WO2006042005A2 - Profilage de l'expression genetique globale de cellules tumorales circulantes - Google Patents

Profilage de l'expression genetique globale de cellules tumorales circulantes Download PDF

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WO2006042005A2
WO2006042005A2 PCT/US2005/035969 US2005035969W WO2006042005A2 WO 2006042005 A2 WO2006042005 A2 WO 2006042005A2 US 2005035969 W US2005035969 W US 2005035969W WO 2006042005 A2 WO2006042005 A2 WO 2006042005A2
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cells
epithelial
circulating tumor
cell
cancer
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PCT/US2005/035969
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WO2006042005A3 (fr
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Mark O'hara Shawn
Denis Smirnov
Daniel Zweitzig
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Immunivest Corporation
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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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—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
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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

Definitions

  • This invention relates generally to gene specific amplification, analysis and profiling of cytosolic biomolecules useful in the fields of oncology, diagnostic testing and pharmacogenomics (personalized medicine).
  • the invention is particularly useful in such fields as cancer screening, selecting (identification and stratification of therapy responders vs non-responders) and monitoring for chemotherapy treatment, or cancer recurrence.
  • the present invention facilitates comprehensive analysis of mRNA and DNA from rare target cells.
  • the invention characterizes a molecular profile for identifying the tissue source or predicting disease status.
  • tumor cells can be identified in peripheral blood. Characterization of these cells offers a unique opportunity to provide insights into the cascade of metastatic events.
  • CTCs Metastatic lesions and not the primary tumors are the leading cause of death in patients with carcinomas.
  • cancerous cells detach from the primary tumor and enter the bloodstream directly or through lymphatic channels.
  • Some of these CTCs migrate to secondary organs to form a metastatic lesion.
  • the presence of CTCs has been associated with poor prognosis in patients with metastatic breast cancer 2 . Similar conclusions are likely to be true for other types of cancer.
  • detailed molecular characterization of CTCs is lacking. This can be attributed to the extremely low frequency of CTCs.
  • the presence of CTCs can be detected by RT-
  • Breast cancer cells are particularly well known to adapt and survive periods of stress such as hypoxia (Knowles, et al., 2001. Hypoxia and Oxidative Stress in Breast Cancer: Hypoxia and tumourigenisis. Br Can Res 3, 318-322; Pugh, et al., 2001. Hypoxia and Oxidative Stress in Breast Cancer: Hypoxia Signalling Pathways. Br Can Res 3, 313-317) accompanied by serum deprivation, provides a means to monitor the expression of a panel of genes identified as breast CTC identification markers. Thus during or after exposure to hypoxia or serum deprivation, increases in selective genes associated with breast CTC would provide a sensative tool in cancer detection. Serum deprivation alone, and especially serum deprivation in combination with hypoxia, lead to a dramatic increase in human anterior gradient-2 (Hag-2, AGR2) and intestinal trefoil factor-3 (TFF3, ITF3) mRNA expression.
  • Hag-2, AGR2 human anterior gradient-2
  • TNF3, ITF3 intestinal trefo
  • the present invention provides methods and compositions for detecting the expression profile in circulating tumor cells from an enriched blood sample, which methods generally comprise: a. obtaining a biological sample containing a mixed population of cells from an individual suspected of having target rare cells; b. enriching said biological sample; c. identifying said target rare cells using immunofluorescent characterization; and d. confirming said target rare cells as a CTC whereby said target rare cell expresses a gene from a group consisting of AGR2, S100A14, S100A16, FABP1 , TFF3, or combinations thereof.
  • the present invention describes an approach for assessing disease status based on the identification of molecular markers that are uniquely expressed in circulating cells, such as circulating tumor cells (CTCs) or circulating endothelial cells (CECs).
  • CTCs circulating tumor cells
  • CECs circulating endothelial cells
  • blood samples from patients with disease such as, but not limited to, colorectal, prostate and breast cancer are identified.
  • Global expression profiles from CTCs are generated from these samples and a list of genes characteristic of specific CTC expression is obtained.
  • the gene expression profiles are used to predict disease status and/or identify the origin of the target circulating cells.
  • Another aspect of the present invention is the incorporation of an integrated platform for immunomagnetic enrichment and immunofluorescent identification to identify genetic expression profiles in target cells such as, but not limited to, circulating tumor cells (CTCs) or circulating endothelial cells (CECs). Accordingly after genetic profiles are generated from target cells, the presence of these select genes is confirmed by comparison with serum expression levels in disease vs normal patient blood using quantitative real-time RT-PCR.
  • target cells such as, but not limited to, circulating tumor cells (CTCs) or circulating endothelial cells (CECs).
  • a further aspect of the present invention incorporates a global expression profile either to predict disease status or to identify the origin of the target circulating cells after identifying genetic expression profiles with the induction of physiological stress on target cells.
  • the global expression profile of CTCs provided through the methods in the present invention enables valuable real-time molecular insight into the process of metastasis for diagnositic and research purposes, identification of new therapeutic target molecules, and the development of novel minimally invasive diagnostics.
  • Figure 1 Results of global CTC expression profiling by microarrays.
  • Log2- transformed fluorescent intensity ratios between CTC-enriched and corresponding CTC-depleted blood samples from patients with breast cancer (BrCa), prostate cancer (PrCa) and colorectal cancer (CoCa) are presented for selected genes in form of a heatmap. Genes are grouped based on their expression pattern and selected for specificity to: all CTCs regardless of tissue of cancer origin (pan cancer group), breast cancer CTCs (breast cancer group), prostate cancer CTCs (prostate cancer group), colon cancer CTCs (colon cancer group). Genes within each category are sorted in descending order by their Iog 2 -transformed fluorescent intensity ratios. Fluorescent intensity ratios for the pan leukocyte marker CD45 and housekeeping (Hskp.) control genes are also presented.
  • Figure 2 Results of confirmatory gene expression profiling of candidates by quantitative real-time RT-PCR. Expression levels of 25 genes were calculated as log 2 (transcript copy number+1) and are presented in form of a heatmap for CTC- enriched blood samples from indicated 30 patients with colorectal cancer, 31 patients with prostate cancer, 13 breast cancer patients and 50 normal donors. Number of detected CTCs per 7.5 ml of peripheral blood is also presented for each cancer patient. Normals had no CTCs detected. Genes that show an expression pattern specific to a particular cancer type(s) are marked as pan cancer, breast cancer, colorectal and prostate cancers.
  • FIG. 3 Expression levels of AGR2 (panel A), TFF3 (panel B), VEGF (panel C) increase when exposed to physiological stress. Panel D confirms an increase in expression with stress induction.
  • CTCs circulating tumor cells
  • a major characteristic required of metastatic cells is the ability to adapt to and survive the insults of pathophysiological stress before, during and after dissemination. Since breast cancer cells are particularly well known to adapt and survive periods of stress, a method to monitor the expression of a panel of genes identified as breast CTC identification markers, during exposure of breast cancer cell lines to hypoxia, serum deprivation or combinations thereof.
  • the present inventiion incorporates serum deprivation alone, and especially serum deprivation in combination with hypoxia, provides a dramatic increase in human anterior gradient-2 (Hag-2, AGR2) and intestinal trefoil factor-3 (TFF3, ITF3) mRNA expression.
  • the present invention provides a method and means for assessing CTC markers and their regulation in vivo, aiding in the design of novel therapies targeted at blocking breast cancer metastasis.
  • CTCs are enriched from 7.5ml_ of blood using magnetic nanoparticles conjugated to monoclonal antibodies against epithelial-cell adhesion molecule (EpCAM). Two aliquots of blood are collected from each cancer patient. The first aliquot is used to enumerate CTCs and the second for gene expression measurements in CTCs.
  • EpCAM epithelial-cell adhesion molecule
  • CTCs are still outnumbered by captured leukocytes.
  • Tumor cells added as controls to blood samples showed that expressed genes can be detected with the Affymetrix GeneChip system if 100 or more CTCs (-800 or more copies of each CTC specific transcript) were present in a background of ⁇ 1 ,000-10,000 leukocytes (data not shown). Based on these criteria, we selected a 7.5 mL blood sample from a patient with colorectal cancer (105 CTC), prostate cancer (647 CTC) and breast cancer (3,700 CTC) to obtain global CTC gene expression profiles for each condition. Gene expression signatures specific to the CTCs were obtained after white blood cell subtraction (PCT/US05/10940) and normalization.
  • PCT/US05/10940 white blood cell subtraction
  • prostate specific antigen KLK3 was up-regulated, and in colon cancer carcinoembryonic antigen (CEACAM 5) was detected.
  • CD45 or PTPRC
  • ACTB ⁇ -actin
  • RPS27A ribosomal protein
  • transcripts for 9 genes TST, ASGR2, MARCO, TFF3, SIL1 , S100A13, MAOB, SLC2A10 and VIL1
  • Figure 2 Genes KRT19 and AGR2 (hAG-2) were expressed in the majority of patient samples regardless of the cancer type.
  • Genes FABP1 and KRT20 showed patterns characteristic of colorectal cancer.
  • KLK2, MSMB, DDC, AR, HPN and KLK3 were characteristic to prostate cancer, while genes SCGB2A1 , SCGB2A2 and PIP were associated with breast cancer.
  • Genes S100A14, S100A16 and CEACAM5 showed expression restricted to colorectal and breast cancers.
  • the support vector machine (SVM) supervised learning algorithm was used to identify sets of genes that provided the highest sensitivity and specificity to discriminate between different sample types. The greatest accuracy of 82% was obtained when a combination of KRT19, AGR2, S100A13 ASGR2 and TST was used to distinguish between cancer and normal samples. The best discrimination between the three groups of cancer and samples from normal donors was obtained with S100A14, KLK3, S100A13, CEACAM5, SCGB2A2, MSMB, KLK2, S100A16, KRT20 and TST. Discrimination within the group of prostate cancer was obtained with KLK3, KLK2, AR, DDC, HPN, MSMB, KRT19, AGR2, FABP1 and VIL1.
  • the present example demonstrates that global gene profiles of CTCs such as AGR2, FABP1 , S100A14, S100A16 and others are expressed in CTCs.
  • Global expression profiling of CTCs provides a means to assessing the progression toward metastasis, especially in the development of non-invasive diagnostic tools and therapeutic targets.

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Abstract

Procédés de génération de profils d'expression génétique globale dans les cellules tumorales circulantes et de génération d'un ensemble de gènes utilisés pour le diagnostic du cancer. Procédés et moyens de diagnostic du cancer par évaluation de l'expression AGR2 ou TFF3 dans les CTC exposées au stress physiologique.
PCT/US2005/035969 2004-10-07 2005-10-06 Profilage de l'expression genetique globale de cellules tumorales circulantes WO2006042005A2 (fr)

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US61676404P 2004-10-07 2004-10-07
US60/616,764 2004-10-07
US62444504P 2004-11-02 2004-11-02
US60/624,445 2004-11-02

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101884794A (zh) * 2010-07-02 2010-11-17 刘云 S100a16基因在制备治疗胰岛素抵抗药物中的应用
US20100297634A1 (en) * 2007-09-19 2010-11-25 Research Foundation Of State University Of N.Y. Gene expression signatures in enriched tumor cell samples
US8071395B2 (en) 2007-12-12 2011-12-06 The Board Of Trustees Of The Leland Stanford Junior University Methods and apparatus for magnetic separation of cells

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DAS R. ET AL: 'Expression Pattern of Fatty Acid-binding Proteins in Human Normal and Cancer Prostate Cells and Tissues' CLINICAL CANCER RESEARCH vol. 7, 2001, pages 1706 - 1715, XP003001884 *
ELLIS W.J. ET AL: 'Detection and Isolation of prostate cancer cells from peripheral blood and bone marrow' UROLOGY vol. 61, no. 2, 2003, pages 277 - 281, XP003001883 *
FURUTA G.T. ET AL: 'Hypoxia-inducibleFactor 1-dependent Induction of Intestinal T refoil Factor Protects Barrier Function during Hypoxia' J. EXP. MED. vol. 193, no. 9, pages 1027 - 1034, XP002982852 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100297634A1 (en) * 2007-09-19 2010-11-25 Research Foundation Of State University Of N.Y. Gene expression signatures in enriched tumor cell samples
US8889361B2 (en) * 2007-09-19 2014-11-18 The Research Foundation For The State University Of New York Gene expression signatures in enriched tumor cell samples
US8071395B2 (en) 2007-12-12 2011-12-06 The Board Of Trustees Of The Leland Stanford Junior University Methods and apparatus for magnetic separation of cells
US9267943B2 (en) 2007-12-12 2016-02-23 The Board Of Trustees Of The Leland Stanford Junior University Apparatus for magnetic separation of cells
CN101884794A (zh) * 2010-07-02 2010-11-17 刘云 S100a16基因在制备治疗胰岛素抵抗药物中的应用
CN101884794B (zh) * 2010-07-02 2011-08-31 刘云 S100a16基因在制备治疗胰岛素抵抗药物中的应用

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