WO2007070784A2 - Detection du cancer par fluorescence et elimination chirurgicale d'une tumeur guidee par fluorescence - Google Patents

Detection du cancer par fluorescence et elimination chirurgicale d'une tumeur guidee par fluorescence Download PDF

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WO2007070784A2
WO2007070784A2 PCT/US2006/061902 US2006061902W WO2007070784A2 WO 2007070784 A2 WO2007070784 A2 WO 2007070784A2 US 2006061902 W US2006061902 W US 2006061902W WO 2007070784 A2 WO2007070784 A2 WO 2007070784A2
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instrument
tissue
tumor
fluorescent
kit
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PCT/US2006/061902
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WO2007070784A3 (fr
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Eben Rosenthal
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The Uab Research Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B18/18—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves
    • A61B18/20—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser
    • A61B18/22—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser the beam being directed along or through a flexible conduit, e.g. an optical fibre; Couplings or hand-pieces therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
    • A61B17/32—Surgical cutting instruments
    • A61B17/3209—Incision instruments
    • A61B17/3211—Surgical scalpels, knives; Accessories therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B18/04—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by heating
    • A61B18/12—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by heating by passing a current through the tissue to be heated, e.g. high-frequency current
    • A61B18/14—Probes or electrodes therefor
    • A61B18/1402—Probes for open surgery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
    • A61B2017/00017—Electrical control of surgical instruments
    • A61B2017/00022—Sensing or detecting at the treatment site
    • A61B2017/00057—Light
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B90/00—Instruments, implements or accessories specially adapted for surgery or diagnosis and not covered by any of the groups A61B1/00 - A61B50/00, e.g. for luxation treatment or for protecting wound edges
    • A61B90/08—Accessories or related features not otherwise provided for
    • A61B2090/0803—Counting the number of times an instrument is used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B90/00—Instruments, implements or accessories specially adapted for surgery or diagnosis and not covered by any of the groups A61B1/00 - A61B50/00, e.g. for luxation treatment or for protecting wound edges
    • A61B90/08—Accessories or related features not otherwise provided for
    • A61B2090/0813—Accessories designed for easy sterilising, i.e. re-usable
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B90/00—Instruments, implements or accessories specially adapted for surgery or diagnosis and not covered by any of the groups A61B1/00 - A61B50/00, e.g. for luxation treatment or for protecting wound edges
    • A61B90/39—Markers, e.g. radio-opaque or breast lesions markers
    • A61B2090/3937—Visible markers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B90/00—Instruments, implements or accessories specially adapted for surgery or diagnosis and not covered by any of the groups A61B1/00 - A61B50/00, e.g. for luxation treatment or for protecting wound edges
    • A61B90/20—Surgical microscopes characterised by non-optical aspects

Definitions

  • the present invention relates to detection and removal of cancerous tissue at the primary or metastatic site. More particularly, the present invention relates to fluorescent imaging to detect cancer and guide surgical removal using one or more fluorescently labeled immunoconjugates, surgical instruments that contain a detectable fluorescent signal distinct from autofluorescent signals from the tissue or instrument, and visualization instruments.
  • Tumor specific methods to detect disease in previous operated fields are of benefit in detecting tumor recurrence which normally require a combination of PET/CT and operative evaluation.
  • Tumor specific tagged antibodies can significantly improve the sensitivity of detecting a tumor and allow earlier detection.
  • HNSCC head and neck squamous cell carcinoma
  • HNSCC currently affects more than 42,000 people in the United States annually with a five year survival that is less than 60% for oral cavity squamous cel ⁇ carcinoma.
  • survival rate has remained unchanged for over 30 years.
  • tumor margins are determined intraoperatively by a combination of palpation, visual inspection, and microscopic assessment frozen tissue sections. With the exception of previously untreated tongue cancers, assessment of tumor margins by intraoperative palpation is limited because tumors are adjacent to bone (such as palate or alveolus), adjacent to cartilage (larynx), in deep tissues (pterygoid muscles) * or in previously irradiated tissues. More accurate assessment of tumor extent could limit the ablative defect size.
  • Intraoperative froze sections have significant deficiencies: frozen sections I) require resection of additional resection of normal tissue for assessment, 2) add between 30 and 60 minutes to the operative time, 3) are difficult to interpret in patients with field cancerizatio ⁇ or previous irradiation, 4) cannot be performed on bone or calcified cartilage, 5) are reversed on permanent section in approximately 5% of cases and 6) fail to detect close margins (>5mm). Intraoperative tumor imaging can provide surgeons with clear delineation of tumor margins and assess residual disease.
  • Radio-guided surgery presents several unwanted and non-beneficial components.
  • tumors are targeted by 1-125 labeled antibodies raised against a common tumor antigen and assessed intraoperatively using a hand-held, gamma detecting probe.
  • Such techniques are not widely used for numerous reasons. First, it requires injecting the patient with radioactive materials which arc associated with high costs and increased patient morbidity, Second, it gives the surgeon little anatomical information about the local extent of the tumor. Such a fack of information leads to one of two results.
  • EGFR epidermal growth factor receptor
  • HNSCC has the highest rates of EGFR overexpression compared to other cancers and is upregulated early in tumor progression.
  • Cetuximab labeled with Tc-99m has been found to have dosimetric properties of a good diagnostic imaging agent.
  • the pooling of cetuximab within the liver in humans was circumvented by administering the labeled antibody after a loading dose of unlabeled antibody.
  • Radiolabeled antibodies arc used intraoperatively for tumor detection; this technique has been popularized in sentinel lymph node assessment (also called radioimmunoguided surgery [RIGS]).
  • RIGS radioimmunoguided surgery
  • tumors are targeted by 1-125 labeled antibody raised against a common tumor antigen (such as carcinoembryonic antigen, or CEA) and assessed intraoperatively using a hand-held, gamma-detecting probe.
  • RIGS radioimmunoguided surgery
  • Tissue autofluorescence may be used in diagnosis of cervical cancer.
  • NIR fluorescence for biomedical imaging allows deep tissue penetration, is biocompatible, and limits tissue autofluorescence. This highly sensitized technique has not yet been applied to tumors of the upper digestive tract.
  • Anti-EGFR antibodies conjugated to quantum dots and gold nanoparticles have been used to identify pre-malignant lesions of the cervix using topical application.
  • Soukos, et al. (Cancer Res 63:1999-2004, 2003) have used EGFR targeting to demonstrate regression of premalignant lesions in the hamster cheek model after intravenous administration. Because hamsters do not express human EGFR protein, the background fluorescence expected in humans may not be detected in this model.
  • Hsu, et al. Photochm Photobiol 79:272-279, 2004
  • used topical application of far-red fluorescent dye conjugated to cetuximab to demarcate abnormal from normal tissue in 500 urn thick multilayer tissue constructs.
  • the present invention provides fluorescence-immuno-detection of tumor at the primary and regional or distant metastatic site and fluorescence-guided surgery.
  • the present invention provides surgical instruments useful for performing a tissue manipulation procedure, wherein the surgical instruments emit a detectable fluorescent signal when excited by light distinct from autofluorescent signals from said tissue.
  • the fluorescent signal emanates from a coating on at least some portion of the surgical instruments.
  • the fluorescent signal emanates from a marking comprising paint, an adhesive sticker, or alloy disposed on the instruments.
  • the fluorescent signal emanates from near-infrared (NIR) fluorescence.
  • NIR near-infrared
  • the surgical instruments provided by the present invention include but are not limited to instruments comprising an electrocautery tip and/or a fiberoptic laser delivery tip, cutting instruments including but not limited to scissors and/or scalpels, grasping instruments including but not limited to forceps and/or clamps, tissue marking devices and/or probes.
  • the surgical instruments are provided as disposable, single-use devices.
  • the surgical instruments are provided as reusable devices capable of repeated cleaning and sterilization.
  • kits for a tissue manipulation procedure comprising a plurality of surgical instruments provided by the present invention.
  • Each surgical instrument provided in the kit emits a unique intensity level of detectable fluorescent signal.
  • the kit provided by the present invention further comprises one or multiple fluorescently labeled immunoconjugates, such as antibodies, in a clinical delivery device, e.g., a preloaded injection syringe.
  • the kit provided by the present invention also comprises an infrared light- emitting light source and a near infrared filter suitable for use with a standard endoscope for fluorescent imaging.
  • the kit provided by the present invention also comprises an adaptor for fluorescent stereomicroscopic imaging. The adaptor can further comprise associated computer software and hardware for fluorescent imaging.
  • the present invention further provides methods of intraoperative tissue manipulation and removal.
  • Such methods comprise administering one or more fluorescently labeled immunoconjugates to a subject, said immunoconjugates being capable of targeting the tissue of interest, visualizing said tissue tagged with said one or more fluorescently labeled immunoconjugates (e.g., antibodies) with a visualization instrument, and performing a tissue manipulation procedure using the surgical instruments provided by the present invention.
  • the fluorescently labeled antibody is a monoclonal antibody capable of targeting said tissue.
  • the fluorescently labeled antibody is monoclonal antibody targeting to epidermal growth factor receptor (EGFR). including cetuximab.
  • the fluorescently labeled antibody is antibody UFA which is the fully humanized form of cetuximab and also reacts with EGFR.
  • the fluorescently labeled antibody comprises a near infrared fluorophorc, including Cy5.5.
  • the fluorescently labeled antibody is Panitumab (Amgen).
  • the present invention provides for detection of distant or regional metastasis through minimally invasive techniques.
  • Fluorescently labeled Immunoconjugates are systemically injected and the relevant body cavity imaged for possible disease using endoscopes adapted for fluorescent imaging. Instruments provided for in this application can then be used to biopsy sites of high fluorescent activity.
  • video assisted thorascopy is used to image for possible pulmonary metastasis within the parenchyma or mediastinal lymphadenopathy.
  • minimally invasive techniques arc used to image for cervical metastasis in a minimally invasive or open technique.
  • the present invention further provides for the use of a visualization instrument which comprises an attachment to a conventional stereomicroscope and/or endoscope fitted with a near infrared filter and a charge coupled device camera particularly designed for use in the surgical procedure.
  • the visualization instrument further comprises a light source, and an optical imaging camera at the end of said visualization instrument.
  • the visualization instrument is a standard endoscope comprising an adaptor for fluorescent imaging, wherein said adaptor further comprise associated computer software.
  • the fluorescently labeled antibody is administered 30 minutes to 72 hours prior to intraoperative visualization.
  • the present invention further provides the use of the surgical instruments with the visualization instrument described above for removal of tumors and affected tumor tissues in a surgical procedure, wherein said surgical instruments emit a detectable fluorescent signal when excited by light distinct from autofluorescent signals from said tumor and affected tumor tissue.
  • the present invention provides a method of intraoperative tumor tissue detection, manipulation and/or removal using fluorescently labeled antibodies, the surgical instruments that emit a unique intensity level of detectable fluorescent signal, and a visualization instrument, all described above, in a surgical procedure.
  • the target tumors suitable under these procedures include, but are not limited to, tumors of the upper digestive tract, such as head and neck tumors, esophageal tumors, and/or other tumors, such as pancreatic tumor, lung tumors, cutanous non-melanoma skin cancers, colon/rectal cancer, any primary or metastasis tumors including, but not limited to tumors in lymph nodes and/or pulmonary metastasis tumors.
  • the target lumor it> a head and neck squamous cell carcinoma (HNSCC) or an orai cavity squamous cell carcinoma (OCSCC).
  • HNSCC head and neck squamous cell carcinoma
  • OCSCC orai cavity squamous cell carcinoma
  • the target tumors are tumors within lymph nodes.
  • the target tumors are pulmonary metastasis tumors.
  • the present invention also provides a device for intraoperative tissue detection, manipulation, and/or removel.
  • a device for intraoperative tissue detection, manipulation, and/or removel comprises one or more fluorescently labeled immunoconjugates including, but not limited to, antibodies that arc capable of targeting said tissue, a stereomicroscope, a light emitting device, a device for receiving fluoresced light emitted from said tissue, one or more fluorescently coated surgical instruments described above, and a camera for displaying and recording the fluoresced light.
  • the light emitting device transmits light capable of exciting the fluorescently labeled tissue and causing said tissue to emit fluorescent light distinguishable from the transmitted light.
  • the device for receiving emitted light is a charge coupled device camera that is capable of displaying images in real-time, and receiving light in the near infrared spectrum.
  • the present invention also provides a method of determining the prognosis of a disease condition in a tissue in vivo.
  • Such method comprises the steps of administering to a patient a detection effective amount of a fluorescently labeled antibody specific for the disease condition in the tissue, and visualizing and comparing a fluorescent pattern in the tissue with a fluorescent pattern in normal tissue to determine the prognosis of the disease condition.
  • the disease tissue comprises tumor tissue from tumors of the upper digestive tract, such as head and neck tumors, esophageal tumors, and/or other tumors, such as pancreatic tumor, lung tumors, cutanous non-melanoma skin cancers, colon/rectal cancer, any primary or metastasis tumors including, but not iimitod to tumors in lymph nodes and/or pulmonary metastasis tumors.
  • the disease tissue is a head and neck squamous cell carcinoma (UNSCC) or an oral cavity squamous cell carcinoma (OCSCC).
  • the present invention a Hows for detection of tumor avidity for the labeled immimoconjugate in order to predict tumor response to the targeted agent or medical treatment.
  • the intensity of tumor fluorescence pr ovides information about antibody uptake in the tumor that translates to therapeutic response to the antibody or medical therapy as a whole. IB comparison to the limited success met with measuring ligand expression in the tumor of interest. Uptake of fluorescent immunocongate guides clinical responses and helps physicians make treatment decisions as to whether patients should continue current treatment or be shunted to a different treatment modality.
  • the present invention further allows for monitoring of therapy with a combination of therapeutic and diagnostic antibodies. More particularly, the present invention provides a method of determining the prognosis and monitoring therapy of a disease condition further comprising administering to the patient a treatment effective amount of an unlabeled antibody specific for the disease tissue and/or tumor at approximately the same time as, or simultaneously with, the fluorcscently labeled same antibody.
  • the combination of fluorescently labeled and unlabeled antibodies in a single preparation and/or administration at about the same time can be used for diagnostic and therapeutic purposes with little added toxicity.
  • the present invention allows the clinician to monitor disease response in the clinic with endoscopic equipment previously mentioned, and further allows the clinician to make a decision on continuation of medical treatment or shunt the patient to alternative treatment including more aggressive medical approaches or surgery.
  • Figure 1 shows the increase in fluorescence of HNSCC tumors following administration of Cy5.5 cetuximab.
  • Figure 2 shows via histological sections the fluorescence of HNSCC tumors following treatment with Cy5.5 cetuximab.
  • Figure 3 shows the increase in fluorescence of 1 INSCC tumors following administration of Cy5.5 cetuximab.
  • FIG. 4 shows the results of Cy5.5-cetuximab injected into SCID mice bearing engrafted human skin and human tumor xenografts.
  • the present invention provides fluorescence-immuno-tissue detection and fluorescence guided surgery. More particularly, the present invention provides surgical instruments useful for performing a tissue manipulation procedure, wherein the surgical instruments emit a detectable fluorescent signal when excited by light distinct from autofluorescent signals from said tissue.
  • surgical instruments refer to any medical instruments, devices and/or tools, in all types and sizes, that are used in a surgical procedure.
  • Exemplary surgical instruments include, but are not limited to, medical instruments comprising an electrocautery tip and/or a fiberoptic laser delivery tip, cutting instruments including but not limited to scissors, scalpels, surgical knifes, and blades, grasping instruments including but not limited to forceps and clamps, tissue marking devices, probes, and other instruments including, but not limited to, curettes, dermatomes, dilators, hemostats, photocoagulators, retractors, snares, and trephins.
  • the surgical instruments are provided as single-use devices, being designed to be disposed of after one single use.
  • the surgical instruments are provided as reusable devices capable of repeated cleaning and sterilization.
  • cleaning and sterilization refers to any acts and procedures that make the surgical instruments free of live bacteria or other microorganisms, usually by heat or any chemical means, and yet maintains the property of emitting a detectable fluorescent signal when excited by light.
  • the fluorescent signal emanates from a coating on at least some portion of the surgical instruments.
  • coating refers to a thin layer covering a portion or the entire surface of the surgical instrument.
  • the coating may be in form of pre-applied label or applied immediately prior to the procedure.
  • the coating can be made by any conventional methods known in the art, including, but not limited to painting, adhesive substrates (labels or stickers), or disposing a metal alloy on the instruments containing the desired type and amount of fluorescence for the particular application.
  • the fluorescent signal emanates from any conventional fluorescence materials and/or dyes, also known as fluorophores.
  • fluorophore is analogous to a chromophore and is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. The amount and wavelength of the emitted energy depend on both the fluorophore and the chemical environment of the tluorophore. Many natural and synthetic compounds that exhibit fluorescence and/or fluorophores are known in the art and can be used in the present invention.
  • a near infrared fluorophore including Cy5.5 (available from Amersham).
  • Cy5.5 available from Amersham
  • This near infrared fluorophore is biocompatible, allows deep tissue penetration, and limits tissue autofluorescence when used for biomedical imaging.
  • the fluorescent signal is excited by broad spectrum iighls, UV, far red spectrum, near infrared spectrum, and any other exciting sources known in the art.
  • the prevent invention further provides a kit for a tissue manipulation procedure comprising a plurality of surgical instruments described above.
  • Each surgical instrument provided in the kit emits a unique intensity level of detectable fluorescent signal.
  • the term "unique intensity level" refers to a serial of or a set of instruments of calibrated fluorescent markings on the surgical instruments.
  • a kit of the present invention provides a set of surgical instruments, e.g. 5 pieces of the same instrument, comprising 1 to 5 units of fluorescence coating and/or alloy disposed on each instrument. When excited, each instrument emits a distinct intensity level of detectable fluorescent signal corresponding to the fluorescent quality coated on that instrument.
  • the kit of the present invention provides a set of adhesive stickers coated with a series of fluorescent materials in various intensity that is calibrated. These fluorescently coated instruments can then be selected by the surgeon to match the intensity of fluorescently labeled tissues in order to better visualize the field of surgery.
  • the kit provided by the present invention further comprises one or more fluorescently labeled immunoconjugates.
  • immunoconjugates refers to antibodies and fragments thereof, but also includes any protein substance produced in the blood or tissues in response to a specific antigen, such as a bacterium or a toxin, that destroys or weakens bacteria and neutralizes organic poisons, thus forming the basis of immunity, or an immunoglobulin present in the blood serum or body fluids as a result of antigenic stimulus and interacting only with the antigen that induced it or with an antigen closely related to it.
  • the antibody used herein is a monoclonal antibody capable of targeting cellular receptors and/or proteins specially expressed or over-expressed by tissue of interest.
  • the fluorescently labeled antibody is monoclonal antibody targeting epidermal growth factor receptor (EGFR), including cetuximab.
  • EGFR epidermal growth factor receptor
  • the fluorescently labeled antibody is antibody UFA. which is the fully humanized version of cetuximab, and also reacts to EGFR.
  • Many other antibodies and immunoconjugates, and their functional fragments thereof, that are capable of targeting tissues of interest, can also be used in the present invention, preferably, such antibodies, and the functional fragments thereof, can be used for therapeutic, diagnostic and detection purposes.
  • the detection refers to a fluorescent imaging of targeting tissues and/or tumors.
  • fluorescently labeled antibodies provided by the kit of the present invention are stored in a clinical delivery device, e.g., a preloaded injection syringe, and are ready for use to be injected into a subject for treatment and fluorescent-guided detection and surgery.
  • a clinical delivery device e.g., a preloaded injection syringe
  • the term "subject” refers to any animals and mammals of interests, preferably., a human being.
  • tissue of interest refers to any bodily tissues, organs, glands, cells that are in either normal or defected conditions.
  • the tissue is a tumor.
  • tumor refers to an abnormal growth of tissue resulting from uncontrolled, progressive multiplication of cells and serving no physiological, but rather pathological, functions.
  • the tumor used herein refers to any tumor and/or cancer in any part of a human body, including, but not limited to, the upper digestive tract tumors, such as the head and neck tumors and esophageal tumors, and other tumors, such as pancreatic tumor, lung tumors, cutanous non-melanoma skin cancers, and colon/rectal cancer.
  • the target tumor is a head and neck squamous cell carcinoma (FfNSCC) or an oral cavity squamous cell carcinoma (OCSCC).
  • the tissue of interest refers to glands, including, but not limited to thyroid and parathyroid glands.
  • tissue of interest also includes metastasized tumor that has spread to distant sites (liver or lung) or to regional sites (lymph nodes). Under these conditions the fluorescently labeled antibody and associated imaging equipment and instruments can be used for diagnostic purposes or therapeutic purposes to image the site(s). Confirmation of distant disease and directed biopsies based on fluorescence can be performed. Alternatively, dissection and sampling of the lymph nodes or fluorescently guided biopsies of distant metastatic sites can be performed.
  • the kit provided by the present invention also comprises an infrared light-emitting light source and a near infrared filter suitable for use with a standard endoscope for fluorescent imaging.
  • the kit provided by the present invention also comprise an adaptor for fluorescent imaging, said adaptor further comprises associated computer software, hardware and light source for fluorescent imaging.
  • the present invention further provides a method of intraoperative tissue detection, manipulation and/or removal.
  • Such method comprises administering one or more fluorescently labeled immunoeonjugates, such as antibodies, to a subject, said immunoconjugates being capable of targeting the tissue of interest, visualizing said tissue tagged with said one or more fluorescently labeled immunoconjugates with a visualization instrument, and performing a tissue manipulation procedure using the surgical instruments provided by the present invention.
  • the term ' ⁇ visualization instrument refers to any instruments that capable of assisting a surgeon to visualize the operation areas in a surgery.
  • the visualization instrument refers to a standard stereomicroscope and/or endoscope fitted with a near infrared filter for fluorescence imaging, and a charge coupled device camera particularly designed for use in the surgical procedure.
  • the visualization inslrument comprises a light source, and an optical imaging camera at the end of said visualization instrument.
  • the visualization instrument is a standard endoscope comprising an adaptor for fluorescent imaging, wherein said adaptor further comprising associated computer software.
  • the visualization instrument is a fluorescent detecting microscope.
  • the fluorescently labeled antibody is administered between 30 minutes to 72 hours, preferably 30, 60, 90 or 120 minutes prior to intraoperative visualization.
  • the present invention provide a method of intraoperative removal of tumors using fluorescently labeled antibodies, the surgical instruments that emit a unique intensity level of detectable fluorescent signal, and a visualization instrument, all described above, in a surgical procedure.
  • a device for intraoperative tissue detection, manipulation and/or removal under ⁇ mrrranofluorescent guidance comprises one or more fluorescently labeled immunoconjugates including, but not limited to, antibodies that are capable of targeting said tissue, a stereomicroscope, a Hghl emitting device, a device for receiving fluoresced light emitted from said tissue, one or more surgical instruments described above, and a camera for displaying and recording the fluoresced light.
  • the light emitting device transmits light capable of exciting the fluorescently labeled tissue and causing said tissue to emit fluorescent light distinguishable from the transmitted light.
  • the device for receiving emitted light is a charge coupled device camera that is capable of displaying images in realtime, and receiving light in the near infrared spectrum.
  • the present invention also provides a method of determining the prognosis of a disease condition in a tissue in vivo comprising administering to a patient a detection effective amount of a fluorescently labeled antibody specific for the disease condition in the tissue, and visualizing and comparing a fluorescent pattern in the disease tissue with a fluorescent pattern in normal tissue to determine the prognosis of the disease condition.
  • prognosis refers to a predicted and/or expected course of a disease including various developments, changes and outcomes of the disease.
  • the term “detection effective amount'" refers to any amount of antibody that is labeled with an amount of fluorescent so that the labeled antibody can be visualized for fluorescent imaging, with and/o ⁇ without a microscope.
  • the present invention further provides a method of monitoring therapy of a disease condition with a combination of fluorescent labeled and unlabeled antibodies for both therapeutic and diagnostic purposes.
  • a treatment effective amount of an unlabeled antibody specific for a disease tissue and/or tumor, and a detection effective amount of a fluorescent labeled same antibody are administered, simultaneously or at approximately the same time, to a patient for both treatment and detection purposes with little added toxicity.
  • the unlabeled and fluorescently labeled antibody are prepared in a single preparation.
  • treatment effective amount refers to any amount of antibody that has therapeutic effects on the disease tissue and/or lurnor such that the disease tissue becomes normal tissue gradually and/or eventually, and the tumor cells are killed or dead with the tumor size reduced and/or disappeared.
  • [O0S2J lmmunofluorescent guided surgery of the present invention improves identification of residual tumor within the resection bed. That is, the tumor beneath the outer level of surrounding tissue which is often the source of residual tumor margin remaining following surgery.
  • the present invention allows rapid assessment of tumor margins after tumor ablation.
  • the lymphatic drainage basins can be assessed intraopcrativcly to identify nests of tumor within or extending out of lymph nodes.
  • routine histology combined with confocal fluorescence can improve the identification of positive or close margins pathologies.
  • the present invention permits immunofluorescence detection and microscopic assisted resection guided by immunofluorescence (MARGIN) for surgical guidance.
  • the present invention further provides a method of increasing the intraoperative visualization of tumors comprising determining one or more antibodies specific to a target tumor, conjugating a fluorophore to said antibodies, and ensuring that fluorescence of surrounding tissue is minimized so as to increase the contrast between said target tumor and said surrounding tissue.
  • the invention further provides a visualization molecule for intraoperative tumor visualization comprising an antibody specific to an antigen expressed or overexpressed on tumor cells conjugated to a fluorophore with known excitation and emission wavelengths.
  • the fluorophore emits light in the near infrared spectrum.
  • the antibody is sequestered by the targeted tumor such that the target tumor fluoresces across the depth of the target tumor.
  • the antibody binds to the target tumor with greater affinity than to surrounding non-tumor tissue.
  • immunofluorescent detection according to the present invention further provides a means of diagnosing HNSCC and other tumors.
  • Fluorescent imaging neoplasm detection (FIND) according to the present invention thus offers a specific and sensitive tool for the detection and resection of cancer.
  • fluorescent imaging tissue detection according to the present invention may be employed in the planning of radiotherapy ports for patients with cancer.
  • Anti-EGFR antibody were fluorescently labeled for imaging.
  • Cetuximab known as C225 or ERBITUX, ImClone Systems Inc, New York, NY
  • Cy5.5 a bisftmctiona! fluorescent dye
  • the fluorescently labeled antibody is referred to ay 'Cy5.5-cetuximab'.
  • ICG insulin glycosides
  • human IgGl isotype control antibody Axxora Life Sciences, San Diego, CA
  • Indocyanine green N-hydroxysulfosuccinimide ester ICG-sulfo-OSu, Daiichi Pure Chemicals, Tokyo
  • Japan can also be used for labeling antibodies. It has a specific fluorescence emission at
  • ICG-IgGl ICG-sulfo-OSu labeled IgGl
  • the flank model was chosen because it allows human skin grafting and human tumor xenografting.
  • Human tumors obtained from patients with previously untreated HNSCC were implanted adjacent to engrafted human skin in immunodeficient 6-week old BALB/c female mice.
  • a sample of tumor used in these experiments was snap frozen in liquid nitrogen at the time of surgical resection and another sample was placed in formalin for analysis of EGFR expression.
  • Minced tumor (four to six l x l x l mm pieces) was inserted subcutaneously and monitored for growth over the course of 4-6 weeks. If the tumor remained viable (about 50% of tumors), initial regression of tumor size was followed by expansion at 4-6 weeks from implantation.
  • Tumors measuring between 2 mm 2 and 10 mm 2 were considered for assessment.
  • Skin grafts between 1 and 2 cm 2 were applied adjacent to tumor sites on the opposite flank.
  • Skin graft tissue was obtained from excess split thickness skin grafts in plastic surgery cases.
  • Skin grafts were secured onto the flank of mice using fine suture (5-0 proline) after removal of a segment of mouse dermis. Skin grafts were bolstered into place with xeroform gauze for 3-4 days and then removed. The viability of skin grafts was confirmed prior to fluorescent imaging studies.
  • Imaging was initially performed at 2, 4, S 3 18, 24, and 48 hours to determine optimal differences in ICG and Cy5.5 fluorescence. Untreated controls (0 ug dose) were also imaged. Note that the two fluorophores were injected together and imaged because there was minimal overlap of excitation and emission between Cy5.5 and ICG. The tumor fluorescence intensity relative to the skin graft was calculated. Tumors and grafts were harvested after imaging and paraffin embedded with serial sectioning to confirm viable tumor, assess the tumor size and for analysis of EGFR expression.
  • Optimal imaging characteristics were determined by the imaging parameters that generated the greatest difference between tumor and skin fluorescence intensity and the time point and dose at which there was the greatest difference between non-specific (IgGl antibody fluorescence) and tumor-specific binding (Cy5.5-cetux ⁇ rnab).
  • mice 100621
  • cetuximab 25 x optimal dose
  • the present invention provides that it is increasingly beneficial if the tumor is fluorescent not only on the surface of the cancerous body, but also internal to the tumor.
  • the benefits of such intratumor fluorescence include increased ability for the surgeon to determine if full resection is complete, the invention provides that the surgeon is able to visually distinguish between the inner portions of the tumor and the surrounding noncancerous tissues, and to more efficiently and accurately remove the tumor.
  • Cetuximab is thought to be internalized following binding to EGFR, therefore, it would be expected that Cy5.5-cetuximab, but not indocyanine green (ICG)-IgGl control antibody would be identified within the tumor cell.
  • Tumors and the skin grafts were harvested at the conclusion of imaging (48 hours) and processed for routine and confocal microscopy. Tissues were paraffin embedded, cut into 8 urn sections and then coverslipped prior to confocal microscopy.
  • a Leica DMIRBE Inverted Microscope was used in conjunction with a Leica SPl Confocal Scanning System to image the tissue.
  • Neon Laser HeNe was used to excite Cy5.5 labeled tissue at 633nm. Emission from the
  • Cy5.5 was captured at a range of 647 to 800 nm using Leica Confocal Software (LCS) and a
  • Tumor specimens were paraffin embedded and histological sections obtained to assess incorporation of fluorescence by confocal microscopy. Incorporation of fluorescence was compared to FGFR expression across cell lines. Irnmunohistocherrustry was also performed on paraffin embedded tumor specimens to confirm that the relative EGFR expression identified in vitro persist after in vivo growth.
  • Immunohistocher ⁇ ical analysis was performed on deparaffinized 8 um sections by incubating anti-EGFR mAb (10 ug/ml; Sigma) and then detected with Anti-Mouse Ig Detection Kit (BD Biosciences). Specimens were incubated with anti-mouse IgG conjugated with horseradish peroxidase at a 1:50 dilution in PBS-BSA solution for 60 min before addition of the DAB substrate. Immunohistochemistry was assessed blinded to the fluorescence data of each specimen. Immunohistochemical reactivity was graded as previously described in Rosenthal et al, Laryngoscope 113:1406-1410, 2003. Total scoring for EGFR reactivity was plotted with the relative intensity of tumor fluorescence corrected for tumor size and weight.
  • mice were anesthetized with isoflurane and evaluated under the stereomicroscope at the optima] imaging time point after injection with Cy5.5-cetuxirnab and ICG-isotype control antibody.
  • a control animal underwent resection without Cy5.5-cetuximab injection.
  • Either visual guidance alone or fluorescence imaging combined with visual inspection was used to perform the tumor resection.
  • Surgical instruments of the present invention can be used with this surgery.
  • Resected specimens were serial sectioned and the distance between the surgical edge (margin) and tumor calculated for each type of resection. Resected tissue was paraffin embedded and serially sectioned in bread loaf fashion, with every other section being stained with H & E. Tumor margins were reviewed in a blinded fashion. The distance between the cut edge and tumor was calculated as 'tumor margin' and has an expected, range 0.1 — 2.0 mm. The number of positive margins (tumor extending beyond surgical specimen) was recorded for each histological section on either side of the resected specimen. Two measurements for each section was obtained (one margin on either side of the tumor specimen).
  • Specimens were paraffin embedded and then sectioned for confocal microscopy (8 um thick) or routine H&E staining (6 urn thick). Note that the tumor treated with Cy5.5-cetuximab was smaller than the untreated tumor. This is consistent with the anti-tumor activity of cetuximab. It should also be noted that H&E sections demonstrate stromal elements within the transplanted tumors which are typical of human HNSCC, but not of cell line xenografts.
  • Cy5.5-cetuximab treated tumors show marked increase in fluorescence over human skin lacking a tumor. This indicates that administration of a tumor specific fluourescently tagged antibody, will distinguish the tumor from the surrounding tissue upon intraoperative visualization.
  • FIG. 3 shows the results of Cy5.5-cetuximab injected into SClD mice bearing engrafted human skin and SCC-I tumor cells. 6-week oid SC ⁇ D mice underwent injection of SCC-I cells into the right flank (5 x 10 5 cells). Skin (1.6 cm 2 ) was obtained as excess/waste tissue from split thickness skin graft procedures and then engrafted onto the opposite flank of the same mouse as described above.
  • the data indicates that the tumor again showed greater than a two-fold increase in fluorescence of the tumor following dosing with Cy5.5- cetuximab.
  • the skin graft showed not only a demonstrably smaller amount of fluorescence than the tumor, but also a statistically insignificant increase following treatment. Therefore, administering Cy5,5-cetuximab promotes a definitive fluorescence increase in targeted tumors but little or no increase in the fluorescence of surrounding, non-diseased tissues.
  • the tumor xenograft showed a greater than two-fold increase in fluorescence following treatment.
  • the skin graft did not show an increase in fluorescence post-treatment and following the treatment shows nearly three-foid less fluorescence than the targeted tumor.
  • Imaging was performed as described above with a custom stereomicroscope and unenhanced digital images captured in grcyscale and near infrared fluorescence and then the imaged fused. The tumor was then dissected from the surrounding tissue and measured. Histological sectioning and H & E staining confirmed the presence of tumor (data not shown).
  • HNSCC tumor cell lines were injected systemically into SClD mice. After 2 or 4 weeks, the animals were euthanized and the thoracic cavity explored. Fluorescent stercomicroscopic imaging was performed as above 48 hours after systemic administration of Cy5.5-cetuximab. Although control mice were found to have some background fluorescence, a distinct pattern of miliary pattern of fluorescence was seen that correlated with histological evidence of pulmonary squamous cell metastasis. This data indicate that the present invention can be used to identify pulmonary metastasis or lung tumors in vivo using minimally invasive techniques.

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Abstract

La présente invention concerne des instruments chirurgicaux qui émettent un signal détectable de fluorescence distinct des signaux d'autofluorescence d'un tissu ou d'un instrument et des procédés d'utilisation des instruments chirurgicaux, d'un ou plusieurs immunoconjugués marqués ou non marqués par fluorescence et d'un instrument de visualisation qui permet de détecter par immunofluorescence et en cours d'opération un tissu cancéreux tant sur le site primaire que sur des sites de métastase, ainsi que la manipulation et l'élimination chirurgicales guidées par fluorescence de ce tissu cancéreux. Des kits qui comprennent des instruments chirurgicaux, des immunoconjugués marqués ou non marqués par fluorescence et des instruments de visualisation qui permettent l'imagerie par fluorescence sont également proposés.
PCT/US2006/061902 2005-12-12 2006-12-12 Detection du cancer par fluorescence et elimination chirurgicale d'une tumeur guidee par fluorescence WO2007070784A2 (fr)

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WO2009089543A2 (fr) * 2008-01-10 2009-07-16 The Ohio State University Research Foundation Système de détection de fluorescence
WO2010076334A3 (fr) * 2008-12-31 2010-09-23 Centre Leon Berard Diagnostic intra-opératoire de tumeurs primaires et de tumeurs secondaires ou de métastases
WO2013162502A1 (fr) * 2012-04-23 2013-10-31 Empire Technology Development Llc Visualisation de tissu pour une résection
CN116385337A (zh) * 2022-12-15 2023-07-04 陕西中科创孚医疗科技有限责任公司 一种基于多光融合的甲状旁腺识别装置及方法
US11871988B1 (en) 2022-12-01 2024-01-16 Michael E. Starzak Mapping and endoscopic excision of a tumor using intracavity laser quenching and emission spectroscopy

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US20020026649A1 (en) * 1997-04-28 2002-02-28 Yuying Tan Metastasis models using green fluorescent protein (GFP) as a marker
US20020029032A1 (en) * 2000-09-07 2002-03-07 Eva Arkin Fluorescent surgical hardware and surgical supplies for improved visualization
US20040006276A1 (en) * 2002-07-05 2004-01-08 The Regents Of The University Of California Autofluorescence detection and imaging of bladder cancer realized through a cystoscope
US20040015062A1 (en) * 2000-11-27 2004-01-22 Vasilis Ntziachristos Fluorescence-mediated molecular tomography
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US5845639A (en) * 1990-08-10 1998-12-08 Board Of Regents Of The University Of Washington Optical imaging methods
US20020026649A1 (en) * 1997-04-28 2002-02-28 Yuying Tan Metastasis models using green fluorescent protein (GFP) as a marker
US20020029032A1 (en) * 2000-09-07 2002-03-07 Eva Arkin Fluorescent surgical hardware and surgical supplies for improved visualization
US20040015062A1 (en) * 2000-11-27 2004-01-22 Vasilis Ntziachristos Fluorescence-mediated molecular tomography
US20040006276A1 (en) * 2002-07-05 2004-01-08 The Regents Of The University Of California Autofluorescence detection and imaging of bladder cancer realized through a cystoscope
US20040241808A1 (en) * 2003-04-14 2004-12-02 Renata Pasqualini Methods for ex vivo hybridoma-free production of polyclonal and monoclonal antibodies and generation of immortalized cell populations

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009089543A2 (fr) * 2008-01-10 2009-07-16 The Ohio State University Research Foundation Système de détection de fluorescence
WO2009089543A3 (fr) * 2008-01-10 2009-10-08 The Ohio State University Research Foundation Système de détection de fluorescence
WO2010076334A3 (fr) * 2008-12-31 2010-09-23 Centre Leon Berard Diagnostic intra-opératoire de tumeurs primaires et de tumeurs secondaires ou de métastases
WO2013162502A1 (fr) * 2012-04-23 2013-10-31 Empire Technology Development Llc Visualisation de tissu pour une résection
US11871988B1 (en) 2022-12-01 2024-01-16 Michael E. Starzak Mapping and endoscopic excision of a tumor using intracavity laser quenching and emission spectroscopy
CN116385337A (zh) * 2022-12-15 2023-07-04 陕西中科创孚医疗科技有限责任公司 一种基于多光融合的甲状旁腺识别装置及方法
CN116385337B (zh) * 2022-12-15 2023-10-17 西安长空医疗科技服务有限公司 一种基于多光融合的甲状旁腺识别装置及方法

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