WO2009051734A1 - Dispositifs à base de micropuce pour capturer des cellules tumorales circulantes et procédés pour leur utilisation - Google Patents

Dispositifs à base de micropuce pour capturer des cellules tumorales circulantes et procédés pour leur utilisation Download PDF

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WO2009051734A1
WO2009051734A1 PCT/US2008/011785 US2008011785W WO2009051734A1 WO 2009051734 A1 WO2009051734 A1 WO 2009051734A1 US 2008011785 W US2008011785 W US 2008011785W WO 2009051734 A1 WO2009051734 A1 WO 2009051734A1
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tumor
cells
tumor cells
circulating
cell
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PCT/US2008/011785
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Sunitha Nagrath
Lecia V. Sequist
Ronald G. Tompkins
Daniel A. Haber
Mehmet Toner
Daniel Irimia
Shyamala Maheswaran
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The General Hospital Corporation
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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
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • 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
    • C12Q2600/156—Polymorphic or mutational 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
    • C12Q2600/158—Expression markers

Definitions

  • CTCs Viable tumor-derived epithelial cells (circulating tumor cells or CTCs) have been identified in peripheral blood from cancer patients and are likely the origin of intractable metastatic disease.
  • 1"4 CTCs represent a potential alternative to invasive biopsies as a source of tumor tissue for the detection, characterization, and monitoring of non-hematologic cancers.
  • 5"8 The ability to identify, isolate, propagate and molecularly characterize CTC subpopulations could further the discovery of cancer stem cell biomarkers and expand the understanding of the biology of metastasis.
  • Current strategies for isolating CTCs are limited to complex analytic approaches that generate very low yield and purity.
  • 9 CTCs are considered to be rare, making up as few as 1 to 10 cells/mL ( ⁇ 1
  • the invention provides devices and methods for capturing rare cells, e.g., CTCs from blood samples.
  • the devices of the invention are capable of capturing large numbers of viable CTCs in a single step from whole blood without pre-dilution, pre-labeling, pre-fixation, or any other processing steps.
  • the techniques described here and the broader application of microfluidic rare cell capture technology to cancer patients hold significant promise for identifying key biological determinants of blood-borne metastases, and for providing a robust platform aimed at early diagnosis and longitudinal monitoring of cancer.
  • the invention features a device for capturing circulating, nonhemopoietic tumor cells including a microfluidic channel to which is bound a tumor specific binding agent; and a pump producing a continuous, unidirectional shear stress of 0.1 to 20 dyn/cm 2 in the channel.
  • the specified shear stress may be localized to those regions where binding agents are located.
  • the invention features a device for capturing circulating, nonhemopoietic tumor cells including a microfluidic channel having a plurality of obstacles to each of which is bound a tumor specific binding agent, wherein the obstacles are disposed in an equilateral triangular arrangement with a 10-100 ⁇ m distance between obstacles and a 10-100 ⁇ m shift between at least two rows.
  • the shift may occur after at least three rows and may be repeated after every three rows.
  • the distance between obstacles and the shift are about 50 ⁇ m.
  • the invention also features a device for capturing circulating, nonhemopoietic tumor cells including a microfluidic channel to which is bound a tumor specific binding agent and a reservoir having a volume of less than 5 mL, wherein the reservoir and the channel are fluidically connected.
  • the reservoir may have a volume of 10 ⁇ L, 20 ⁇ L, 50 ⁇ L, 100 ⁇ L, 250 ⁇ L, 500 ⁇ L, 1 mL, up to 5 mL. These reservoirs may also be employed in conjunction with other devices of the invention.
  • the channels may include a plurality of obstacles, e.g., to which are bound the tumor specific binding agent, or the walls of the channel may be substantially planar, unless otherwise stated.
  • tumor specific binding agent may be disposed in a region of the channel having a volume of 10 ⁇ L-20 mL, e.g., 100 ⁇ L-15 mL, 100 ⁇ L-10 mL, 100 ⁇ L-5 mL, 100 ⁇ L-1 mL, or 100 ⁇ L-0.5 mL.
  • the shear stress produced in a channel may in general be between the range of 0.1 to 20 dyn/cm 2 , e.g., less than 15, 10, 5, 1, or 0.5 dyn/cm 2 .
  • Shear stress is not necessarily constant throughout a channel or region containing binding moieties, although it may be.
  • sample may be transported through the channel at a rate of 0.1 mL to 30 mL/hr. Typical flow rates will be less than 20, 15, 10, 5, 1, or 0.5 mL/hr.
  • samples will be passed through the channel at a constant rate, but a constant rate is not required.
  • An exemplary binding agent binds to EpCAM, e.g., anti- EpCAM.
  • Exemplary tumors producing CTCs are those of epithelial origin.
  • kits including any device of the invention and a reagent for obtaining genetic information, e.g., presence or absence of a mutation, level of gene expression, presence of absence of a protein, or level of protein expression, from a circulating, nonhemopoietic tumor cell.
  • exemplary reagents lyse tumor cells, amplify nucleic acids from tumor cells, bind to a nucleic acid sequence in a tumor cell, or bind to a protein.
  • Particular genetic information that may be obtained includes: for lung cancer, the presence or absence of an EGFR mutation; for prostate cancer, the presence or absence of a TMPRSS2-ERG fusion or level of PSA; and for breast cancer, genetic information on HER2.
  • Exemplary EGFR mutations are a deletion in exon 19, T790M, L858R, L861Q, G719(S, A, or C), S768I, an insertion in exon 20, and a combination thereof.
  • Exemplary TMPRSS2-ERG fusions are T3/E4, T2/E2, Tl/E3_5, T2/E5, Tl/E234_6, T1/E2, T1/E3, T2/E4, T1/E5, T1/E6, Tl/E_IIIa_4, Tl_I/E_IIIb_4, Tl/E_IIIc_4, T5/E4, T4/E4, T4/E5, and a combination thereof.
  • CTCs may also be assayed for cancer stem cell markers, as described herein. Molecular diagnostics for other types of cancer are also encompassed by the invention.
  • the invention features a method for capturing circulating, nonhemopoietic tumor cells by introducing a blood sample into a device of the invention so that circulating tumor cells in the blood sample bind to the binding agent in the device.
  • Another method of the invention obtains genetic information from a subject with a tumor by introducing a blood sample from the subject into a microfluidic channel to which is bound a tumor specific binding agent so that circulating tumor cells in the blood sample bind to the binding agent; lysing cells bound to the channel; and obtaining genetic information from the lysate, wherein the lysate is not purified prior to obtaining the genetic information.
  • Particular genetic information that may be obtained includes: for lung cancer, the presence or absence of an EGFR mutation; for prostate cancer, the presence or absence of a TMPRSS2-ERG fusion or level of PSA; and for breast cancer, genetic information on HER2.
  • Exemplary EGFR mutations are a deletion in exon 19, T790M, L858R, L861Q, G719(S, A, or C), S768I, an insertion in exon 20, and a combination thereof.
  • Exemplary TMPRSS2-ERG fusions are T3/E4, T2/E2, Tl/E3_5, T2/E5, Tl/E234_6, T1/E2, T1/E3, T2/E4, T1/E5, T1/E6, Tl/E_IIIa_4, Tl_I/E_IIIb_4, Tl/E_IIIc_4, T5/E4, T4/E4, T4/E5, and a combination thereof.
  • This method may further include treatment selection based on the obtained genetic information.
  • the treatment may include, for example, tyrosine kinase inhibitors.
  • a further method of the invention for capturing circulating, nonhemopoietic tumor cells includes passing a blood sample of less than 4 mL through a microfluidic channel to which is bound a tumor specific binding agent so that circulating tumor cells in said blood sample bind to said binding agent.
  • the invention also features a method of obtaining genetic information from a subject with a tumor by obtaining 1 to 1500 circulating tumor cells from a blood sample of 1 mL or less from the subject and assaying the cells for genetic information. For example, at 5, 10, 25, 50, 100, 200, 500, or 1000 CTCs may be obtained per mL.
  • Yet another method of the invention is for diagnosing a carcinoma in a subject and includes introducing a blood sample from the subject into a microfluidic channel to which is bound a tumor specific binding agent so that circulating tumor cells in the blood sample bind to the binding agent, wherein the presence of one circulating tumor cell is diagnostic for the presence of the carcinoma, and the absence of any circulating tumor cells is diagnostic for the absence of the carcinoma.
  • the blood sample assayed may have a volume of 50 ⁇ L or more, e.g., 100 ⁇ L, 250 ⁇ L, 500 ⁇ L, 1 mL, or 5 mL. In other embodiments, the sample may have a volume of less than 4 mL.
  • Preferred samples are anticoagulated whole blood.
  • CTCs capture during any of the methods of the invention may be enumerated. This enumeration (and any assaying for genetic information) may be repeated over time, e.g., a period of days, weeks, months, or years.
  • a change in the number of tumor cells over time may be indicative of the prognosis of the nonhemopoietic tumor. For example, a negative slope of the number of tumor cells as a function of time is indicative of a positive prognosis, and a positive slope or no slope of the number of tumor cells as a function of time is indicative of a negative prognosis.
  • CTCs obtained by the methods of the invention may be assayed for genetic information, for example, as described in connection with kits of the invention.
  • Cells may be assayed for changes in genetic information over time as well as or in the alternative to enumeration, e.g., to monitor for the appearance of mutations that indicate a change in therapy is advisable.
  • the blood sample is from a subject at risk for a clinically localized tumor, wherein the presence of tumor cells bound in the device is diagnostic for the clinically localized tumor.
  • exemplary clinically localized tumors occur in prostate cancer, renal cancer, and bone cancer.
  • the methods of the invention may also include lysing CTCs bound to the binding agent.
  • the tumor specific binding agent may be disposed in a region of the microfluidic channel, and the lysing may include applying a lysing agent to a portion of the region where a majority of the circulating tumor cells are bound.
  • the tumor specific binding agent is disposed in a region of the microfluidic channel, and the lysing includes applying a lysing agent to the entire region.
  • Methods of the invention may further include washing the bound tumor cells at a higher shear stress or volume than that used in the introducing step to increase purity, e.g., by reducing the number on weakly bound or non-specifically bound cells in the device compared to bound CTCs.
  • Information on CTCs obtained using the methods of the invention may also in general be used for diagnosis, prognosis, therapy selection, triage, or long term surveillance of subjects. In particular, analysis of CTCs from prostate cancer patients may be used to determine whether the tumor is indolent or aggressive.
  • lysis is meant disruption of a cell membrane sufficient to allow extracellular access to cellular nucleic acids. Lysis may occur by any means, e.g., chemical, thermal, optical, or mechanical.
  • cancer a tumor confined to its tissue of origin, as determined by radiographic measures.
  • micro fluidic is meant having at least one dimension of less than 1 mm.
  • nonhemopoietic cell any cell not of hemopoietic origin, that is excluding blood cells and immune cells.
  • nonhemopoietic cells include epithelial cells, endothelial cells, neurons, hepatocytes, nephrons, glial cells, muscle cells, skin cells, adipocytes, fibroblasts, chondrocytes, osteocytes, and osteoblasts.
  • tumor specific binding agent any agent that binds to a nonhemopoietic cell that can form a tumor, either benign or malignant.
  • the binding agent may bind to a cell surface marker that is specific for a type of cell that can form a tumor and that is not normally found in circulating blood.
  • the binding agent may bind to a cell surface marker that is specific for a transformed cell.
  • Such agents may also bind to healthy cells circulating in blood from non-pathogenic origins, e.g., venipuncture or trauma.
  • FIG. 1 CTC capture and enumeration
  • the plot represents number of cells spiked versus number of cells recovered, (d-k) Higher magnification (2Ox) images of captured CTCs and hematologic cells from NSCLC patients, stained with DAPI, CK, and CD45. Merged images identify CTCs in panels d-g and hematologic cells in panels h-k.
  • FIG. 3 Enumeration of CTCs from cancer patients, (a) Summary of samples and CTC counts per 1 mL of blood in patients with various advanced cancers and localized prostate cancer, (b) Frequency of CTCs per 1 mL of blood, by diagnosis. The box plot presents the median, lower, and upper quartiles (25th, 75th percentiles). Data points that lie outside the 10th and 90th percentiles are shown as outliers, (c) Purity of captured CTCs (ratio of CTCs to total nucleated cells), by diagnosis, (d-i) Serial CTC assessment.
  • FIG. 4 Characterization of CTCs with tumor specific molecular markers
  • a-b CTCs from a prostate cancer patient stained positive for DAPI and PSA expression
  • c RT-PCR amplification of PSA transcript is seen in two patients with prostate cancer (PCa), but not in two patients with lung cancer (LuCa), and only in blood fractions enriched for CTCs as opposed to non-enriched fractions (non- CTC).
  • d-e CTCs from a NSCLC patient stained for DAPI and TTF-I.
  • RT-PCR shows the expression of TTF-I in two patients with lung cancer (LuCa), but not in two patients with prostate cancer (PCa), and only when RNA was eluted from blood fractions enriched for CTCs as opposed to non-enriched fractions (non-CTC).
  • FIG. 1 Microfluidic approach to isolate circulating tumor cells, (a) One-step process for point-of-care isolation of CTCs from peripheral blood, (b) Schematic of the manifold assembly. The microfluidic chip is sealed from above with a biological grade adhesive tape and placed in the manifold, (c) Scanning electron micrograph (SEM) image of the microposts array.
  • SEM Scanning electron micrograph
  • Figure 6 Design criteria and computational analysis of hydrodynamics in the microfluidic chip, (a) Comparison of the hydrodynamic efficiency of different post array arrangements by distance between the posts using square, diagonal square, and equilateral triangular arrays, (b) Computational analysis of the micropost array.
  • Cell trajectories (solid lines) are based on particle tracings and the end positions of the cells are indicated by red dots. Cells starting from positions between the microposts followed the streamline and never came into contact with the microposts, an observation predicting detrimental impact on target cell capture. To address this concern, a vertical periodic shift of 50 ⁇ m was incorporated after every 3 rows of microposts, forcing cells to change their trajectory and hence enhancing the probability of collision with microposts.
  • the area covered in each image is lmm x lmm.
  • Spiked cells are pre-labeled with cell tracker dye to fluoresce in orange.
  • the scale bar indicates 1 OO ⁇ m.
  • CTCs green arrows indicate leukocytes.
  • the top left inset shows a magnified view of a CTC and the lower left inset shows a magnified view of a leukocyte,
  • e Comparison of capture efficiency between whole blood and lysed RBC blood
  • f Concordance experiment to test experimental variability of split samples analyzed under identical conditions.
  • Figure 9 Gallery of CTC images captured from various metastatic epithelial cancers.
  • the first column shows low magnification fluorescent images of CK+ cells in lung, prostate, pancreas, and colon cancers.
  • the scale bar indicates 1 OO ⁇ m.
  • the remaining columns show higher magnification images in which the scale bar indicates 1 O ⁇ m.
  • g-j Prostate cancer CTC cluster (2 cells) stained DAPI+, CK+, CD45-, and the merged image of DAPI and CK fluorescent images
  • l-o Pancreatic cancer CTC stained DAPI+, CK+, CD45-, and the merged image of DAPI and CK fluorescent images
  • q-t Colon cancer CTC cluster (3 cells) stained DAPI+, CK+, CD45-, and the merged image of DAPI and CK fluorescent images
  • v-y Fluorescent images of a leukocyte (PBMC) stained DAPI+, CK-, CD45+, and the merged image of DAPI and CD45.
  • PBMC leukocyte
  • FIG. 10 Serial assessments of patients using both the CTC-chip and standard radiographic monitoring.
  • CTC quantity (cells/mL) depicted in red, and tumor size (sum of measurable diameters in cm) in blue, are well correlated over the course of anti-cancer treatments for nine individual patients.
  • Three of the patients are shown here, whose diagnoses and specific therapies are as follows: (a) NSCLC: lst- line carboplatin, paclitaxel, and an experimental agent, (b) Colorectal cancer: lst-line infusional 5FU, oxaliplatin, and bevacizumab.
  • NSCLC lst-line carboplatin
  • paclitaxel paclitaxel
  • Colorectal cancer lst-line infusional 5FU, oxaliplatin, and bevacizumab.
  • One of the samples had 0 CTC/mL, which may be due to insufficient volume
  • Esophageal cancer lst-
  • Figure 11 is a series of images of gels and sequencing runs from prostate cancer molecular diagnostics.
  • Figure 12 is a schematic depiction of lysis of cells in specific regions of a device of the invention.
  • FIG. 13A is a schematic representation of CTC-chip analysis.
  • Whole blood is collected from the patient and passed through the CTC-chip, containing 78,000 microposts coated with antibody to the epithelial surface antigen EpCAM.
  • EpCAM epithelial surface antigen
  • captured cells are stained in situ using antibody to cytokeratin; for molecular studies, captured cells are lysed on the chip and eluted DNA undergoes the desired analysis.
  • Figure 13B is a fluorescent photomicrograph of CTCs captured against the sides of the functionalized microposts (dashed lines superimposed on images). DNA staining is used to identify all nucleated cells within a field. Cells here are also stained with rhodamine-conjugated antibody to cytokeratin (red) or fluorescein-conjugated antibody to EGFR (green); magnification 200X
  • Figure 13C is a scanning electron microscopic image of a single CTC captured from a patient with NSCLC (arrow).
  • Figure 14 A is a table showing allele-specific SARMS analysis of EGFR mutations in NSCLC tumor samples. Tumor specimens subjected to standard EGFR sequencing analysis were reanalyzed using the highly sensitive allele specific SARMS assay, and correlated with clinical outcome.
  • SARMS Scorpion Amplification Refractory Mutation System
  • mo months
  • Del deletion
  • Und undetected
  • SD stable disease
  • PR partial response
  • CR complete response
  • PD progressive disease
  • NA not applicable
  • Figure 14B is a graph showing progression-free survival estimates for patients treated with gefitinib or erlotinib therapy.
  • Figure 15A is a series of graphs showing serial analyses of CTC numbers, genotypes, and radiographic tumor burden. Dynamic quantification of isolated CTCs per milliliter (dotted) and radiographic tumor burden in centimeters (triangles) in four patients with EGFZ?-mutant NSCLC, measured at multiple time points during the course of treatment with gefinitib, chemotherapy or experimental agents (Expt). Time in days is displayed along the x-axis. Duration of each therapy is indicated by blue bars. CTC-genotypes determined by SARMS assay are shown in boxes at various time points. Bracketed mutations indicate those present at low allele frequencies.
  • Figure 15B is a series of graphs showing SARMS analysis of EGFR genotypes in patient 9 demonstrating increased allelic abundance of the T790M drug resistance allele at the time of disease progression.
  • Arrows denote the cutoff for amplification cycles (Ct) required for detection of the primary mutation (Deletion) and the T790M mutation, compared with the Exon 2 control.
  • ⁇ Ct reflects the difference in allele frequency between the primary mutation and T790M in the tumor tissue biopsy, the CTCs isolated at the time of gefitinib-responsive disease, and the CTCs isolated at the time of disease progression.
  • Figure 15C are graphs showing nucleotide sequencing tracings from patient 2 in which the tumor tissue biopsy analysis demonstrates a T751_I759delinsS mutation that is distinct from the Del 746 750 mutation present in the CTC analysis.
  • the 27 nucleotides deleted in the tumor are present in the CTC DNA (lower box, upper panel), while the 15 nucleotide deletion in the CTC DNA (upper box, lower panel) is present in the tumor DNA (lower box, lower panel).
  • the CTC tracing represents direct nucleotide sequencing of DNA lysed from cells captured on the CTC-chip, indicating a high degree of captured tumor cell purity.
  • CTC-chip a unique microfluidic platform capable of efficient and selective separation of viable CTCs from peripheral whole blood samples, preferably mediated by the interaction of target CTCs with antibody
  • the devices of the invention are unique in that they are able to sort rare cells directly from whole blood in a single step. From a technical perspective, this is possible because the device is the first microfluidic device that can successfully process milliliter volumes of whole blood, although, as described herein, the high number of CTCs recovered using the devices allows for the use of lower volumes of blood. This contrasts with magnetic bead-based systems ° that require multiple "bulk” semi-automated preparatory steps (centrifugation, washing, and incubation), resulting in loss and/or destruction of a significant proportion of the rare cells.
  • the present devices are readily adaptable for potential use in various clinical scenarios, including changes in throughput and in the functionalized binding agent on the channel, allowing capture of other types of rare circulating cells.
  • the invention's one-step potential and versatility make it conducive to point-of-care use and rapid integration into clinical practice.
  • the devices are also distinctive in that their gentle nature (e.g., maximum shear stress may be 0.4 dynes/cm 2 ) allows for isolation of viable cells, whereas magnetic bead-based approaches can only isolate fixed, nonviable cells.
  • the stationary nature of the captured cells in the present invention allows wash-out of non-specifically bound cells, e.g., leukocytes, resulting in a 10 6 -fold enrichment, a level of purity that is two orders of magnitude higher than existing technologies.
  • the capacity to isolate concentrated, viable CTCs makes the present invention an ideal tool for molecular access to rare CTC subpopulations such as metastatic precursor cells or cancer stem cells.
  • the devices and methods of the invention achieve capture of CTCs at high sensitivity (defined as the percentage of patients having a tumor identified as having CTCs); high specificity (defined as the percentage of patients not having a tumor identified as not having CTCs); and high purity (defined as the percentage of CTCs relative to other cells retained by the device).
  • high sensitivity defined as the percentage of patients having a tumor identified as having CTCs
  • high specificity defined as the percentage of patients not having a tumor identified as not having CTCs
  • high purity defined as the percentage of CTCs relative to other cells retained by the device.
  • the devices and methods of the invention are also highly efficient, capturing on average 155 ⁇ 236 (mean ⁇ s.d.) CTCs/mL for NSCLC, 16 to 292 (86 ⁇ 78) for metastatic prostate, 25 to 174 (94 ⁇ 63) among localized prostate cancer, 9 to 831 (196 ⁇ 228) for pancreatic, 5 to 176 (79 ⁇ 52) for breast, and 42 to 375 (121 ⁇ 127) for colorectal, well above that typically obtained with other techniques, such as magnetic enrichment.
  • the invention is capable of utilizing whole, anticoagulated blood (although not limited thereto) without any further sample treatment steps, such as dilution, centrifugation, red blood cell lysis, cell fixation, or cell labeling.
  • a CTC-chip of the invention includes a microfluidic channel having tumor specific binding agents bound to the surface of the channel and which is capable of supporting fluid flow at the desired shear stress.
  • the microfluidic channel may include posts or other interior structure to increase the surface area of the channel and, in some instances, increase the probability that a given cell passing through the channel will come into contact with a binding agent.
  • the channel walls are substantially planar. When channel walls are substantially planar, the height of the channel may be designed so that CTCs readily contact the binding moieties.
  • Devices may, or may not, include regions that allow for optical or visual inspection of the channels.
  • Fluid pumps capable of producing desired shear stress in the device are also known in the art. Examples of pumps include syringe pumps, peristaltic pumps, and vacuum sources. Methods for coupling pumps to devices are known in the art.
  • the device may be configured for substantially constant shear stress in any given channel or variable shear stress in a given channel. Exemplary devices are described herein.
  • the CTC-chip (Fig. Ib) includes an array of microposts (Fig. 5c) that are chemically functionalized with antiepithelial cell adhesion molecule (EpCAM) antibodies.
  • EpCAM provides specificity for CTC capture from unfractionated blood as it is frequently overexpressed by carcinomas of lung, colorectal, breast, prostate, head and neck, and hepatic origin. 23 " 24 A description of a manifold for use with a CTC chip is found in International Publication No. WO 2006/108101. Two essential parameters that determine the efficiency of cell capture on the
  • CTC-chip are (1) flow velocity, as it influences the duration of cell-channel (e.g., post) contact, and (2) shear force, which must be sufficiently low to ensure maximum cell-channel (e.g., post) attachment.
  • flow velocity as it influences the duration of cell-channel (e.g., post) contact
  • shear force which must be sufficiently low to ensure maximum cell-channel (e.g., post) attachment.
  • Fig. 6b-d linear shear stress chamber studies
  • the volume of the channel or the region having the binding agents may also be altered depending on the volume of the blood sample being employed.
  • the volume of the channel (defined as that portion through which cells may pass) may be larger than the size of the sample.
  • a transporting fluid which may be miscible or immiscible with the sample, may be employed to ensure that the sample comes into contact with the binding agents.
  • Suitable transporting fluids include air and buffer. At least two variables can be manipulated to control the shear stress applied to the channel: the cross sectional area of the chamber and the fluid pressure applied to the chamber.
  • Pumps that produce suitable shear forces in combination with channels of the invention preferably produce a unidirectional shear stress, i.e., there is no reversal of direction of flow, and/or substantially constant shear stress. Either unidirectional or substantially constant shear stress may be maintained only during the time in which a sample is passed through a channel. Washing or labeling steps after target cells have bound to the device may utilize reversals of flow or changes in shear stress.
  • the shear stress is not necessarily constant but is kept below a critical value for the duration of binding of target cells to a channel.
  • the flow rate will typically be between 0.1 mL to 30 mL/hr. Dilution of blood may be employed at high flow rates, e.g., above 10 mL/hr.
  • the channel may include one or more binding agents (e.g., 1, 2, 3, 4, 5, or more). Multiple binding agents may bind to the same or different cells and may be placed in the same or different channels. For example, binding agents to multiple cell surface markers that occur on a desired cell may be disposed in one channel.
  • binding agents e.g., 1, 2, 3, 4, 5, or more.
  • channels are arranged in series, (e.g., 2, 3, 4, 5, or more channels).
  • each channel isolates one or more types of cells, which may or may not be the cells of interest.
  • the shear stress applied to each of the channels can be different (achieved for example by varying the cross sectional area of the channels) or the shear stress can be the same.
  • each channel can contain binding agents that bind to different cell surface markers or the same cell surface markers.
  • the methods may also be employed to isolate various types of analytes in parallel, e.g., by passing aliquots of the same sample through separate devices or one device including multiple channels in parallel. Different samples may also be assayed in parallel.
  • Devices of the invention may be fabricated using techniques known in the art.
  • fabrication techniques employed will depend on the material used to make the device. Examples of fabrication techniques include molding, photolithography, electroforming, and machining. Exemplary materials include glass, polymers (e.g., polystyrene, silicones such as polydimethylsiloxane, epoxy, polymethylmethacrylate, and urethanes), silicon and other semiconductors, and metals.
  • polymers e.g., polystyrene, silicones such as polydimethylsiloxane, epoxy, polymethylmethacrylate, and urethanes
  • silicon and other semiconductors e.g., silicon and other semiconductors.
  • Devices of the invention may be combined with pumps, detectors, and other laboratory components.
  • the devices of the invention may include one or more inlets, e.g., to deliver two or more different fluids simultaneously or at different times.
  • fluids that may be introduced into a device include washing buffers, e.g., to remove nonspecifically bound cells or unused reagents, lysing reagents, or labeling reagents, e.g., extracellular or intracellular stains.
  • devices of the invention are designed to have removable covers to allow access to all of the region in which cell may be bound or a portion thereof. With these devices, it is possible to apply reagents, e.g., labeling reagents or lysing reagents, to specific regions. Individual cells may also be removed from such devices, e.g., using a pipette.
  • the device has more than one inlet and outlet to allow the introduction of more than one fluid to the device, typically at different times.
  • fluids may be introduced simultaneously in the device to lyse or otherwise manipulate bound cells in specified regions. The size of these regions may be controlled based on the location of the inlets and outlets and the relative volumetric flow rates from the inlets and outlets, under the principles of laminar flow occurring in microfluidic channels.
  • the devices may in principle be employed for any rare cell separation that employs a selective binding agent, i.e., an agent that binds to the target cell and not to (or at least not to the same extent) to a non-target cell.
  • a preferred rare cell is a circulating tumor cell of epithelial origin from peripheral blood.
  • Other rare cells include organisms potentially found in peripheral blood (e.g., bacteria, viruses, protists, or fungi), other nonhemopoietic cells not normally found in blood (e.g., endothelial cells or fetal cells), and even cells of hemopoietic origin (e.g., platelets, sickle cell red blood cells, and subpopulations of leukocytes).
  • binding agent or agents employed will depend on the type of cell or cells being targeted. In general, specific binding agents for these cells are known in the art. Exemplary types of binding agents include antibodies, antibody fragments (e.g., Fc fragments), oligo- or polypeptides, nucleic acids, cellular receptors, ligands, aptamers, MHC-peptide monomers or oligomers, biotin, avidin, oligonucleotides, coordination complexes, synthetic polymers, and carbohydrates. Binding moieties may be attached to chambers using methods known in the art. The method employed will depend on the binding moiety and the material used to construct the device. Examples of attachment methods include non-specific adsorption to the surface, either of the binding moiety or a compound to which the binding moiety is attached or chemical binding, e.g., through self assembled monolayers or silane chemistry.
  • An exemplary binding agent is anti-EpCAM antibody, which is specific for epithelial cells.
  • circulating epithelial cells may provide clinical and diagnostic information relevant to tumors, even those considered clinically localized.
  • Cancers that may be detected using the devices of the invention include prostate, lung, adenocarcinoma, adenoma, adrenal cancer, basal cell carcinoma, bone cancer, brain cancer, breast cancer, bronchi cancer, cervical dysplasia, colon cancer, epidermoid carcinoma, Ewing's sarcoma, gallbladder cancer, gallstone tumor, giant cell tumor, glioblastoma multiforma, head cancer, hyperplasia, hyperplastic corneal nerve tumor, in situ carcinoma, intestinal ganglioneuroma, islet cell tumor, Kaposi's sarcoma, kidney cancer, larynx cancer, leiomyoma tumor, liver cancer, malignant carcinoid, malignant hypercalcemia, malignant melanomas, marfanoid habitus tumor, medullary carcinoma
  • the invention provides methods in which the cells isolated may be used to provide additional information.
  • cells isolated using the methods and devices of the invention can be further assayed using additional methods of the invention.
  • cells that are isolated using the methods and devices of the invention are counted.
  • Cells can be counting by any method known in the art, including optical, e.g., visual inspection, automated counting, microscopy based detection, and FACS, and electrical detection, e.g., Coulter counters.
  • Counting of the cells, or other analytes, isolated using the methods and devices of the invention can be useful for diagnosing diseases, monitoring the progress of disease, and monitoring or determining the efficacy of a treatment.
  • Cell, or other analyte, counting may also be of use in non-medical applications, e.g., for determination of the amount, presence, or type of contaminants in environmental samples (e.g., water, air, and soil), pharmaceuticals, food, or cosmetics.
  • environmental samples e.g., water, air, and soil
  • cells isolated using the methods and devices of the invention can be lysed, and one or more properties of the cells, or portions thereof, can be measured.
  • biological properties that can be measured in isolated cells include mRNA expression, protein expression, and DNA quantification.
  • the DNA of cells isolated by the methods of the invention can be sequenced, or certain sequence characteristics (e.g., polymorphisms and chromosomal abnormalities) can be identified using standard techniques, e.g., FISH or PCR.
  • sequence characteristics e.g., polymorphisms and chromosomal abnormalities
  • the chemical components of cells, and other analytes may also be assayed after isolation.
  • Cells may also be assayed without lysis, e.g., using extracellular or intracellular stains or by other observation, e.g., morphology or growth characteristics in various media.
  • lysis e.g., CTCs may be lysed while still bound to the chip, e.g., with any other cells nonspecifically retained.
  • the ability to lyse CTCs on chip and obtain useful genetic information is made possible by the high purity of samples (typically greater than 50%) using the devices and methods of the invention.
  • Particular genetic information that may be obtained from a tumor cell captured by a CTC-chip includes identification or enumeration of particular genomic DNA, cDNA, or mRNA sequences; identification or enumeration of cell surface markers; and identification or enumeration of proteins or other intracellular contents that is indicative of the type or presence of a particular tumor.
  • CTCs may be analyzed to determine the tissue of origin, the stage or severity of disease, or the susceptibility to a particular treatment.
  • a diagnostic indicator for lung cancer and other cancers is the presence or absence of certain mutations in EGFR
  • PSA prostate specific antigen
  • RNA extracted from the CTCs is detectable by protein expression staining using antibodies that measure PSA expression within cells captured on the chip (immunofluorescence and immunohistochemistry), and it is also detectable by reverse transcription polymerase chain reaction (RT-PCR) in RNA extracted from the CTCs.
  • RT-PCR reverse transcription polymerase chain reaction
  • TMPRSS2-ERG prostate cancer-specific chromosome translocation
  • the translocation results in the abnormal expression of a transcription factor (gene regulator), which, as a result of the translocation, is now driven by androgen.
  • a transcription factor gene regulator
  • androgen testosterone
  • CTCs captured by the devices and methods described herein may also be assayed for the presence of markers indicative of cancer stem cells.
  • markers include CDl 33, CD44, CD24, epithelial-specific antigen (ESA), Nanog, and BMIl.
  • Example 1 Metastatic lung, prostate, pancreas, breast, and colon cancer
  • a CTC -chip successfully identified CTCs in the peripheral blood of patients with metastatic lung, prostate, pancreas, breast, and colon cancer in 115 of 116 (99%) samples, with a range of 5-1281 CTC/mL and approximately 50% purity.
  • CTCs were isolated in 7/7 patients with early stage prostate cancer. Given the high sensitivity and specificity of the CTC-chip, we tested its potential utility in monitoring response to anti-cancer therapy. In a small cohort of patients with metastatic cancer undergoing systemic treatment, temporal changes in CTC numbers correlated reasonably well with the clinical course of disease as measured by standard radiographic methods.
  • NSCLC NSCLC cells
  • PCBS phosphate buffered saline
  • NSCLC cells were visually evident about EpCAM-coated microposts, whereas no cancer cells were seen following flow through uncoated posts (Fig. 8a-c).
  • the calculated capture efficiency was 65% and decreased significantly at flow rates above 2.5 mL/h (Fig. 2a), presumably due to increased shear stress, consistent with our simulation predictions.
  • the efficiency of capture was not enhanced at flow rates less than 0.75mL/h, leading us to select a flow rate of 1-2 mL/h for subsequent studies.
  • CTCs captured from a group of patient samples were identified using a comprehensive image analysis algorithm, consisting of staining with DAPI for DNA content, rhodamine-conjugated anti-cytokeratin (CK) antibodies for epithelial cells, and fluorescein-conjugated anti-CD45 antibodies for hematologic cells (Fig. 3d-k, Fig. 9) in order to confirm the validity of CK stain. Thereafter, cells captured by anti- EpCAM-coated microposts and staining for CK were scored as CTCs, while CD45- positive cells were scored as contaminating normal hematologic cells.
  • DAPI rhodamine-conjugated anti-cytokeratin
  • CD45-positive cells were scored as contaminating normal hematologic cells.
  • the morphologic characteristics exhibited by the captured CTCs were consistent with malignant cells, including large cellular size with a high nuclear: cytoplasmic ratios and visible nucleoli (Fig. 2d-g).
  • CTCs were identified in 115 of 116 (>99%) patient samples analyzed, with the single negative specimen being a small volume sample (0.9mL) from a colorectal patient.
  • the number of CTCs isolated ranged from 5 to 1,281/mL for NSCLC (155 ⁇ 236 (mean ⁇ s.d.) CTCs/mL), 16 to 292 (86 ⁇ 78) for metastatic prostate, 25 to 174 (94 ⁇ 63) among localized prostate cancer, 9 to 831 (196 ⁇ 228) for pancreatic, 5 to 176 (79 ⁇ 52) for breast, and 42 to 375 (121 ⁇ 127) for colorectal (Fig. 3a, b).
  • the identification of CTCs in subjects with clinically localized prostate cancer at numbers approximating those in metastatic prostate cancer patients is a novel finding enabled by the high sensitivity of our technique.
  • PSA prostate specific antigen
  • TTF-I thyroid transcription factor- 1
  • FIG. Ia The microfluidic system described in these examples (Fig. Ia) consists of a microfluidic chip etched in silicon (Fig. Ib), a manifold to enclose the chip (Fig. Ic, Fig. 5b), and a pneumatic pump (Fig. 1 a) to establish the flow through the chip (Fig. Ic).
  • the schematic of the microfluidic system is depicted in Figure 5b.
  • the dimensions of the chip are 25mm x 66mm, with an active capture area of 19mm x 51mm. It contains an equilateral triangular array of microposts, lOO ⁇ m tall and lOO ⁇ m in diameter with an average 50 ⁇ m gap between microposts (Fig. 5c).
  • microposts were fabricated with deep reactive ion etching (DRIE) by Silex (Stockholm, Sweden).
  • DRIE deep reactive ion etching
  • Blood samples were drawn from patients with advanced stage solid tumors before, during, and after chemotherapy at Massachusetts General Hospital under an IRB-approved protocol. Blood specimens were also drawn from healthy donors after obtaining informed consent. All specimens were collected into vacutainer tubes containing the anticoagulant EDTA and were processed within 24hrs. Between sample collection and sample processing, whole blood specimens were stored at 4°C on a rocking platform to prevent cell settling. For experiments using lysed blood, NH 4 Cl was added to whole blood in 10:1 v/v ratio and mixed for 15-20 minutes at room temperature. Following centrifugation at 1050 rpm (10°C) for 5 minutes, the supernatant was removed, and the pellet re-suspended in an equivalent volume of buffer and stored on a lab mixer at 4°C.
  • the silicon chips were purged with nitrogen and sealed with pressure-sensitive adhesive tape (3M, St. Paul, MN.)-
  • the sealed microfluidic devices were then placed in a transparent 2 inch x3 inch plastic manifold consisting of a base, top cover and a spacer (Fig. 5b).
  • the base has inlet and outlet ports for fluid handling.
  • the manifold base also has six guiding metal pillars, each lmm in height, to hold the device in place and in alignment with the inlet and outlet ports.
  • a metal spacer placed between the base and the top cover prevents mechanical stress on the device.
  • the base and top cover attach by screws, providing a leak-proof assembly with minimum dead-volume. For ease of operation the port dimensions are such that standard Luer fittings can be used.
  • a pneumatic macrofluidic drive system was specifically designed to control flow through the microfluidic CTC-chip, as shown in Figure Ia. It uses a pneumatic pump, pressure regulators, and a digital pressure display to control the pressure of the air used to drive blood from a sealed sample container into the CTC-chip.
  • a rocker assembly provides sample mixing throughout the experiment. Prior to running samples through the chip, the device was purged with 3.0 mL of buffer. A 5 mL aliquot of blood sample was measured into a conical tube, sealed, placed on the rocker unit, and connected to the chip with low dead volume fittings (Fig Ia). The sample was allowed to mix on the rocker for at least 5 minutes before running the experiment. The pneumatic pump was turned on, and the pressure adjusted according to the required flow rate. After the experiment, the CTC-chip was flushed with 10.0OmL PBS at lOmL/hr to remove any non-specifically bound cells.
  • Olympus SZX Olympus America Inc., NY
  • ProScan stage Primary Scientific Inc., MA
  • Captured images at 10Ox total magnification were carefully examined, and the objects that met predetermined criteria were counted. Color, brightness and morphometric characteristics such as cell size, shape, and nuclear size were considered in identifying potential CTCs and excluding cell debris and non-specific cells.
  • Cell viability was determined with the LIVE/DEAD viability assay kit. This assay is based on intracellular esterase activity of live cells and plasma membrane integrity of dead cells. Briefly, captured CTCs were incubated at room temperature for 30 minutes in a solution of 2 ⁇ M calcein AM and 4 ⁇ M ethidium bromide prepared in PBS. At the end of the incubation period, the chip was washed with ImL of 1 x PBS and visualized under microscope.
  • the 509 base pair human PSA coding region was amplified from circulating prostate tumor cell cDNA using the following primers pairs (sense and antisense, 5 '-3'): primary PCR: (TTGTGGGAGGCTGGGAGTG and CCTTCTGAGGGTGAACTTGCG; SEQ ID NO: 1), secondary PCR: (GGCAGGTGCTTGTGGCCTCTCGTGG and GTCATTGGAAATAACATGGAGGTCC; SEQ ID NO: 2).
  • the TTF-I transcript was amplified using the following primer pairs (sense and antisense, 5 '-3'): primary PCR: (CTGCAACGGCAACCTGGGCAACATG ; SEQ ID NO: 3 and CAGGTACTTCTGTTGCTTGAAGCG; SEQ ID NO: 4), secondary PCR: (CAGGACACCATGAGGAACAGCGCCTC; SEQ ID NO: 5 and CAGGTACTTCTGTTGCTTGAAGCG; SEQ ID NO: 6).
  • Micropost geometry and the arrangement of the micropost array were systematically explored in the process of designing the CTC-chip.
  • Three different micropost distributions and arrangements were tested: a square array, a diagonal square array, and an equilateral triangular array.
  • the area occupied by the microposts for the square and triangular distribution is given by: For a square array,
  • the analysis indicated an equilateral triangular micropost arrangement with a 50 ⁇ m distance between microposts and with a 50 ⁇ m shift after every 3 rows of microposts to be the most efficient micropost geometric arrangement and spacing.
  • the CTC-chip surface was functionalized with EpCAM antibodies using Avidin-Biotin chemistry.
  • the surface of the chip was modified with 4% (v/v) 3- mercaptopropyl trimethoxysilane in ethanol at room temperature for 45 min, then treated with the coupling agent N- ⁇ -maleimidobutyryloxy succinimide ester (GMBS, l ⁇ M) resulting in GMBS attachment to the microposts.
  • GMBS, l ⁇ M N- ⁇ -maleimidobutyryloxy succinimide ester
  • the chip was treated with lO ⁇ g/mL of Neutravidin at room temperature for 30 min leading to immobilization onto GMBS, and then flushed with PBS to remove excess Avidin.
  • biotinylated EpCAM antibody at a concentration of lO ⁇ g/mL in phosphate buffered solution (PBS) with 1% (w/v) BSA and 0.09% (w/v) sodium azide was allowed to react for 15-30 minutes before washing with PBS.
  • PBS phosphate buffered solution
  • the chip was air dried and stored at ambient temperature for up to three weeks until use.
  • the human non-small-cell lung cancer (NSCLC) cell line NCI-H1650 was maintained and grown to confluence in RPMI- 1640 medium containing 1.5mM L- glutamine supplemented with 10% fetal bovine serum at 37°C in 5% CO 2 with humidity. Growth medium was aspirated, and cells incubated with trypsin for 10 minutes. A protein buffer was added to quench protease activity. Cells were then pre- labeled with cell tracker orange using the standard protocol provided by the manufacturer. The cell titer was determined by counting with a hemocytometer. The desired concentration of cells was then prepared by serial dilution of the original cell suspension in PBS. Labeled cells were spiked into whole blood.
  • Captured cells were fixed by flowing 0.9mL of 1 % PFA in PBS, through the device at 3.0mL/hr for 20 minutes. The chip was subsequently washed with a solution of 0.9mL of 0.2% Triton X-100 in PBS for 10 minutes to induce cellular permeability and allow for intracellular staining. To identify any bound lymphocytes, 0.9mL of anti-CD45 stock solution (50 ⁇ L of antibody stock solution in ImL of PBS) was passed through the chip at 3mL/hr for 30 minutes, followed by a PBS wash to remove excess antibody.
  • 0.9 mL of anti-cytokeratin stock solution 50 ⁇ L of antibody stock solution in ImL of PBS was passed through the chip at 3mL/hr for 30 minutes, followed by a PBS wash.
  • 0.9mL of DAPI solution (lO ⁇ l of DAPI reagent in ImL of DI water) was passed through the chip at 3mL/hr, for 15 minutes followed by a PBS wash.
  • the chip was removed from the manifold, wiped dry near the fluid ports and stored in the dark at 4°C until imaging. Shear stress studies using linear shear Hele-Shaw chambers
  • An optimum shear stress should be applied such that one can capture maximum number of cancer cells at high enough flow rates.
  • the geometry of these chambers (Fig. 6d) is such that the shear stress varies linearly along the chamber length (Fig. 6e), permitting the study of a wide range of shear stresses for a given flow rate.
  • Cultured lung cancer cells were spiked into PBS solution, and then passed through the Hele-Shaw chambers functionalized with EpCAM Ab at a constant flow rate.
  • the shear stress decreased along the channel, the density of the cells adhering to the micropost surface increased (Fig. 6, a-c).
  • the effect of shear stress on cell adhesion through EpCAM antibody-antigen binding (Fig. 6f) indicated that 8 dyn/cm 2 was the optimum shear rate, resulting in the capture of 200 cells/mm 2 of functionalized capture surface.
  • the coupling agent GMBS N- ⁇ - maleimidobutyryloxy succinimide ester
  • NHS-LC-LC-biotin succinimidyl-6 1 - [biotinamido]-6-hexanamido hexanoate
  • fluorescein-conjugated NeutrAvidin were obtained from Pierce Biotechnology (Rockford, IL).
  • Biotinylated mouse anti- human anti-EpCAM was obtained from R&D Systems (Minneapolis, MN).
  • Human non-small-cell lung cancer line NCI-H1650, prostate cell line PC3-9, breast cancer cell line SKBr-3 and bladder cancer cell line T-24 were purchased from American Type Culture Collection (Manassas, VA), and RPMI- 1640 growth medium was purchased from Invitrogen Corporation.
  • Orange [5- (and 6-)-(((4-chloromethyl)- benzoyl) amino) tetrarnethyl-rhodamine, CMTMR] and green [5- chloromethylfluorescein diacetate, CMFDA] cell tracker dyes were obtained from Molecular Probes (Eugene, OR).
  • Anti-Cytokeratin PE CAM 5.2, conjugated with phycoerythrin
  • CD45 FITC the fluorescent nucleic acid dye nuclear dye 4',6- diamidino-2-phenylindole (DAPI) and 1OmL vacutainer tubes was purchased from BD Biosciences (San Jose, CA).
  • Figure 11 shows the results of RT-PCR of prostate CTCs run on a gel and the sizes of different isoforms. Most bands were one size (Tl :E4), but in the GU34 fraction one band was shorter, corresponding to Tl :E5. Sequencing chromatograms below the gel illustrate the different breakpoints (Tl :E4 and Tl :E5) at the DNA level.
  • Example 2 EGFR mutations in NSCLC
  • NSCLC non-small cell lung cancer
  • the genomic DNA extracted from CTCs was amplified twice using the TransPlex nucleic acid amplification kit (Rubicon Genomics) according the manufacturer's protocol.
  • the EGFR mutations were detected in the amplified material using the EGFR Mutation Test Kit from DxS Ltd. This assay can detect EGFR mutations in a background of large quantities of wild type genomic DNA.
  • the present invention in combination with the fractionation method to isolate genomic DNA and RNA offers a blood-based molecular diagnostics that provides a new and exciting approach to monitor genetic lesions in circulating tumor cells, possibly circumventing the requirement for serial biopsies of inaccessible solid tumors.
  • this approach has tremendous potential to provide an ideal tool that will enable cancer biologists unprecedented molecular access to rare CTC subpopulations.
  • Blood samples were obtained from 23 patients with £GFi?-mutant advanced NSCLC, including 5 treatment-na ⁇ ve patients, 10 previously treated with either erlotinib or gefitinib, and 8 previously treated with chemotherapy or multiple regimens including TKIs and chemotherapy.
  • the strategy used for microfluidic CTC isolation from whole blood is schematically depicted, along with representative images of captured cells in Figs. 13A-C.
  • CTCs were identified in all patients, with a mean of 131 cells/ml (range 5 to 771 cells/ml), which was not different from the quantity identified in patients with EGFR wild-type NSCLC (Table 4).
  • Tumor burden was measured by unidimensional diameter as per RECIST.
  • the allele-specific SARMS assay which is designed to detect 7 types of EGFR mutations, including the multiple in-frame exon 19 deletions (collectively analyzed as "Del" mutations) and the L858R missense mutation, which together account for 90% of sensitizing EGFR mutations.
  • the assay also detects the recurrent T790M mutation associated with acquired TKI resistance. Since the SARMS assay has not previously been compared to standard sequencing analysis of paraffin-embedded tumor samples, we first analyzed 26 NSCLC tumors previously identified as EGFi?-mutant and 8 tumors reported as wild-type by sequencing analysis (Fig 14A).
  • SARMS analysis and nucleotide sequencing identified the same mutation in 25 cases, while all 8 wild-type cases were confirmed negative yielding a sensitivity of 96% and a specificity of 100%.
  • the single discrepancy was due to a unique deletion mutation that is not within the detection capacity of the SARMS assay.
  • Presence of the drug resistance mutation at such low frequency did not preclude significant responses to TKI therapy, but it was associated with a striking difference in progression-free survival (PFS) with a median PFS of 7.7 months in cases with a detectable T790M allele, compared to 16.5 months in cases lacking T790M (PO.001) (Fig 14B).
  • PFS progression-free survival
  • the preexisting drug resistance allele may be rapidly selected following TKI therapy, possibly accounting for some of the known variation in response duration among EGFi?-mutant NSCLC.
  • T790M allele was detectable in CTCs from 2/6 patients responding to TKI therapy and 9/14 patients with clinical progression.
  • SARMS Scorpion Amplification Refractory Mutation System
  • Del deletion
  • na sample not available for analysis
  • NA not applicable due to unavailable tumor for comparison
  • Und undetected
  • SARMS assay The detection of T790M by SARMS assay is indicated by "+” or "-”.
  • CTCs CTCs
  • P free plasma DNA analysis
  • the SARMS assay groups all variant breakpoints of the in-frame EGFR deletion mutations as a single "Del" mutation, and all mutations at codon 719 as "G719X”.
  • the detection of T790M by SARMS assay is indicated by "+” or "-”.
  • the detection of other mutations by SARMS assay is listed only when present.
  • the mutation identified by sequencing in patient 2 is not within the detection capacity of the SARMS assay, and therefore SARMS analysis would be expected to be negative.
  • EGFR TKI therapy was either gefitinib or erlotinib and was administered as l st -line therapy for advanced disease except where indicated by " " (EGFR TKI therapy was given 2 nd - or 3 rd -line), or by "none" (no EGFR TKI was given). Duration of therapy was measured in months, and when preceded by ">" indicates ongoing therapy. Best clinical response is defined per RECIST.
  • CTC genotypes evolved during the course of therapy, with consistent presence of the primary EGFR activating mutation, but emergence of the T790M drug resistance mutation. While this mutation was present at very low allele frequency in the initial tumor specimen, as determined by the relative number of cycles required for amplification ( ⁇ Ct), serial analysis indicated increased prevalence over time consistent with acquisition of clinical resistance (Fig 15B). Remarkably, some cases also showed the emergence of additional EGFR activating mutations. While in most cases, these additional mutations were less prevalent than the primary mutation, at least one case clearly demonstrates the potential for evolution in the dominant tumor genotype (Fig. 15C). In this case, sufficient DNA was isolated from captured CTCs to allow direct nucleotide sequencing of EGFR, confirming that the dominant mutation in CTCs differs from that present in the original tumor specimen.
  • CTC-chip technology By studying patients with advanced EGFZ?-mutant NSCLC, we have shown that the CTC-chip technology reproducibly identifies CTCs in sufficient quantity and with sufficient purity to allow molecular analyses that are relevant to clinical management. CTCs were readily identified in all cases, in numbers that are approximately 100-fold higher than with currently other technology. In cases studied longitudinally, CTC numbers over time showed a significant decline associated with tumor response to EGFR TKIs, with rising numbers as drug resistance emerged. CTC genotypes were highly concordant with the mutational status of tumor biopsy specimens, and provided additional information as they were serially repeated during the course of the disease. Taken together, these molecular studies provide novel insight into the progression of EGi*7?-mutant NSCLC, and illustrate the potential impact of CTC-based serial noninvasive monitoring in epithelial cancers.
  • T790M mutation In addition to the primary activating EGFR mutation, we also identified the secondary T790M mutation associated with acquired TKI-resistance. Consistent with results from serial biopsy and autopsy studies, the T790M mutation was commonly observed in CTCs from patients progressing on ⁇ GFR TKI therapy. Unexpectedly, use of the highly sensitive allele-specific assay showed that a subset of NSCLCs harbor rare T790M alleles prior to TKI exposure. The T790M allele is thought to emerge through selective pressure during therapy, although it has been reported in rare cases without prior drug exposure and has been shown to encode additional transforming properties when combined in cis with the more common EGFR activating mutations.
  • the T790M allele may initially arise by virtue of its oncogenicity, and rapidly emerge as a dominant allele following drug treatment. Presence of rare T790M alleles in pre-treatment tumor specimens did not preclude dramatic clinical responses to TKIs, but did have a very significant impact on the progression-free survival. This molecular marker may therefore be a major determinant distinguishing patients likely to have a prolonged response to single agent erlotinib or gefitinib, from those whose response is likely to be short-lived and who may be appropriate candidates for second-generation irreversible TKIs or combination targeted therapy regimens.
  • Amplification of the gene encoding the growth factor receptor MET has recently been reported as a second mechanism of acquired resistance to EGFR TKIs 19 ' 20 .
  • the number of tumor biopsy specimens available for comparison of EGFR sequencing and SARMS analysis was extended by inclusion of Group B patients, who had participated in a multi-center phase II clinical trial utilizing first-line gefitinib therapy. Medical charts of all patients were reviewed for demographics and clinical history, with tumor burden at various time points quantified as the sum of the unidimensional size of all measurable tumor sites (as per RECIST) via central review. Patients who had received therapy with an EGFR TKI (gefitinib or erlotinib) were assessed for length of therapy and best clinical response using RECIST. The correlation between CTC quantity and computed tomography (CT scan) measurements was analyzed using the Spearman correlation coefficient. The relationship between progression-free survival on EGFR TKI therapy (time from start of therapy until tumor progression by RECIST or death, whichever was sooner) was analyzed using the Kaplan-Meier method and the Log-Rank test. CTC capture and enumeration
  • the number of CTCs/ml was determined via comprehensive image analysis, scanning the entire chip (Olympus SZX microscope, Olympus America Inc., NY) and identifying CTCs based on cell size, morphology, and fluorescence staining (Hoechst, CK positive). For demonstration of EGFR expression, captured cells were stained with a mouse monoclonal antibody (Vector Laboratories). Molecular analysis of CTCs DNA was eluted from captured CTCs using the Pico Pure DNA Extraction Kit (Molecular Devices) and subjected to two rounds of linear amplification using the Transplex amplification kit (Rubicon Genomics).
  • Free plasma DNA was isolated using Vacutainer PPT Plasma Preparation Tubes and the QIAmp DNA Blood Midi Kit (Fisher Scientific/ DNA was prepared from paraffin-embedded tumor blocs using standard Proteinase K isolation.
  • SARMS assay DxS Delivery Pharmacogenomics
  • 1.5 ng of DNA was analyzed using the ABI 7500 Detection System.
  • the assay detects grouped deletions within exon 19, insertions within exon 20 and mutations affecting codon 719 (G719X), as well as the individual mutations T790M, L858R, L861Q, and S768I.
  • the rate of amplification of these mutant alleles is compared with a control amplification of EGFR exon 2.
  • Standard bidirectional nucleotide sequencing was performed using a Capillary ABI 3100 sequencer (Applied Biosystems, Foster City, CA).

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Abstract

L'invention propose, d'une façon générale, des dispositifs et des procédés pour capturer des cellules rares, par exemple des CTC à partir d'échantillons de sang. Les dispositifs de l'invention sont capables de capturer de grands nombres de CTC viables en une seule étape à partir de sang entier sans pré-dilution, pré-marquage, préfixation ou tout autre étape de traitement. Les techniques décrites ici et l'application plus large de la technologie microfluidique de capture de cellules rares pour des patients présentant un cancer sont significativement prometteuses pour identifier des déterminants biologiques clés de métastases à diffusion hématogène et pour fournir une plateforme robuste visant à un diagnostic précoce et à une surveillance longitudinale du cancer.
PCT/US2008/011785 2007-10-17 2008-10-16 Dispositifs à base de micropuce pour capturer des cellules tumorales circulantes et procédés pour leur utilisation WO2009051734A1 (fr)

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US99926607P 2007-10-17 2007-10-17
US60/999,266 2007-10-17
US99119307P 2007-11-29 2007-11-29
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