript is GSATII, TAR1 and/or SST1, and the presence of a level of GSATII, TAR1 and/or SST1 satellite transcripts below the reference level indicates that subject has a tumor.
  • the invention provides methods, e.g., in vitro methods, for evaluating the efficacy of a treatment for cancer in a subject.
  • the methods include determining a level of LINE-1 in a first sample from the subject to obtain a first value; administering a treatment for cancer to the subject; determining a level of LINE-1 in a subsequent sample obtained from the subject at a later time, to obtain a treatment value; and comparing the first value to the treatment value.
  • a treatment value that is below the first value indicates that the treatment is effective.
  • the invention provides methods, e.g., in vitro methods, for evaluating the efficacy of a treatment for cancer in a subject.
  • the methods include determining a level of satellite transcripts in a first sample from the subject to obtain a first value; administering a treatment for cancer to the subject; determining a level of satellite transcripts in a subsequent sample obtained from the subject at a later time, to obtain a treatment value; and comparing the first value to the treatment value, wherein a treatment value that is below the first value indicates that the treatment is effective.
  • the satellite transcripts comprise one or more of ALR, HSATII, GSATII, TAR1, and SST1.
  • the first and second samples are known or suspected to comprise tumor cells, e.g., blood samples known or suspected of comprising circulating tumor cells (CTCs), or biopsy samples known or suspected of comprising tumor cells.
  • the sample comprises free RNA in serum or RNA within exosomes in blood.
  • the treatment includes administration of a surgical intervention, chemotherapy, radiation therapy, or a combination thereof.
  • the subject is a mammal, e.g., a human or veterinary subject, e.g., experimental animal.
  • the cancer is a solid tumor of epithelial origin, e.g., pancreatic, lung, breast, prostate, renal, ovarian or colon cancer.
  • the methods described herein include measuring a level of LINE-1 transcript.
  • the level of a LINE-1 transcript or satellite is determined using a branched DNA assay.
  • FIG. 1B is a graphical representation of sequence read contributions from major satellite among all primary tumors, cancer cell lines, and normal tissues.
  • FIG. 2A shows the results of Northern blot analysis of three KrasG12D, Tp53lox/+ pancreatic primary tumors (Tumors 1-3) and a stable cell line (CL3) derived from Tumor 3.
  • FIG. 2B shows the results of Northern blot analysis of CL3 before (0) and after (+) treatment with the DNA hypomethylating agent 5-azacitadine (AZA).
  • FIG. 2C shows the results of Northern blot analysis of total RNA from multiple adult and fetal mouse tissues. All Northern blots exposed for approximately 30 minutes.
  • FIG. 2D is a pair of photomicrographs showing the results of RNA in-situ hybridization (ISH) of normal pancreas (left) and primary pancreatic ductal adenocarcinoma (right), hybridized with a 1 kb major satellite repeat probe.
  • ISH RNA in-situ hybridization
  • FIG. 2E is a set of three photomicrographs showing the results of ISH analysis of preneoplastic PanIN (P) lesion, adjacent to PDAC (T) and normal pancreas (N), showing positive staining in PanIN, with increased expression in full carcinoma. Higher magnification (40 ⁇ ) of PanIN (left) and PDAC (right) lesions.
  • FIG. 3A is a bar graph showing the Total satellite expression in human pancreatic ductal adenocarcinoma (PDAC), normal pancreas, other cancers (L—lung, K—kidney, O—ovary, P—prostate), and other normal human tissues (1—fetal brain, 2—brain, 3—colon, 4—fetal liver, 5—liver, 6—lung, 7—kidney, 8—placenta, 9—prostate, and 10—uterus) quantitated by DGE. Satellite expression is shown as transcripts per million aligned to human genome.
  • PDAC pancreatic ductal adenocarcinoma
  • FIG. 4A shows the results of multiple linear correlation analysis of major satellite to other cellular transcripts among all mouse tumors and normal tissues as depicted by a heat map.
  • X-axis is samples ordered by expression of major satellite and y-axis is genes ordered by linear correlation to major satellite expression.
  • Light grey (High) and dark grey (Low) color is log 2 (reads per million).
  • FIG. 4B is a dot graph showing the Median distance of transcriptional start sites of all genes to Line-1 elements ordered by linearity to satellite expression (Dark gray; highest linearity to the left) or by random (Light gray). Plotted by genes binned in 100 s.
  • FIG. 4C is a dot graph showing Top genes with highest linearity (R>0.85) defining satellite correlated genes or SCGs plotted by frequency against distance of transcriptional start site to LINE-1 elements (Dark gray) compared to the expected frequency of these genes (Light gray).
  • FIG. 4D is a set of four photomicrographs showing the results of immunohistochemistry of mouse PDAC (KrasG12D, Tp53 lox/+) for the neuroendocrine marker chromogranin A. Tumors are depicted as a function of increasing chromogranin A staining (dark grey), with the relative level of major satellite expression noted for each tumor at the bottom of each image (percentage of all transcripts)
  • FIG. 5 is a bar graph indicating fold change expression of the indicated genes in CTC Device vs. control device.
  • the subjects were newly diagnosed metastatic pancreatic adenocarcinoma patients. LINE-1 expression was seen in all patients at some point.
  • FIG. 6B is an image of RNA in situ hybridization of HSATII using Affymetrix ViewRNA of a potential human pancreatic circulating tumor cell captured on the HB-chip.
  • HSATII lightest areas; yellow in original
  • DAPI nuclear stain medium grey areas, blue in original.
  • Scale bar 20 ⁇ m.
  • the present invention is based, at least in part, on the identification of a massive generation of LINE-1 protein and bidirectional ncRNAs from the major satellite repeat in mouse tumor models and from ALR and HSATII satellite repeats in human cancers.
  • the exceptional magnitude of satellite levels in these cancers is unprecedented. This is likely to result from a general derepression of chromosomal marks affecting both satellites and LINE-1 retrotransposons, with proximity to LINE-1 activation potentially affecting the expression of selected cellular mRNAs. Together, the very high expression of satellites may affect chromosomal integrity and genetic stability, while the co-deregulated coding sequences may affect cell fates and biological behavior of cancer cells.
  • the methods described herein can be used to diagnose the presence of, and monitor the efficacy of a treatment for, cancer, e.g., solid tumors of epithelial origin, e.g., pancreatic, lung, breast, prostate, renal, ovarian or colon cancer, in a subject.
  • cancer e.g., solid tumors of epithelial origin, e.g., pancreatic, lung, breast, prostate, renal, ovarian or colon cancer
  • hyperproliferative refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.
  • Hyperproliferative disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state.
  • pathologic i.e., characterizing or constituting a disease state
  • non-pathologic i.e., a deviation from normal but not associated with a disease state.
  • the term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • a “tumor” is an abnormal growth of hyperproliferative cells.
  • Cancer refers to pathologic disease states, e.g., characterized by malignant tumor growth.
  • cancer e.g., solid tumors of epithelial origin, e.g., as defined by the ICD-O (International Classification of Diseases-Oncology) code (revision 3), section (8010-8790), e.g., early stage cancer
  • ICD-O International Classification of Diseases-Oncology
  • section (8010-8790) e.g., early stage cancer
  • the methods can include the detection of expression levels of satellite repeats in a sample comprising cells known or suspected of being tumor cells, e.g., cells from solid tumors of epithelial origin, e.g., pancreatic, lung, breast, prostate, renal, ovarian or colon cancer cells.
  • the methods can include the detection of increased levels of LINE-1 in a sample, e.g., a sample known or suspected of including tumor cells, e.g., circulating tumor cells (CTCs), e.g., using a microfluidic device as described herein.
  • CTCs circulating tumor cells
  • Cancers of epithelial origin can include pancreatic cancer (e.g., pancreatic adenocarcinoma or intraductal papillary mucinous carcinoma (IPMN, pancreatic mass)), lung cancer (e.g., non-small cell lung cancer), prostate cancer, breast cancer, renal cancer, ovarian cancer, or colon cancer.
  • pancreatic cancer e.g., pancreatic adenocarcinoma or intraductal papillary mucinous carcinoma (IPMN, pancreatic mass)
  • lung cancer e.g., non-small cell lung cancer
  • prostate cancer e.g., breast cancer, renal cancer, ovarian cancer, or colon cancer.
  • the present methods can be used to distinguish between benign IPMN, for which surveillance is the standard treatment, and malignant IPMN, which require resection, a procedure associated with significant morbidity and a small but significant possibility of death.
  • the methods described herein can be used for surveillance/monitoring of the subject, e.g., the methods can be repeated at selected intervals (e.g., every 3, 6, 12, or 24 months) to determine whether a benign IPMN has become a malignant IPMN warranting surgical intervention.
  • the methods can be used to distinguish bronchioloalveolar carcinomas from reactive processes (e.g., postpneumonic reactive processes) in samples from subjects suspected of having non-small cell lung cancer.
  • the methods in a sample from a subject who is suspected of having breast cancer, can be used to distinguish ductal hyperplasia from atypical ductal hyperplasia and ductal carcinoma in situ (DCIS).
  • DCIS ductal carcinoma in situ
  • the methods can be used to distinguish between atypical small acinar proliferation and malignant cancer.
  • the methods in subjects suspected of having bladder cancer, can be used to detect, e.g., transitional cell carcinoma (TCC), e.g., in urine specimens.
  • TCC transitional cell carcinoma
  • subjects diagnosed with Barrett's Esophagus in subjects diagnosed with Barrett's Esophagus (Sharma, N Engl J Med.
  • the methods can be used for distinguishing dysplasia in Barrett's esophagus from a reactive process.
  • the clinical implications are significant, as a diagnosis of dysplasia demands a therapeutic intervention.
  • Other embodiments include, but are not limited to, diagnosis of well differentiated hepatocellular carcinoma, ampullary and bile duct carcinoma, glioma vs. reactive gliosis, melanoma vs. dermal nevus, low grade sarcoma, and pancreatic endocrine tumors, inter alia.
  • the methods include obtaining a sample from a subject, and evaluating the presence and/or level of LINE-1 or satellites in the sample, and comparing the presence and/or level with one or more references, e.g., a control reference that represents a normal level of LINE-1 or satellites, e.g., a level in an unaffected subject or a normal cell from the same subject, and/or a disease reference that represents a level of LINE-1 or satellites associated with cancer, e.g., a level in a subject having pancreatic, lung, breast, prostate, renal, ovarian or colon cancer.
  • a control reference that represents a normal level of LINE-1 or satellites, e.g., a level in an unaffected subject or a normal cell from the same subject
  • a disease reference that represents a level of LINE-1 or satellites associated with cancer, e.g., a level in a subject having pancreatic, lung, breast, prostate, renal, ovarian or colon cancer.
  • the present methods can also be used to determine the stage of a cancer, e.g., whether a sample includes cells that are from a precancerous lesion, an early stage tumor, or an advanced tumor. For example, the present methods can be used to determine whether a subject has a precancerous pancreatic, breast, or prostate lesion.
  • the markers used are LINE-1, or satellite transcript ALR and/or HSATII
  • increasing levels are correlated with advancing stage.
  • satellite transcripts GSATII, TAR1 and/or SST1 decreasing levels are correlated with increasing stage.
  • levels of LINE-1 and satellite ALR and/or HSATII may be prognostic and predictive to clinical outcomes.
  • the sample is or includes blood, serum, and/or plasma, or a portion or subfraction thereof, e.g., free RNA in serum or RNA within exosomes in blood.
  • the sample comprises (or is suspected of comprising) CTCs.
  • the sample is or includes urine or a portion or subfraction thereof.
  • the sample includes known or suspected tumor cells, e.g., is a biopsy sample, e.g., a fine needle aspirate (FNA), endoscopic biopsy, or core needle biopsy; in some embodiments the sample comprises cells from the pancreatic, lung, breast, prostate, renal, ovarian or colon of the subject.
  • FNA fine needle aspirate
  • the sample comprises lung cells obtained from a sputum sample or from the lung of the subject by brushing, washing, bronchoscopic biopsy, transbronchial biopsy, or FNA, e.g., bronchoscopic, fluoroscopic, or CT-guided FNA (such methods can also be used to obtain samples from other tissues as well).
  • FNA fluoroscopic, or CT-guided FNA
  • the sample is frozen, fixed and/or permeabilized, e.g., is an formalin-fixed paraffin-embedded (FFPE) sample.
  • FFPE formalin-fixed paraffin-embedded
  • the level of satellite transcripts is detected, e.g., in a sample known or suspected to include tumor cells. In some embodiments, the level of satellite transcripts in a known or suspected tumor cell, e.g., a test cell, is compared to a reference level.
  • the methods include detecting levels of alpha (ALR) satellite transcripts (D. Lipson et al., Nat Biotechnol 27, 652 (July, 2009)) or HSATII satellite transcripts (J. Jurka et al., Cytogenet Genome Res 110, 462 (2005)); in some embodiments, those levels are compared to a reference.
  • ALR alpha
  • the reference level is a level of ALR and/or HSATII satellite transcripts in a normal (non-cancerous) cell, e.g., a normal cell from the same subject, or a reference level determined from a cohort of normal cells; the presence of levels of ALR and/or HSATII in the test cell above those in the normal cell indicate that the test cell is a tumor cell (e.g., the subject from whom the test cell came has or can be diagnosed with cancer).
  • a normal (non-cancerous) cell e.g., a normal cell from the same subject, or a reference level determined from a cohort of normal cells
  • the presence of levels of ALR and/or HSATII in the test cell above those in the normal cell indicate that the test cell is a tumor cell (e.g., the subject from whom the test cell came has or can be diagnosed with cancer).
  • the reference level of ALR and/or HSATII transcripts is a threshold level, and the presence of a level of ALR and/or HSATII satellite transcripts above the threshold level indicates that the cell is a tumor cell (e.g., the subject from whom the test cell came has or can be diagnosed with cancer).
  • the methods include detecting levels of GSATII, TAR1 and/or SST1 transcripts; in some embodiments, those levels are compared to a reference.
  • the reference level is a level of GSATII, TAR1 and/or SST1 satellite transcripts in a normal (non-cancerous) cell, e.g., a normal cell from the same subject, or a reference level determined from a cohort of normal cells; the presence of levels of GSATII, TAR1 and/or SST1 in the test cell below those in the normal cell indicate that the test cell is a tumor cell (e.g., the subject from whom the test cell came has or can be diagnosed with cancer).
  • the reference level of GSATII, TAR1 and/or SST1 transcripts is a threshold level, and the presence of a level of GSATII, TAR1 and/or SST1 satellite transcripts below the threshold level indicates that the cell is a tumor cell (e.g., the subject from whom the test cell came has or can be diagnosed with cancer).
  • the levels of the satellite transcripts are normalized to a relatively non-variant transcript such as GAPDH, actin, or tubulin, e.g., the level of expression of the satellite is compared to the non-variant transcript.
  • a ratio of expression levels can be calculated, and the ratio can be compared to the ratio in a normal (non-cancerous) cell.
  • the presence of a ratio of ALR:GAPDH of over 10:1, e.g., over 50:1, over 100:1, or over 150:1, indicates that the test cell is a cancer cell; in some embodiments the presence of a ratio of ALR:GAPDH of about 3:1 or 5:1 indicates that the test cell is a normal cell.
  • the presence of a ratio of HSATII satellites:GAPDH transcripts of over 10:1, e.g., 20:1, 30:1, 40:1, or 45:1, indicates that the test cell is a cancer cell.
  • the presence of significant (e.g., more than about 100 transcripts per million aligned) levels of HSATII indicates that the test cell is a cancer cell.
  • the absence or presence of very low levels (e.g., less than about 20 transcripts per million aligned) of HSATII indicates that the test cell is a normal cell.
  • LINE Long interspersed nucleotide element
  • Singer, Cell 28 (3): 433-4 (1982) are a group of genetic elements that are found in large numbers in eukaryotic genomes, and generate insertion mutations, contribute to genomic instability and innovation, and can alter gene expression.
  • the canonical, full-length LINE-1 element is about 6 kilobases (kb) in length and includes a 5′ untranslated region (UTR) with an internal RNA polymerase II promoter (Swergold, Mol Cell Biol. 10(12):6718-29 (1990)), two open reading frames (designated ORF1 and ORF2) and a 3′ UTR containing a polyadenylation signal ending with an oligo dA-rich tail of variable length (Babushok and Kazazian, Hum Mutat. 28(6):527-39 (2007)).
  • UTR 5′ untranslated region
  • ORF1 and ORF2 two open reading frames
  • 3′ UTR containing a polyadenylation signal ending with an oligo dA-rich tail of variable length
  • Exemplary LINE-1 sequences include GenBank Ref. No. NM — 001164835.1 (nucleic acid) and NP — 001158307.1 (protein) for variant (1); and GenBank Ref. No. NM — 019079.4 (nucleic acid) and NP — 061952.3 (protein) for variant 2, which is the shorter transcript.
  • Variant 2 differs in the 5′ UTR compared to variant 1, but both variants 1 and 2 encode the same protein. See also Gene ID: 54596.
  • the methods for diagnosing cancer described herein include determining a level of LINE-1 mRNA in a cell, e.g., in CTCs present in blood of a subject to obtain a LINE-1 value, and comparing the value to an appropriate reference value, e.g., a value that represents a threshold level, above which the subject can be diagnosed with cancer.
  • the reference can also be a range of values, e.g., that indicate severity or stage of the cancer in the subject.
  • a suitable reference value can be determined by methods known in the art.
  • the reference level is a level of LINE-1 transcripts in a normal (non-cancerous) cell, e.g., a normal cell from the same subject, or a reference level determined from a cohort of normal cells; the presence of levels of LINE-1 in the test cell above those in the normal cell indicate that the test cell is a tumor cell (e.g., the subject from whom the test cell came has or can be diagnosed with cancer).
  • the reference level of LINE-1 transcripts is a threshold level, and the presence of a level of LINE-1 transcripts above the threshold level indicates that the cell is a tumor cell (e.g., the subject from whom the test cell came has or can be diagnosed with cancer).
  • RNA expression assays e.g., microarray analysis, RT-PCR, deep sequencing, cloning, Northern blot, and quantitative real time polymerase chain reaction (qRT-PCR).
  • qRT-PCR quantitative real time polymerase chain reaction
  • the level of the LINE-1 protein is detected.
  • the presence and/or level of a protein can be evaluated using methods known in the art, e.g., using quantitative immunoassay methods such as enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, immunohistochemistry, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis.
  • ELISAs enzyme linked immunosorbent assays
  • IA enzyme immunoassay
  • RIA radioimmunoassay
  • the methods include contacting an agent that selectively binds to a biomarker, e.g., to a satellite transcript or LINE-1 mRNA or protein (such as an oligonucleotide probe, an antibody or antigen-binding portion thereof) with a sample, to evaluate the level of the biomarker in the sample.
  • a biomarker e.g., to a satellite transcript or LINE-1 mRNA or protein (such as an oligonucleotide probe, an antibody or antigen-binding portion thereof)
  • the agent bears a detectable label.
  • the term “labeled,” with regard to an agent encompasses direct labeling of the agent by coupling (i.e., physically linking) a detectable substance to the agent, as well as indirect labeling of the agent by reactivity with a detectable substance.
  • detectable substances examples include chemiluminescent, fluorescent, radioactive, or colorimetric labels.
  • detectable substances can include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, quantum dots, or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • antibodies can be used.
  • Antibodies can be polyclonal, or more preferably, monoclonal.
  • An intact antibody, or an antigen-binding fragment thereof (e.g., Fab or F(ab′) 2 ) can be used.
  • high throughput methods e.g., protein or gene chips as are known in the art (see, e.g., Ch. 12, “Genomics,” in Griffiths et al., Eds. Modern genetic Analysis, 1999, W. H. Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999; 17:217-218; MacBeath and Schreiber, Science 2000, 289(5485):1760-1763; Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 2002; Hardiman, Microarrays Methods and Applications: Nuts & Bolts, DNA Press, 2003), can be used to detect the presence and/or level of satellites or LINE-1.
  • a kit for performing this assay is commercially-available from Affymetrix (ViewRNA).
  • microfluidic (e.g., “lab-on-a-chip”) devices can be used in the present methods. Such devices have been successfully used for microfluidic flow cytometry, continuous size-based separation, and chromatographic separation.
  • methods in which expression of satellites or LINE-1 is detected in circulating tumor cells (CTCs) can be used for the early detection of cancer, e.g., early detection of tumors of epithelial origin, e.g., pancreatic, lung, breast, prostate, renal, ovarian or colon cancer.
  • the devices can be used for separating CTCs from a mixture of cells, or preparing an enriched population of CTCs.
  • such devices can be used for the isolation of CTCs from complex mixtures such as whole blood.
  • a device can include an array of multiple posts arranged in a hexagonal packing pattern in a microfluidic channel upstream of a block barrier.
  • the posts and the block barrier can be functionalized with different binding moieties.
  • the posts can be functionalized with anti-EPCAM antibody to capture circulating tumor cells (CTCs); see, e.g., Nagrath et al., Nature 450:1235-1239 (2007), optionally with downstream block barriers functionalized with to capture LINE-1 nucleic acids or proteins, or satellites. See, e.g., (S. Maheswaran et al., N Engl J Med. 359, 366 (Jul.
  • Processes for enriching specific particles from a sample are generally based on sequential processing steps, each of which reduces the number of undesired cells/particles in the mixture, but one processing step may suffice in some embodiments.
  • Devices for carrying out various processing steps can be separate or integrated into one microfluidic system.
  • the devices include devices for cell/particle binding, devices for cell lysis, devices for arraying cells, and devices for particle separation, e.g., based on size, shape, and/or deformability or other criteria.
  • processing steps are used to reduce the number of cells prior to introducing them into the device or system.
  • the devices retain at least 75%, e.g., 80%, 90%, 95%, 98%, or 99% of the desired cells compared to the initial sample mixture, while enriching the population of desired cells by a factor of at least 100, e.g., by 1000, 10,000, 100,000, or even 1,000,000 relative to one or more non-desired cell types.
  • Some devices for the separation of particles rely on size-based separation with or without simultaneous cell binding.
  • Some size-based separation devices include one or more arrays of obstacles that cause lateral displacement of CTCs and other components of fluids, thereby offering mechanisms of enriching or otherwise processing such components.
  • the array(s) of obstacles for separating particles according to size typically define a network of gaps, wherein a fluid passing through a gap is divided unequally into subsequent gaps.
  • Both sieve and array sized-based separation devices can incorporate selectively permeable obstacles as described above with respect to cell-binding devices.
  • Devices including an array of obstacles that form a network of gaps can include, for example, a staggered two-dimensional array of obstacles, e.g., such that each successive row is offset by less than half of the period of the previous row.
  • the obstacles can also be arranged in different patterns. Examples of possible obstacle shapes and patterns are discussed in more detail in WO 2004/029221.
  • the device can provide separation and/or enrichment of CTCs using array-based size separation methods, e.g., as described in U.S. Pat. Pub. No. 2007/0026413.
  • the devices include one or more arrays of selectively permeable obstacles that cause lateral displacement of large particles such as CTCs and other components suspended in fluid samples, thereby offering mechanisms of enriching or otherwise processing such components, while also offering the possibility of selectively binding other, smaller particles that can penetrate into the voids in the dense matrices of nanotubes that make up the obstacles.
  • Devices that employ such selectively permeable obstacles for size, shape, or deformability based enrichment of particles, including filters, sieves, and enrichment or separation devices, are described in International Publication Nos.
  • 60/668,415 devices useful for arraying cells, e.g., those described in International Publication No. 2004/029221, U.S. Pat. No. 6,692,952, and U.S. application Ser. Nos. 10/778,831 and 11/146,581; and devices useful for fluid delivery, e.g., those described in U.S. application Ser. Nos. 11/071,270 and 11/227,469.
  • Two or more devices can be combined in series, e.g., as described in International Publication No. WO 2004/029221. All of the foregoing are incorporated by reference herein.
  • a device can contain obstacles that include binding moieties, e.g., monoclonal anti-EpCAM antibodies or fragments thereof, that selectively bind to particular cell types, e.g., cells of epithelial origin, e.g., tumor cells. All of the obstacles of the device can include these binding moieties; alternatively, only a subset of the obstacles include them.
  • Devices can also include additional modules, e.g., a cell counting module or a detection module, which are in fluid communication with the microfluidic channel device. For example, the detection module can be configured to visualize an output sample of the device.
  • a detection module can be in fluid communication with a separation or enrichment device.
  • the detection module can operate using any method of detection disclosed herein, or other methods known in the art.
  • the detection module includes a microscope, a cell counter, a magnet, a biocavity laser (see, e.g., Gourley et al., J. Phys. D: Appl. Phys., 36: R228-R239 (2003)), a mass spectrometer, a PCR device, an RT-PCR device, a microarray, RNA in situ hybridization system, or a hyperspectral imaging system (see, e.g., Vo-Dinh et al., IEEE Eng. Med. Biol.
  • a computer terminal can be connected to the detection module.
  • the detection module can detect a label that selectively binds to cells, proteins, or nucleic acids of interest, e.g., LINE-1 DNA, mRNA, or proteins, or satellite DNA or mRNA.
  • the microfluidic system includes (i) a device for separation or enrichment of CTCs; (ii) a device for lysis of the enriched CTCs; and (iii) a device for detection of LINE-1 DNA, mRNA, or proteins, or satellite DNA or mRNA.
  • a population of CTCs prepared using a microfluidic device as described herein is used for analysis of expression of LINE-1 and/or satellites using known molecular biological techniques, e.g., as described above and in Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Laboratory Press; 3rd edition (Jan. 15, 2001)); and Short Protocols in Molecular Biology, Ausubel et al., eds. (Current Protocols; 52 edition (Nov. 5, 2002)).
  • devices for detection and/or quantification of expression of satellites or LINE-1 in an enriched population of CTCs are described herein and can be used for the early detection of cancer, e.g., tumors of epithelial origin, e.g., early detection of pancreatic, lung, breast, prostate, renal, ovarian or colon cancer.
  • cancer e.g., tumors of epithelial origin, e.g., early detection of pancreatic, lung, breast, prostate, renal, ovarian or colon cancer.
  • a treatment e.g., as known in the art, can be administered.
  • the efficacy of the treatment can be monitored using the methods described herein; an additional sample can be evaluated after (or during) treatment, e.g., after one or more doses of the treatment are administered, and a decrease in the level of LINE-1, and/or ALR and/or HSATII satellites expression, or in the number of LINE-1-, and/or ALR and/or HSATII satellite-expressing cells in a sample, would indicate that the treatment was effective, while no change or an increase in the level of LINE-1, and/or ALR and/or HSATII satellite expression or LINE-1-, and/or ALR and/or HSATII satellite-expressing cells would indicate that the treatment was not effective (the converse would of course be true for levels of GSATII, TAR1 and/or SST1 satellites).
  • the methods can be repeated multiple
  • the methods can be repeated at selected intervals, e.g., at 3, 6, 12, or 24 month intervals, to monitor the disease in the subject for early detection of progression to malignancy or development of cancer in the subject.
  • DGE digital gene expression
  • mice with pancreatic cancer of different genotypes were bred as previously described in the Bardeesy laboratory (Bardeesy et al., Proc Natl Acad Sci USA 103, 5947 (2006)). Normal wild type mice were purchased from Jackson laboratories. Animals were euthanized as per animal protocol guidelines. Pancreatic tumors and normal tissue were extracted sterilely and then flash frozen with liquid nitrogen. Tissues were stored at ⁇ 80° C. Cell lines were generated fresh for animals AH367 and AH368 as previously described (Aguirre et al., Genes Dev 17, 3112 (2003)) and established cell lines were cultured in RPMI-1640+10% FBS+1% Pen/Strep (Gibco/Invitrogen). Additional mouse tumors from colon and lung were generously provided by Kevin Haigis (Massachusetts General Hospital) and Kwok-Kin Wong (Dana Farber Cancer Institute).
  • Fresh frozen tissue was pulverized with a sterile pestle in a microfuge tube on dry ice.
  • Cell lines were cultured and fresh frozen in liquid nitrogen prior to nucleic acid extraction.
  • RNA and DNA from cell lines and fresh frozen tumor and normal tissues were all processed in the same manner.
  • RNA was extracted using the TRIzol® Reagent (Invitrogen) per manufacturer's specifications.
  • DNA from tissue and cell lines was extracted using the QIAamp Mini Kit (QIAGEN) per manufacturer's protocol.
  • DGE Digital Gene Expression
  • DNA sequencing sample prepping protocol from Helicos that has been previously described (Pushkarev, N. F. Neff, S. R. Quake, Nat Biotech 27, 847 (2009)). Briefly, genomic DNA was sheared with a Covaris S2 acoustic sonicator producing fragments averaging 200 bps and ranging from 100-500 bps. Sheared DNA was then cleaned with SPRI. DNA was then denatured and a poly-A tail was added to the 3′ end using terminal transferase.
  • the levels of satellite transcripts in tumor tissues were about 8,000-fold higher than the abundant mRNA Gapdh.
  • a second independent pancreatic tumor nodule from the same mouse showed a lower, albeit still greatly elevated, level of satellite transcript (4.5% of total cellular transcripts).
  • the composite distribution of all RNA reads among coding, ribosomal and other non-coding transcripts showed significant variation between primary tumors and normal tissues ( FIG. 1A ), suggesting that the global cellular transcriptional machinery is affected by the massive expression of satellite transcripts in primary tumors.
  • Immortalized cell lines established from 3 primary pancreatic tumors displayed minimal expression of satellite repeats, suggesting either negative selection pressure during in vitro proliferation or reestablishment of stable satellite silencing mechanisms under in vitro culture conditions ( FIG. 1A ).
  • the composite distribution of all RNA reads among coding, ribosomal and other non-coding transcripts shows significant variation with that of normal tissues ( FIG. 1B ), suggesting that the cellular transcriptional machinery is affected by the massive expression of satellite transcripts in these tumors.
  • Satellite Transcripts are of Various Sizes Depending on Tissue Type and Expression Levels are Linked to Genomic Methylation and Amplification
  • Northern Blot analysis of mouse primary pancreatic tumors was carried out as follows. Northern Blot was performed using the NorthernMax-Gly Kit (Ambion). Total RNA (10 ug) was mixed with equal volume of Glyoxal Load Dye (Ambion) and incubated at 50° C. for 30 min. After electrophoresis in a 1% agarose gel, RNA was transferred onto BrightStar-Plus membranes (Ambion) and crosslinked with ultraviolet light. The membrane was prehybridized in ULTRAhyb buffer (Ambion) at 68° C. for 30 min.
  • the mouse RNA probe (1100 bp) was prepared using the MAXIscript Kit (Ambion) and was nonisotopically labeled using the BrightStar Psoralen-Biotin Kit (Ambion) according to the manufacturer's instructions.
  • the membrane was hybridized in ULTRAhyb buffer (Ambion) at 68° C. for 2 hours. The membrane was washed with a Low Stringency wash at room temperature for 10 min, followed by two High Stringency washes at 68° C. for 15 min.
  • the BrightStar BioDetect Kit was used according to the manufacturer's instructions.
  • the single molecule sequencing platform was exceptionally sensitive for quantitation of small repetitive ncRNA fragments, each of which is scored as a unique read.
  • High level expression of the mouse major satellite was evident in all cells within the primary tumor ( FIG. 2D ), as shown by RNA in situ hybridization (ISH).
  • ISH RNA in situ hybridization
  • FIG. 2E RNA in situ hybridization
  • Clearly defined metastatic lesions to the liver ware strongly positive by RNA ISH, as were individual PDAC cells within the liver parenchyma that otherwise would not have been detected by histopathological analysis ( FIG. 2F ).
  • Low level diffuse expression was evident in liver and lung, as shown by whole mount embryo analysis, but no normal adult or embryonic tissues demonstrated satellite expression comparable to that evident in tumor cells.
  • the index AH284 tumor was analyzed using next generation DNA digital copy number variation (CNV) analysis as described above for genomic DNA sequencing.
  • CNV next generation DNA digital copy number variation
  • ALR alpha RI
  • normal pancreatic tissue has a much higher representation of GSATII, TAR1 and SST1 classes (26.4%, 10.6%, and 8.6% of all satellite reads), while these were a small minority of satellite reads in pancreatic cancers.
  • cancers express high levels of HSATII satellites (4,000 per 10 6 transcripts; 15% of satellite reads), a subtype whose expression is undetectable in normal pancreas ( FIG. 3B ).
  • mice sample reads were aligned to a custom made library for the mouse major satellite (sequence from UCSC genome browser). Human samples were aligned to a custom made reference library for all satellite repeats and LINE-1 variants generated from the Repbase library (Pushkarev et al., Nat Biotech 27, 847 (2009)). In addition, all samples were subjected to the DGE program for transcriptome analysis. Reads were normalized per 10 6 genomic aligned reads for all samples.
  • Line-1 the autonomous retrotransposon Line-1 had the highest expression level in mouse samples of diverse tissue types.
  • Mouse pancreatic tumors have a mean Line-1 expression 30,690 tpm (range 183-120,002), representing an average of 330-fold higher levels compared to Gapdh (Table 5).
  • SCGs satellite Correlated Genes
  • Histone modifications including H3K9 trimethylation (P. A. Cloos, J. Christensen, K. Agger, K. Helin, Genes Dev 22, 1115 (May 1, 2008)), combined with Dicer1 and Piwi-related protein-mediated ncRNA processing (A. A. Aravin, G. J. Hannon, J. Brennecke, Science 318, 761 (Nov. 2, 2007)) have been linked to maintenance of repression of satellite repeats.
  • To search for candidate regulators of satellite derepression in primary tumor specimens we first measured the quantitative DGE of known epigenetic regulators and RNA processing genes in mouse tumors, as a function of increasing major satellite expression.
  • a targeted gene expression analysis of demethylases and RNA processing enzymes was carried out in mouse and human PDAC samples.
  • a list of demethylases and RNA processing enzymes were generated from two recent publications (Cloos et al., Genes Dev 22, 1115 (2008); Aravin et al., Science 318, 761 (2007)).
  • Mouse PDACs with Kras G12D and Tp53 loss were used for this analysis.
  • Mouse tumors were separated into high vs low satellite levels using the median satellite expression (7%). A total of 37 genes were evaluated between high and low satellite tumors and fold change was calculated. Analysis of the population means was compared using the 2-tailed student t-test assuming equal variance.
  • mouse pancreatic tumors with satellite expression above the median had higher expression of the demethylases Hspbap1, Jmjd1B, Jmjd4, Jarid1d, Jmjd3, and Fbxl10 as well as the RNA processing enzyme Dicer1.
  • Hspbap1, Jmjd1B, Jmjd4, Jarid1d, Jmjd3, and Fbxl10 as well as the RNA processing enzyme Dicer1.
  • HSPBAP1, FBXL10 and DICER1 overexpression was also observed in human pancreatic adenocarcinomas (Table 7, p ⁇ 0.05, student t-test).
  • LINE-1 may drive expression of specific cellular mRNAs through its insertion upstream of their transcriptional start sites (T. Kuwabara et al., Nat Neurosci 12, 1097 (September, 2009)) or through alterations in flanking chromatin marks (J. A. Bailey, L. Carrel, A. Chakravarti, E. E. Eichler, Proceedings of the National Academy of Sciences of the United States of America 97, 6634 (Jun. 6, 2000, 2000); D. E.
  • the transcriptional start sites tissues and cell lines were determined (UCSC genome browser (D. Karolchik et al., Nucleic Acids Res 32, D493 (Jan. 1, 2004)) as well as the position of all Line-1 elements in the mouse genome with a threshold of 1 Kbp in length. Line-1 closest distance upstream of the transcriptional start sites of all annotated genes with a minimum expression level of 5 transcripts per million were calculated. Genes were then rank ordered according to the Pearson coefficient for linear regression. Genes were binned in 100 s and plotted by Excel. Randomization of all genes, followed by binning, and plotting was done as a control.
  • LINE-1 sequences within the proximity of cellular transcripts may contribute to their overexpression in primary tumors, in striking correlation with the expression of both LINE-1 and satellite repeats.
  • the consequence of increased expression of these cellular transcripts remains to be defined.
  • the high prevalence of genes linked to stem-like and neurogenic fates, along with the frequency of HOX and zinc finger transcriptional regulators raises the possibility that at least a subset of these may contribute to tumor-related phenotypes.
  • LINE-1 is a Specific and Sensitive Marker of CTCs
  • Satellite levels are most strongly linked with the expression of the autonomous retrotransposon Line-1, which has recently been shown to be a major cause for genomic variation in normal and tumor tissues (J. Berretta, A. Morillon, EMBO Rep 10, 973 (September, 2009); A. Jacquier, Nat Rev Genet 10, 833 (December, 2009); M. Guenatri, D. Bailly, C. Maison, G. Almouzni, J Cell Biol 166, 493 (Aug. 16, 2004)). Aberrant expression of cellular transcripts linked to stem cells and neural tissues is also highly correlated with satellite transcript levels, suggesting alteration of cell fate through derepression of a coordinated epigenetic program.
  • CTCs circulating tumor cells
  • HB herringbone chip
  • EpCAM epithelial cell adhesion molecule
  • RNA was extracted from the devices using the Qiagen RNeasy MinElute kit.
  • Krt Keratins
  • the HSATII satellite is overexpressed in pancreatic cancer and was confirmed to be overexpressed in human preneoplastic pancreatic lesions ( FIG. 6A ) using a branched DNA detection assay (QuantiGene® ViewRNA Assay, Affymetrix).
  • a branched DNA detection assay QuantantiGene® ViewRNA Assay, Affymetrix.
  • Breast cancer samples were also tested for HSATII using this method and were found to have significant expression compared to normal breast tissues.
  • Extension of this technique to potential circulating tumor cells captured on the HB-chip FIG. 6B ) has been accomplished indicating that HSATII may be used as a blood based diagnostic for epithelial cancers.
  • Serum was extracted from the blood of 8 metastatic pancreatic cancer patients by using Ficoll buffy coat method.
  • Serum RNA (cell free RNA), which includes exosomes, was purified using the Trizol method and then purified using Qiagen RNA MinElute columns kits. RNA was then subjected to Helicos DGE sequencing preparation and sequenced on a HeliScope next generation sequencer. Results of this data are summarized in Table 1.
  • HSATII was specific for cancer and GSATII was found to correlate with normal tissues. Therefore the ratio of HSATII to GSATII was evaluated as a marker for identifying cancer burden and potentially an early detection marker. In this case, one patient who had stable disease had the lowest HSATII/GSATII ratio as predicted (see Table 8).
  • Table 8 A total of 8 metastatic cancer patients with clinical status, total satellites, HSATII, and GSATII in transcripts per million aligned to genome (tpm) and the ratio of HSATII/GSATII in cell free RNA sequenced.
  • RNA cell free
  • HSATII and GSATII did not perform as well as expected, though the presence of.
  • other satellites like TAR1 seemed to be better predictors of “cancer” compared to “non-cancer” status as shown in Table 9.
  • Table 9 Average total satellites, HSATII, GSATII, HSATII/GSATII, and TAR1 (tpm) in a total of 8 metastatic PDAC patients and 4 healthy donors with cell free RNA sequenced. Student t-test was used to calculate significance.

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US11142800B2 (en) 2021-10-12
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US20170356054A1 (en) 2017-12-14

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