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WO2020160006A1 - Identification rapide de marges chirurgicales proches sur des échantillons chirurgicaux - Google Patents

Identification rapide de marges chirurgicales proches sur des échantillons chirurgicaux Download PDF

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Publication number
WO2020160006A1
WO2020160006A1 PCT/US2020/015436 US2020015436W WO2020160006A1 WO 2020160006 A1 WO2020160006 A1 WO 2020160006A1 US 2020015436 W US2020015436 W US 2020015436W WO 2020160006 A1 WO2020160006 A1 WO 2020160006A1
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Prior art keywords
tumor
specimen
surgical
fluorescence
margin
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Eben Lloyd ROSENTHAL
Stan VAN KEULEN
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • FSA frozen section analysis
  • kits, and systems for identifying suspicious areas in surgical specimens that potentially contain cancerous cells are provided.
  • the methods utilize fluorescence imaging of a surgical specimen with a fluorescent contrast agent that selectively localizes to cancerous cells to identify fluorescence peak-intensity regions corresponding to locations in the specimen where the distance between the tumor and the margin border is the closest.
  • the methods disclosed herein are useful in identifying areas of a tumor specimen that should be selected for further examination and provide intraoperative guidance to allow a surgeon to take corrective action during procedures.
  • a method of detecting a close or positive margin in a surgical tumor specimen comprising: a) administering a fluorescent contrast agent to a subject, wherein the fluorescent contrast agent selectively localizes to cancerous cells of a tumor in the subject; b) performing a resection of the tumor to produce the surgical tumor specimen; c) illuminating the surgical tumor specimen with light at a fluorescence excitation wavelength of the fluorescent contrast agent; d) performing fluorescence imaging on a tumor margin of the surgical tumor specimen by detecting fluorescent signals emitted from the fluorescent contrast agent localized to the cancerous cells in the tumor specimen; e) plotting a two-dimensional map or a three-dimensional map of the fluorescent signals; f) quantifying the fluorescent signals; g) identifying at least one fluorescence peak-intensity area in the two- dimensional map or the three-dimensional map, wherein the fluorescence peak-intensity area corresponds to a specimen location where the surgical tumor specimen has a close
  • the method further comprises collecting a tissue section of the surgical tumor specimen comprising tissue from the specimen location where the surgical tumor specimen has a close margin; and detecting cancerous cells if present in the tissue section to determine if the tissue section comprises a positive margin.
  • tissue at the specimen location where the surgical tumor specimen has a close margin is analyzed for cancerous cells without first sectioning the tumor specimen.
  • cancerous cells are detected by histopathological examination or fluorescent examination of the tissue.
  • this method is performed intraoperatively or postoperatively to provide guidance to a surgeon for removal of additional cancerous tissue from the subject.
  • the method further comprises surgically removing additional cancerous tissue from the subject if cancerous cells are detected in the tissue section indicating that the tissue section comprises a positive margin, wherein the location of the positive margin in the tumor specimen is used to identify the location of the cancerous tissue in the subject in need of surgical removal.
  • the fluorescence peak-intensity area has the highest fluorescence intensity, second highest fluorescence intensity, third highest fluorescence intensity, or fourth highest fluorescence intensity of all detected fluorescence peak-intensity areas in the two-dimensional map or three-dimensional map.
  • the imaging is performed on a tumor margin of less than or equal to about 7 mm in length, such as a tumor margin of about 7, 6, 5, 4, 3, 2, or 1 mm, or any length in between.
  • the tumor margin is a close margin of less than or equal to about 5 mm in length, such as a tumor margin of about 5, 4, 3, 2, or 1 mm, or any length in between.
  • the tumor margin is a positive margin of less than or equal to about 2 mm in length, such as a tumor margin of about 2, 1 .5, 1 , 0.5, or 0 mm, or any length in between.
  • the method further comprises measuring a surgical margin thickness at the specimen location corresponding to the fluorescence peak-intensity area.
  • the method further comprises identifying the closest surgical margin corresponding to a fluorescence peak-intensity area in the two-dimensional map or three-dimensional map (e.g., surgical margin less than 5 mm from the tumor).
  • the fluorescent signals are quantified with or without scaling relative to fluorescent signals emitted from specimen regions known to have tumor or normal tissue (e.g., see Examples).
  • the fluorescent contrast agent comprises a fluorophore conjugated to a binding agent that specifically binds to a tumor marker or antigen in or on the cancerous cells of interest.
  • binding agents include antibodies, antibody fragments, antibody mimetics, ligands, and aptamers that specifically bind to the tumor marker or antigen.
  • the fluorescent contrast agent comprises a fluorescent dye (e.g., 5-aminolevulinic acid (5-ALA) and indocyanine green (ICG) dyes and derivatives) that selectively localizes to cancerous cells.
  • a fluorescent dye e.g., 5-aminolevulinic acid (5-ALA) and indocyanine green (ICG) dyes and derivatives
  • ICG indocyanine green
  • the fluorescent contrast agent comprises a fluorophore conjugated to a delocalized lipophilic cation (e.g., triphenylphosphonium derivatives and rhodamine derivatives) that selectively localizes to cancerous cells.
  • a delocalized lipophilic cation e.g., triphenylphosphonium derivatives and rhodamine derivatives
  • the fluorescent contrast agent comprises a fluorophore that emits visible, near-infrared, or near-infrared II fluorescent light.
  • the fluorescent contrast agent is a panitumumab-IRDye800 conjugate.
  • Fluorescence imaging may be performed using, for example, an open- or closed-field fluorescence imaging system. In some embodiments, fluorescence imaging is performed with a medical fluorescence imaging device.
  • a fluorescence image of the fluorescent signals from the contrast agent is recorded with a digital microscope, a charged coupled device (CCD) image sensor, a complementary metal-oxide-semiconductor (CMOS) image sensor, a digital camera, a fiber-optic imaging system, or a medical fluorescence imaging system.
  • CCD charged coupled device
  • CMOS complementary metal-oxide-semiconductor
  • the method further comprises superimposing a photographic image of the surgical tumor specimen and the fluorescence image of the surgical tumor specimen to determine the specimen location corresponding to the fluorescence peak- intensity area.
  • a method of detecting a close or positive margin in a surgical specimen comprising: a) obtaining the surgical specimen comprising at least a portion of a tumor; b) contacting the surgical specimen with a fluorescent contrast agent, wherein the fluorescent contrast agent selectively localizes to cancerous cells in the tumor specimen; c) illuminating the surgical tumor specimen with light at a fluorescence excitation wavelength of the fluorescent contrast agent; d) performing fluorescence imaging on a tumor margin of the surgical tumor specimen by detecting fluorescent signals emitted from the fluorescent contrast agent localized to the cancerous cells of the tumor specimen; e) plotting a two-dimensional map or a three-dimensional map of the fluorescent signals; f) quantifying the fluorescent signals; g) ) identifying at least one fluorescence peak-intensity area in the two-dimensional map or the three-dimensional map, wherein the fluorescence peak-intensity area corresponds to a specimen location where the surgical specimen has a close margin; and h)
  • the method further comprises collecting a tissue section of the surgical tumor specimen comprising tissue from the specimen location where the surgical tumor specimen has a close margin; and detecting cancerous cells if present in the tissue section to determine if the tissue section comprises a positive margin.
  • tissue at the specimen location where the surgical tumor specimen has a close margin is analyzed for cancerous cells without first sectioning the tumor specimen.
  • cancerous cells are detected by histopathological examination or fluorescent examination of the tissue.
  • this method is performed intraoperatively or postoperatively to provide guidance to a surgeon for removal of additional cancerous tissue from the subject.
  • the method further comprises surgically removing additional cancerous tissue from the subject if cancerous cells are detected in the tissue section indicating that the tissue section comprises a positive margin, wherein the location of the positive margin in the tumor specimen is used to identify the location of the cancerous tissue in the subject in need of surgical removal.
  • a computer implemented method for identifying a close or positive surgical margin in a tumor specimen comprising: a) receiving inputted fluorescence imaging data of the tumor specimen, wherein the fluorescence imaging data comprises fluorescent signals from a fluorescent contrast agent localized to cancerous cells in the tumor specimen; b) plotting a two-dimensional map or a three-dimensional map of the fluorescent signals; c) quantifying the fluorescent signals; d) identifying a fluorescence peak-intensity area in the two-dimensional map or the three- dimensional map, wherein the fluorescence peak-intensity area corresponds to a specimen location where the surgical tumor specimen has a close margin; e) mapping at least one fluorescence peak-intensity area to a specimen location; f) marking a photographic image of the tumor specimen to identify the specimen location corresponding to the fluorescence peak- intensity area; and g) displaying the marked photographic image of the tumor specimen, wherein the specimen location corresponding to the fluorescence
  • the specimen location corresponding to the fluorescence peak- intensity area can be marked to identify it in the photographic image of the tumor specimen by labeling, outlining, coloring, or highlighting it.
  • the computer implemented method further comprises sending the marked photographic image to a surgeon or pathologist.
  • the computer implemented method further comprises storing the marked photographic image of the tumor specimen in a database.
  • a non-transitory computer-readable medium comprising program instructions that, when executed by a processor in a computer, causes the processor to perform a computer implemented method for identifying a close or positive surgical margin in a tumor specimen, as described herein.
  • kits comprising the non-transitory computer-readable medium described herein.
  • the kit may further comprise instructions for identifying a close or positive surgical margin in a tumor specimen according to the methods described herein.
  • the kit further comprises a fluorescent contrast agent.
  • the fluorescent contrast agent in the kit may be a fluorescently labeled antibody that specifically binds to a tumor antigen.
  • the fluorescent contrast agent emits visible, near-infrared, or near-infrared II fluorescence.
  • the kit comprises a panitumumab-IRDye800 conjugate.
  • a system for identifying a close or positive surgical margin in a tumor specimen using a computer implemented method, described herein comprising: a) a storage component for storing data, wherein the storage component has instructions for identifying a close or positive surgical margin in a tumor specimen based on analysis of fluorescence imaging data of the tumor specimen stored therein; b) a computer processor for processing the fluorescence imaging data, wherein the computer processor is coupled to the storage component and configured to execute the instructions stored in the storage component in order to receive the inputted fluorescence imaging data and analyze the data according to the computer implemented method described herein; and c) a display component for displaying a marked photographic image of the tumor specimen, wherein the specimen location corresponding to the fluorescence peak-intensity area is identified.
  • the system further comprises a light source capable of illuminating a surgical specimen at a fluorescence excitation wavelength of the fluorescent contrast agent that is localized to cancerous cells of the surgical specimen.
  • the system further comprises a photodetector capable of detecting fluorescent light emitted by the fluorescent contrast agent.
  • the photodetector may include without limitation a charged coupled device (CCD) image sensor, a complementary metal-oxide-semiconductor (CMOS) image sensor, a digital camera, a fiber-optic imaging system, or a medical fluorescence imaging system.
  • CCD charged coupled device
  • CMOS complementary metal-oxide-semiconductor
  • kits comprising a system for identifying a close or positive surgical margin in a tumor specimen, as described herein.
  • the kit may further comprise instructions for identifying a close or positive surgical margin in a tumor specimen according to the methods described herein.
  • the kit further comprises a fluorescent contrast agent.
  • the fluorescent contrast agent in the kit may be a fluorescently labeled antibody that specifically binds to a tumor antigen.
  • the fluorescent contrast agent emits visible, near-infrared, or near-infrared II fluorescence.
  • the kit comprises a panitumumab-IRDye800 conjugate.
  • FIG. 1 shows an overview of the workflow.
  • Numbers 1 -3 allocate the fluorescence peaks in order of first appearance throughout the whole figure
  • Side view on the entire fluorescence surface map of deep margin (white arrow image (b) indicates angle)
  • (d-f) Identification of highest fluorescence intensity-peaks on the deep surface (with color bar).
  • the white dotted lines and asterisks (red/blue) indicate the orientation in which the hematoxylin & eosin (H&E) slides where cut.
  • FIG. 2A shows a graph of mean fluorescence intensity, showing the relative increase in MFI of all areas of no significance (ANS) to peak intensity areas (PI As) per patient.
  • FIG. 2B shows a graph illustrating the difference in tumor depth for ANS and PIAs per patient.
  • FIG. 3A shows the peak-intensity versus tumor depth. Representative brightfield (top) and fluorescence (middle) images of the deep margin are shown with identification of highest fluorescence intensity-peaks on the deep surface (bottom). Numbers 1 -3 allocate the fluorescence peaks in order of first appearance.
  • FIG. 3B shows a graph of the tumor depth at the 1 st , 2 nd and 3 rd peak-intensity area per patient.
  • FIG. 4 shows a schematic of the workflow.
  • PIA peak-intensity area
  • SCC squamous cell carcinoma
  • H&E hematoxylin & eosin.
  • FIG. 5 shows annotation of a suspicious area by suture.
  • Bright field (a) and corresponding fluorescence (b) images of the specimen’s deep margin after maxillectomy are shown.
  • the white solid line indicates peak intensity area in the bright field (a) and fluorescence (b) images.
  • a white dotted line delineates the infiltrated left concha inferior (nasal cavity).
  • a hematoxylin & eosin slide of the suture marked area c, black solid line delineates tumor from healthy tissue
  • an isolated fluorescence peak-intensity area where the suture was placed (d) are also shown.
  • Methods, kits, and systems for identifying suspicious areas in surgical specimens that potentially contain cancerous cells are provided.
  • the methods utilize fluorescence imaging of a surgical specimen with a fluorescent contrast agent that selectively localizes to cancerous cells to identify fluorescence peak-intensity regions corresponding to locations in the specimen where the distance between the tumor and the margin border is the closest.
  • the methods disclosed herein are useful in identifying suspicious sections of a tumor specimen, potentially containing cancerous cells, which should be selected for further examination. Additionally, these methods aid in providing intraoperative guidance to allow a surgeon to take corrective action during surgical procedures.
  • contrast agent includes a plurality of such contrast agents and reference to “the contrast agent” includes reference to one or more contrast agents and equivalents thereof, such as fluorescently labeled tumor binding agents (e.g., fluorescently labeled antibodies specific for tumor antigens), known to those skilled in the art, and so forth.
  • fluorescently labeled tumor binding agents e.g., fluorescently labeled antibodies specific for tumor antigens
  • fluorescence characteristics means an ability to emit fluorescence by irradiation of excitation light.
  • parameters of fluorescence characteristics include fluorescence intensity, excitation wavelength, fluorescence wavelength, and pH sensitivity.
  • Fluorescent emissions may include fluorescent light, for example, at wavelengths in the visible, near-infrared, or near-infrared II regions of the light spectrum.
  • tissue margin refers to tissue surrounding a tumor in a surgical specimen, including tissue between the specimen edge and the tumor.
  • close margin is meant, when referring to a surgical specimen, that the distance between the specimen edge and tumorous tissue is less than or equal to 5 mm.
  • positive margin is meant, when referring to a surgical specimen, that the tissue between the specimen edge and the tumor comprises cancerous cells.
  • isolated when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type.
  • isolated with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
  • an “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen.
  • the term includes polyclonal, monoclonal, chimeric, single domain (Dab) and bispecific antibodies.
  • antibody or antibody molecule contemplates recombinantly generated intact immunoglobulin molecules and immunologically active portions of an immunoglobulin molecule such as, without limitation: Fab, Fab', F(ab') 2 , F(v), scFv, scFv 2 , scFv-Fc, minibody, diabody, tetrabody, single variable domain (e.g., variable heavy domain, variable light domain), bispecific, Affibody molecules (Affibody, Bromma, Sweden), and peptabodies (Terskikh et al. (1997) PNAS 94:1663-1668). Dabs can be composed of a single variable light or heavy chain domain.
  • variable light domain and/or variable heavy domain specific for MISIIR are inserted into the backbone of the above-mentioned antibody constructs.
  • Methods for recombinantly producing antibodies are well known in the art.
  • commercial vectors comprising constant genes to make IgGs from scFvs are provided by Lonza Biologies (Slough, United Kingdom).
  • the phrase "Affibody molecule” may also refer to "Affibody. RTM. molecule.”
  • Affibody is a trademark owned by Affibody AB.
  • Affibody is a trademark registered in Sweden, Europe, and the United States and under trademark application in Japan.
  • Fv is an antibody fragment which contains an antigen-recognition and -binding site.
  • This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non- covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although often at a lower affinity than the entire binding site.
  • Single-chain Fv or “scFv” antibody fragments comprise the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and ⁇ domains which enables the scFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) on the same polypeptide chain (V H -V L ).
  • V H heavy-chain variable domain
  • V L light-chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11 161 ; and Holliger et al tension (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-6448.
  • binding agents e.g., antibodies, aptamers
  • binding agents e.g., antibodies, aptamers
  • binding agents e.g., antibodies, aptamers
  • binding agents e.g., antibodies, aptamers
  • specific or selective binding will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • affibody molecule refers to a molecule that consists of three alpha helices with 58 amino acids and has a molar mass of about 6 kDa.
  • a monoclonal antibody, for comparison, is 150 kDa, and a single-domain antibody, the smallest type of antigen-binding antibody fragment, 12-15 kDa.
  • conjugated refers to the joining by covalent or noncovalent means of two compounds or agents (e.g., binding agent specific for a tumor antigen conjugated to a fluorophore).
  • treatment used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • treatment encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and/or symptom(s).
  • Those in need of treatment include those already inflicted (e.g., those with cancer) as well as those in which prevention is desired (e.g., those with increased susceptibility to cancer, those suspected of having cancer, those with a risk of recurrence, etc.).
  • tumor refers to a cell or population of cells whose growth, proliferation or survival is greater than growth, proliferation or survival of a normal counterpart cell, e.g., a cell proliferative, hyperproliferative or differentiative disorder. Typically, the growth is uncontrolled.
  • malignancy refers to invasion of nearby tissue.
  • metastasis or a secondary, recurring or recurrent tumor, cancer or neoplasia refers to spread or dissemination of a tumor, cancer or neoplasia to other sites, locations or regions within the subject, in which the sites, locations or regions are distinct from the primary tumor or cancer.
  • Neoplasia, tumors and cancers include benign, malignant, metastatic and non-metastatic types, and include any stage (I, II, III, IV or V) or grade (G1 , G2, G3, etc.) of neoplasia, tumor, or cancer, or a neoplasia, tumor, cancer or metastasis that is progressing, worsening, stabilized or in remission.
  • carcinomas such as squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, and small cell carcinoma
  • cancers such as, but are not limited to, head and neck cancer, skin cancer, breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorders, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumors, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor
  • substantially purified generally refers to isolation of a substance (compound, drug, polynucleotide, protein, polypeptide) such that the substance comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample.
  • Techniques for purifying substances of interest are well-known in the art and include, for example, ion- exchange chromatography, affinity chromatography and sedimentation according to density.
  • the terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • "Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc.
  • the mammal is human.
  • Methods for identifying suspicious areas in surgical specimens that potentially contain cancerous cells utilize fluorescence imaging of a surgical specimen with a fluorescent contrast agent that selectively localizes to cancerous cells to identify fluorescence peak-intensity regions corresponding to locations in the specimen where the distance between the tumor and the margin border is the closest.
  • the methods disclosed herein are useful in identifying suspicious sections of a tumor specimen containing close or positive tumor margins that should be selected for further examination.
  • the methods of the present invention utilize fluorescence imaging of tumor specimens with fluorescent contrast agents that selectively localize to cancerous cells.
  • the fluorescent contrast agent may comprise a fluorophore having fluorescent light emissions at wavelengths, for example, in the visible, near-infrared, or near-infrared II regions of the light spectrum.
  • the fluorophore has a fluorescence emission in the visible region of the light spectrum, which ranges from about 400 nm to about 700 nm.
  • fluorophores that emit fluorescent light in the visible region of the light spectrum include, but are not limited to, CAL Fluor dyes such as CAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, and CAL Fluor Red 635; Quasar dyes such as Quasar 570, Quasar 670, and Quasar 705; Alexa Fluor dyes such as Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 594, Alexa 633, and Alexa Fluor 647; cyanine dyes such as Cy 3, Cy3.5, Cy5, Cy5.5, and Cy7, fluorescein and derivatives such as 2', 4', 5', 7'-tetrachloro
  • the fluorophore has a fluorescence emission in the near- infrared (NIR) region of the light spectrum, which ranges from about 700 nm to about 1700 nm.
  • NIR fluorophores is advantageous in minimizing interference from tissue autofluorescence and enhancing tissue penetration compared to other fluorophores.
  • the fluorescent emission wavelength is in a region of the light spectrum where blood and tissue absorb minimally, and tissue penetration is maximal, such as in the range from 700 nm to 1000 nm.
  • NIR fluorophore with an emission in the NIR region of the light spectrum may be used, including, but not limited to, fluorophores with fluorescence emissions at about 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, or 1000 nm, or any wavelength in between.
  • Exemplary NIR fluorophores include IRDye dyes (e.g., IRDye 800CW, IRDye 680RD, IRDye 750, and IRDye 800RS), CF dyes (e.g., CF680, CF680R, CF750, CF770, and CF790), Tracy dyes (e.g., Tracy 645 and Tracy 652), cyanine dyes (e.g., Cy7 and Cy7.5), thienothiadiazole dyes, phthalocyanine dyes, squaraine dyes, rhodamine dyes and analogues (e.g., Si-pyronine, Si-rhodamine, Te-rhodamine, and Changsha), borondipyrromethane (BODIPY) dyes, seminaphthofluorone xanthene dyes, benzo[c]heterocycle dyes (e.g., isobenzofuran dyes),
  • the fluorophore has an emission in the NIR II region of the light spectrum (i.e., about 1000 nm to about 1700 nm), such as at about 1000, 1050, 1 100, 1 150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, or 1700 nm, or any wavelength in between.
  • NIR II fluorophores include benzobisthiadiazole (BBTD) derivatives, ECX, FD-1080, CH1055, SCH1 100, CQS1000, H2a-4T, polymethine dyes, cyanine dyes, quantum dots, rare-earth (RE)-doped nanoparticles (RENPs), and single-walled carbon nanotubes (SWCNTs).
  • BBTD benzobisthiadiazole
  • ECX benzobisthiadiazole
  • FD-1080 CH1055
  • SCH1 100 CQS1000, H2a-4T
  • polymethine dyes cyanine dyes
  • quantum dots quantum dots
  • RE rare-earth-doped nanoparticles
  • SWCNTs single-walled carbon nanotubes
  • the contrast agent comprises a dye that selectively localizes to cancerous cells.
  • a dye that selectively localizes to cancerous cells For example, 5-aminolevulinic acid (5-ALA) and indocyanine green (ICG) dyes and derivatives thereof selectively accumulate in tumors.
  • 5-aminolevulinic acid (5-ALA) and indocyanine green (ICG) dyes and derivatives thereof selectively accumulate in tumors.
  • ICG is a NIR cyanine dye, which selectively accumulates in tumors, in part, because of the increased permeability of tumor vessels and neovascularization around tumors.
  • ICG binds to serum proteins such as albumin to form complexes that are accumulated and retained in tumors (see, e.g., Intes et al. (2003) Med Phys. 30:1039-1047, Licha et al. (2000) Photochem. Photobiol. 72:392- 398).
  • 5-ALA uptake by tumor cells results in biosynthesis and accumulation of fluorescent protoporphyrin IX (PplX).
  • PplX fluorescent protoporphyrin IX
  • 5-ALA selectively accumulates in tumors, in part, because the metabolism of ALA to PplX in mitochondria is impaired in cancerous cells.
  • such dyes may be conjugated to tumor targeting agents or binding agents, such as antibodies, antibody mimetics, peptide ligands, aptamers, and the like, as discussed further below.
  • the fluorescent contrast agent comprises a fluorophore conjugated to a delocalized lipophilic cation that selectively localizes to cancerous cells.
  • Delocalized lipophilic cations DLCs
  • Delocalized lipophilic cations include, but are not limited to, alkyltriphenylphosphonium (e.g., triphenylphosphonium) derivatives and rhodamine derivatives (see, e.g., Zielonka et al. (2017) Chem Rev. 1 17(15):10043-10120, Yang et al. (2017) Cell Death Dis.
  • the fluorescent contrast agent comprises a fluorophore conjugated to a binding agent that specifically binds to a tumor marker or antigen of interest (i.e., target antigen on or in the tumor that is resected).
  • the binding agent binds to a tumor antigen of interest with high affinity.
  • binding agents include, without limitation, antibodies, antibody fragments, antibody mimetics, ligands, and aptamers.
  • the conjugates of the subject methods include at least one fluorophore attached to the binding agent. Fluorescent dyes with different excitation and emission wavelengths can be conjugated to different binding agents that specifically bind to different tumor markers or antigens. It is expected that simultaneous binding to multiple tumor markers or antigens will increase the sensitivity and specificity of detecting cancerous cells by fluorescence imaging.
  • binding agents may be designed to selectively bind to cell surface molecules or receptors that are highly expressed in tumor cells and/or the tumor environment to increase localization of the contrast agent to the tumor. Binding of targeted fluorophores to cell surface receptors may result in cellular internalization in tumor cells (Becker et al. (2001 ) Nat. Biotechnol. 19:327-331 .
  • Various cell surface molecules including, but not limited to, epidermal growth factor receptor (EGFR), Her-2/Neu, alphaVbeta-3 integrin, urokinase plasminogen activator receptor, mucin 1 , carcinoembryonic antigen, and the folate receptor can be targeted by binding agents (Moon et al.
  • the binding agent comprises an antibody that specifically binds to the tumor antigen of interest.
  • any type of antibody may be used, including polyclonal and monoclonal antibodies, hybrid antibodies, altered antibodies, chimeric antibodies and, humanized antibodies, as well as: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991 ) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab') 2 and F(ab) fragments; F v molecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc. Natl. Acad. Sci. USA 69:2659-2662; and Ehrlich et al. (1980) Biochem.
  • sFv singlechain Fv molecules
  • sdAb single-domain antibodies
  • minibodies see, e.g., Pack et al. (1992) Biochem 31 :1579-1584; Cumber et al.
  • the binding agent comprises an aptamer that specifically binds to the tumor antigen of interest.
  • aptamer Any type of aptamer may be used, including a DNA, RNA, xeno-nucleic acid (XNA), or peptide aptamer that specifically binds to the tumor antigen.
  • XNA xeno-nucleic acid
  • Such aptamers can be identified, for example, by screening a combinatorial library.
  • Nucleic acid aptamers e.g., DNA or RNA aptamers
  • that bind selectively to a target tumor antigen can be produced by carrying out repeated rounds of in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX).
  • Peptide aptamers that bind to a target tumor antigen may be isolated from a combinatorial library and improved by directed mutation or repeated rounds of mutagenesis and selection.
  • Aptamers Tools for Nanotherapy and Molecular Imaging (R.N. Veedu ed., Pan Stanford, 2016)
  • Nucleic Acid and Peptide Aptamers Methods and Protocols (Methods in Molecular Biology, G. Mayer ed., Humana Press, 2009)
  • Nucleic Acid Aptamers Selection, Characterization, and Application (Methods in Molecular Biology, G.
  • the binding agent comprises an antibody mimetic.
  • Any type of antibody mimetic may be used, including, but not limited to, affibody molecules (Nygren (2008) FEBS J. 275 (1 1 ):2668-2676), affilins (Ebersbach et al. (2007) J. Mol. Biol. 372 (1 ):172- 185), affimers (Johnson et al. (2012) Anal. Chem. 84 (15):6553-6560), affitins (Krehenbrink et al. (2008) J. Mol. Biol.
  • Fluorophores may be conjugated to binding agents by any suitable method.
  • the fluorophore and binding agent may be directly linked, e.g., via a single bond, or indirectly linked e.g., through the use of a suitable linker, e.g., a polymer linker, a chemical linker, or one or more linking molecules or moieties.
  • attachment of the fluorophore and binding agent may be by way of one or more covalent interactions.
  • the fluorophore or binding agent may be functionalized, e.g., by addition or creation of a reactive functional group.
  • Functionalized fluorophores or binding agents may be modified to contain any convenient reactive functional group for conjugation such as an amine functional group, a carboxylic functional group, a sulfhydryl group, a thiol functional group, and the like.
  • Any convenient method of bioconjugation may be used including, but not limited to, glutaraldehyde crosslinking, carbodiimide crosslinking, succinimide ester crosslinking, imidoester, crosslinking, maleimide crosslinking, iodoacetamide crosslinking, benzidine crosslinking, periodate crosslinking, isothiocyanate crosslinking, and the like.
  • Such conjugation methods may optionally use a reactive sidechain group of an amino acid residue of the binding agent (e.g., a reactive side-chain group of a Lys, Cys, Ser, Thr, Tyr, His or Arg amino acid residue of the protein, i.e., a polypeptide linking group may be amino-reactive, thiol-reactive, hydroxyl-reactive, imidazolyl-reactive or guanidinyl-reactive).
  • a chemoselective reactive functional group may be utilized.
  • conjugation reagents that can be used include, but are not limited to, e.g., homobifunctional conjugation reagents (e.g., (bis(2-[succinimidooxycarbonyloxy]ethyl) sulfone, l,4-Di-(3'-[2'pyridyldithio]-propionamido) butane, disuccinimidyl suberate, disuccinimidyl tartrate, sulfodisuccinimidyl tartrate, dithiobis(succinimidyl propionate), 3,3'-dithiobis(sulfosuccinimidyl propionate), ethylene glycol bis(succinimidyl succinate), and the like), heterobifunctional conjugation reagents (e.g., m- maleimidobenzoyl-N-hydroxysuccinimide ester, m-maleimidobenzoyl-N
  • a functional linker refers to any suitable linker that has one or more functional groups for the attachment of one molecule to another.
  • the functional linker comprises an amino functional group, a thiol functional group, a hydroxyl functional group, an imidazolyl functional group, a guanidinyl functional group, an alkyne functional group, an azide functional group, or a strained alkyne functional group.
  • Further exemplary functional groups and methods of crosslinking and conjugation are described in, e.g., Hermanson Bioconjugate Techniques (Academic Press, 3 rd edition, 2013), herein incorporated by reference in its entirety.
  • the tumor antigen targeted by a binding agent is the epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • a number of anti-EGFR antibodies are available including panitumumab, cetuximab, zalutumumab, nimotuzumab, and matuzumab, which can be conjugated to suitable fluorophores for use as fluorescent contrast agents, according to the methods described herein, for fluorescence imaging of cancerous cells expressing EGFR in a tumor specimen, including those tumor specimens comprising head and neck cancer, colorectal cancer, lung cancer, ovarian cancer, breast cancer, endometrial cancer, cervical cancer, bladder cancer, gastric cancer, and esophageal cancer.
  • the tumor antigen targeted by a binding agent is HER2.
  • a number of anti-HER2 antibodies are also available including trastuzumab, pertuzumab, and margetuximab, which can be conjugated to suitable fluorophores for use as fluorescent contrast agents, according to the methods described herein, for fluorescence imaging of cancerous cells expressing HER2 in a tumor specimen, including those tumor specimens comprising breast cancer, ovarian cancer, stomach cancer, lung cancer, uterine cancer, gastric cancer, colon cancer, head and neck cancer, and salivary duct carcinoma.
  • the tumor antigen targeted by a binding agent is the epithelial cell adhesion molecule (EpCAM) 17-1 A.
  • EpCAM 17-1 A antibodies are also available including edrecolomab, catumaxomab, and nofetumomab, which can be conjugated to suitable fluorophores for use as fluorescent contrast agents, according to the methods described herein, for fluorescence imaging of cancerous cells expressing EpCAM 17-1 A in tumor specimens, including those tumor specimens comprising epithelial-derived neoplasms and various carcinomas, such as lung cancer, gastrointestinal cancer, breast cancer, ovarian cancer, pancreatic cancer, renal cancer, cervical cancer, colorectal cancer, and bladder cancer.
  • the tumor antigen targeted by a binding agent is CD20.
  • a number of anti-CD20 antibodies are also available including rituximab, tositumomab, ocrelizumab, obinutuzumab, ocaratuzumab, ofatumumab, ibritumomab tiuxetan, ublituximab, and veltuzumab, which can be conjugated to suitable fluorophores for use as fluorescent contrast agents, according to the methods described herein, for fluorescence imaging of cancerous cells expressing CD20 in tumor specimens, including those tumor specimens comprising lymphoma such as, but not limited to, marginal zone lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma; leukemia such as, but not limited to, chronic lymphocytic leukemia, acute lymphoblastic leukemia, myelogenous leukemia, and chemotherapy-resistant hairy cell
  • the tumor antigen targeted by a binding agent is CD52.
  • CD52 antibodies are also available including alemtuzumab, which can be conjugated to suitable fluorophores for use as fluorescent contrast agents, according to the methods described herein, for fluorescence imaging of cancerous cells expressing CD52 in tumor specimens, including those tumor specimens comprising lymphoma such as, but not limited to, cutaneous T-cell lymphoma (CTCL) and T-cell lymphoma and chronic lymphocytic leukemia (CLL).
  • CTCL cutaneous T-cell lymphoma
  • CLL chronic lymphocytic leukemia
  • the tumor antigen targeted by a binding agent is CD22.
  • a number of anti-CD22 antibodies are also available including inotuzumab, which can be conjugated to suitable fluorophores for use as fluorescent contrast agents, according to the methods described herein, for fluorescence imaging of cancerous cells expressing CD22 in tumor specimens, including those tumor specimens comprising leukemia such as, but not limited to, lymphoblastic leukemia and hairy cell leukemia; lymphoma, and lung cancer.
  • the tumor antigen targeted by a binding agent is CD19.
  • a number of anti-C19 antibodies are also available including blinatumomab, MEDI-551 and MOR-208, which can be conjugated to suitable fluorophores for use as fluorescent contrast agents, according to the methods described herein, for fluorescence imaging of cancerous cells expressing CD19 in tumor specimens, including those tumor specimens comprising 13- cell neoplasms, non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM).
  • NHL non-Hodgkin lymphoma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • MM multiple myeloma
  • Tissue containing a tumor or cancerous cells can be contacted with a fluorescent contrast agent, such that the fluorescent contrast agent selectively localizes to the tumor or cancerous cells.
  • a detectably effective amount of a fluorescent contrast agent is administered to a subject before surgical resection of a tumor; that is, an amount that is sufficient to yield an acceptable fluorescence image using the fluorescence imaging equipment that is available for clinical use.
  • a detectably effective amount of the fluorescent contrast agent may be administered in more than one injection if needed.
  • the detectably effective amount of the fluorescent contrast agent needed for an individual may vary according to factors such as the degree of susceptibility to uptake into the target area of interest, the age, sex, and weight of the individual, and the particular fluorescence imaging device used. Optimization of such factors is within the level of skill in the art.
  • the surgical specimen is contacted with a detectably effective amount of a fluorescent contrast agent after removal from the subject.
  • the tumor specimen is illuminated with light at a fluorescence excitation wavelength of the fluorescent contrast agent.
  • Any suitable light source may be used for fluorescence excitation.
  • Exemplary light sources include halogen lamps, light-emitting diodes (LEDs), and laser diodes.
  • band-pass filters can be used to produce multiple excitation channels from a light source and narrow the band of excitation.
  • a fluorescence image of the tumor or cancerous cells in a surgical specimen is recorded using a fluorescence imaging device or photodetector capable of detecting the fluorescence emitted by the fluorescent contrast agent.
  • Fluorescence images may be recorded by any suitable method.
  • a CCD image sensor e.g., intensified CCD (ICCD) or electron multiplying CCD (EMCCD)
  • EMCCD electron multiplying CCD
  • CMOS image sensor e.g., CMOS image sensor
  • digital camera may be used to capture images.
  • the image may be a still photo or a video in any format (e.g., bitmap, Graphics Interchange Format, JPEG file interchange format, TIFF, or mpeg).
  • images may be captured by an analog camera and converted into an electronic form.
  • fluorescence can be detected by a fluorescence microscope, a closed-field fluorescence imaging system, a fiber-optic fluorescence imaging system, or a medical fluorescence imaging device (e.g., a handheld fluorescence microscope, a laparoscope, an endoscope, or a microendoscope).
  • a fluorescence microscope e.g., a fluorescence microscope, a closed-field fluorescence imaging system, a fiber-optic fluorescence imaging system, or a medical fluorescence imaging device (e.g., a handheld fluorescence microscope, a laparoscope, an endoscope, or a microendoscope).
  • fluorescence imaging is performed with near-infrared (near-IR) light, which has the advantage that it can penetrate several millimeters to centimeters into living tissues and be used to visualize cancerous tissue and cells below the surface. Because tissue exhibits almost no autofluorescence in the near-IR spectrum, interfering background fluorescence is minimal.
  • near-IR near-infrared
  • An imaging system that can simultaneously detect fluorescence at multiple wavelengths can be used for detection of fluorescence from multiple fluorescent contrast agents that emit fluorescence at different wavelengths. For example, two or more fluorescent contrast agents targeting different tumor antigens can be used.
  • the excitation light source and photodetector are integrated into the fluorescence imaging device. In other devices, the excitation light source and/or photodetector reside apart and are used with remote delivery of excitation light. For a review of fluorescence imaging devices, see, e.g., Gray et al. (2012) Biomed. Opt. Express. 3(8):1880-1890; Flusberg et al. (2005) Nat.
  • the invention includes a computer implemented method for identifying a close or positive surgical margin in a tumor specimen.
  • the computer performs steps comprising: a) receiving inputted fluorescence imaging data of a tumor surgical specimen, wherein the fluorescence imaging data comprises fluorescent signals from a fluorescent contrast agent localized to cancerous cells in the surgical specimen; b) plotting a two-dimensional map or a three-dimensional map of the fluorescent signals; c) quantifying the fluorescent signals; d) identifying a fluorescence peak-intensity area in the two-dimensional map or the three-dimensional map, wherein the fluorescence peak-intensity area corresponds to a specimen location where the surgical tumor specimen has a close margin; e) mapping the fluorescence peak-intensity area to a specimen location; f) marking a photographic image of the tumor surgical specimen to identify the specimen location corresponding to the fluorescence peak-intensity area; and g) displaying the marked photographic image of the tumor surgical specimen, wherein the specimen
  • the fluorescence peak-intensity area having the highest fluorescence intensity, second highest fluorescence intensity, third highest fluorescence intensity, or fourth highest fluorescence intensity of all detected fluorescence peak-intensity areas in the two-dimensional map or the three-dimensional map is mapped to a location in the tumor surgical specimen, and a photographic image of the tumor surgical specimen is marked to identify the specimen location corresponding to that fluorescence peak- intensity area.
  • multiple fluorescence peak-intensity areas are mapped to locations in the tumor surgical specimen and marked on a photographic image of the tumor surgical specimen to identify the specimen locations corresponding to the fluorescence peak- intensity areas.
  • the fluorescence peak-intensity area having the highest fluorescence intensity, second highest fluorescence intensity, third highest fluorescence intensity, and fourth highest fluorescence intensity of all detected fluorescence peak-intensity areas in the two-dimensional map or the three-dimensional map can be mapped to locations in the tumor surgical specimen, and a photographic image of the tumor surgical specimen can be marked to identify the specimen locations corresponding to those fluorescence peak- intensity areas.
  • the fluorescent signals are quantified with or without scaling relative to fluorescent signals emitted from specimen regions known to have tumor or normal tissue (e.g., see Examples).
  • the computer implemented method further comprises calculating a surgical margin thickness at a specimen location corresponding to a fluorescence peak-intensity area.
  • the computer implemented method further comprises identifying the closest surgical margin corresponding to a fluorescence peak-intensity area (e.g., surgical margin less than 5 mm from the tumor).
  • the computer implemented method further comprises storing a marked photographic image of the tumor specimen (i.e., having at least one specimen location corresponding to a fluorescence peak-intensity area identified) in a database. In certain embodiments, the computer implemented method further comprises sending a marked photographic image to a surgeon or pathologist.
  • the method can be implemented in digital electronic circuitry, or in computer software, firmware, or hardware.
  • the disclosed and other embodiments can be implemented as one or more computer program products, i.e., one or more modules of computer program instructions encoded on a computer readable medium for execution by, or to control the operation of, a data processing apparatus.
  • the computer readable medium can be a machine-readable storage device, a machine-readable storage substrate, a memory device, a composition of matter effecting a machine-readable propagated signal, or any combination thereof.
  • a computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment.
  • a computer program does not necessarily correspond to a file in a file system.
  • a program can be stored in a portion of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, sub programs, or portions of code).
  • a computer program can be deployed to be executed on one computer or on multiple computers that are located at one site or distributed across multiple sites and interconnected by a communication network.
  • a system for performing the computer implemented method, as described includes a computer containing a processor, a storage component (i.e., memory), a display component, and other components typically present in general purpose computers.
  • the storage component stores information accessible by the processor, including instructions that may be executed by the processor and data that may be retrieved, manipulated or stored by the processor.
  • the storage component includes instructions for analyzing fluorescence imaging data of a surgical specimen to identify suspicious regions (e.g., a close or positive surgical margin in need of further assessment to determine if cancerous cells are present). For example, the storage component includes instructions for identifying a close or positive surgical margin in a tumor specimen based on analysis of fluorescence imaging data stored therein.
  • the computer processor is coupled to the storage component and configured to execute the instructions stored in the storage component in order to receive fluorescence imaging data of a surgical specimen and analyze the data according to one or more algorithms as described herein.
  • the display component displays a marked photographic image of the tumor specimen, wherein the specimen location corresponding to the fluorescence peak-intensity area is identified. For example, the specimen location corresponding to the fluorescence peak-intensity area can be marked to identify it in the photographic image of the tumor specimen by labeling, outlining, coloring, or highlighting it.
  • the storage component may be of any type capable of storing information accessible by the processor, such as a hard-drive, memory card, ROM, RAM, DVD, CD-ROM, USB Flash drive, write-capable, and read-only memories.
  • the processor may be any well-known processor, such as processors from Intel Corporation. Alternatively, the processor may be a dedicated controller such as an ASIC.
  • the instructions may be any set of instructions to be executed directly (such as machine code) or indirectly (such as scripts) by the processor.
  • the terms "instructions,” “steps” and “programs” may be used interchangeably herein.
  • the instructions may be stored in object code form for direct processing by the processor, or in any other computer language including scripts or collections of independent source code modules that are interpreted on demand or compiled in advance.
  • Data may be retrieved, stored or modified by the processor in accordance with the instructions.
  • the data may be stored in computer registers, in a relational database as a table having a plurality of different fields and records, XML documents, or flat files.
  • the data may also be formatted in any computer-readable format such as, but not limited to, binary values, ASCII or Unicode.
  • the data may comprise any information sufficient to identify the relevant information, such as numbers, descriptive text, proprietary codes, pointers, references to data stored in other memories (including other network locations) or information which is used by a function to calculate the relevant data.
  • the processor and storage component may comprise multiple processors and storage components that may or may not be stored within the same physical housing.
  • some of the instructions and data may be stored on removable CD- ROM and others within a read-only computer chip. Some or all of the instructions and data may be stored in a location physically remote from, yet still accessible by, the processor.
  • the processor may comprise a collection of processors which may or may not operate in parallel.
  • the system further comprises a light source capable of illuminating a surgical specimen at a fluorescence excitation wavelength of the fluorescent contrast agent used in fluorescently labeling cancerous cells of the surgical specimen and a switch configured to control the switching on and off of the light source.
  • a light source capable of illuminating a surgical specimen at a fluorescence excitation wavelength of the fluorescent contrast agent used in fluorescently labeling cancerous cells of the surgical specimen and a switch configured to control the switching on and off of the light source.
  • Exemplary light sources that can be included in the system include halogen lamps, LEDs, and laser diodes.
  • the system further comprises a photodetector and a controller configured to automate recording of photographic or fluorescent images of a tumor specimen.
  • exemplary photodetectors that may be used in the system include a charged coupled device (CCD) image sensor, a complementary metal-oxide-semiconductor (CMOS) image sensor, a digital camera, a fiber-optic imaging system, and a medical fluorescence imaging system.
  • CCD charged coupled device
  • CMOS complementary metal-oxide-semiconductor
  • Kits are also provided for carrying out the methods described herein.
  • the kit comprises software for carrying out the computer implemented methods for identifying a close or positive surgical margin in a tumor specimen using fluorescence imaging data, as described herein.
  • the kit comprises a system for identifying a close or positive surgical margin in a tumor specimen as described herein.
  • the kit further comprises at least one fluorescent contrast agent that selectively localizes to cancerous cells.
  • Compositions can be in liquid form or can be lyophilized and contained in one or more containers. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic.
  • a container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the kit can further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery devices.
  • a pharmaceutically- acceptable buffer such as phosphate-buffered saline, Ringer's solution, or dextrose solution.
  • the delivery device may be pre-filled with the compositions.
  • the kit can also comprise a package insert containing written instructions for methods of using the compositions comprising fluorescent contrast agents for fluorescence imaging of a surgical specimen according to the methods described herein.
  • the package insert can be an unapproved draft package insert or can be a package insert approved by the Food and Drug Administration (FDA) or other regulatory body.
  • FDA Food and Drug Administration
  • the kit further comprises a light source capable of illuminating a surgical specimen at a fluorescence excitation wavelength of the fluorescent contrast agent used in fluorescently labeling cancerous cells of the surgical specimen.
  • the kit further comprises a photodetector capable of recording photographic or fluorescent images of a tumor specimen.
  • the methods described herein are useful for accurately determining surgical margins for tumor resection (i.e., the distance from a tumor border to healthy tissue). Surgeons typically remove a tumor along with a 5 mm margin that is presumed to be cancer-free healthy tissue surrounding the tumor. However, this margin is sometimes insufficient and failure to remove all cancerous cells results in locoregional recurrence and poor overall survival rates of patients. Tumor borders within deeper tissue layers are especially difficult to determine, and deep margins are consequently more prone to develop local recurrence. Although frozen section analysis (FSA) can detect cancer, typically only a very small percent of the wound bed is sampled, and surgeons struggle to identify which suspicious areas are appropriate for histopathological assessment.
  • FSA frozen section analysis
  • the methods of the present invention address the need for better ways of assessing tumor margins for cancer surgery.
  • the methods disclosed herein consistently detect the smallest margin of “normal” tissue surrounding a tumor in surgical specimens and provide strategical guidance in identifying potentially positive or close margins that need further assessment by a surgeon or pathologist.
  • the methods of the invention can be performed intraoperatively to provide guidance on whether or not additional surgery is required to remove additional cancerous tissue from a subject.
  • the methods can further direct a surgeon to the appropriate tissue location in a patient wherein additional cancerous tissue needs removal.
  • a surgeon can surgically remove additional cancerous tissue from a subject if cancerous cells are detected in a tissue section of a tumor specimen indicating that the tissue section comprises a positive margin, wherein the location of the positive margin in the tumor specimen is used to identify the location of the cancerous tissue remaining in the subject.
  • the methods disclosed herein can be used to evaluate margins in surgical tumor specimens for a wide variety of cancers, including, but not limited to, head and neck cancer, skin cancer, breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorders, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumors, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gestational trophoblastic tumors, hairy cell leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal
  • PEARL closed-field fluorescence imaging device
  • the specimen was formalin-fixed and cut in 5mm tissue sections.
  • the specimen was then reconstructed from the 5mm sections and re-imaged. Thereafter, the 5mm tissue sections were processed and paraffin-embedded. From each 5mm section, a representative 5pm section was cut for routine hematoxylin and eosin (H&E) staining for diagnosis.
  • H&E hematoxylin and eosin
  • ImageJ version 1 .50i, National Institute of Health, Washington D.C., Maryland, USA
  • ImageJ plugin interactive 3D surface plot, Internationale stipulateinformatik, Berlin, Germany
  • PIAs peak-intensity areas
  • the PIAs were numbered in order of appearance ranging from 1 st to 4 th (d-g). Further isolation than the 3 rd or 4 th PI A was deemed unnecessary given the objective to find the closest tumor margin on the deep surface. In addition, because some specimen had no clear 4 th PIA or presented with a simultaneous 3 rd and 4 th peak, we choose to average the values (i.e. fluorescence intensity and tumor depth) of these peaks. Subsequently, for each PIA, an opposing area of no significance (ANS) was randomly assigned 10-15 mm from the PIA as reference.
  • ANS opposing area of no significance
  • MFIs mean fluorescence intensities
  • [001 1 1 ] A total of 18 patients underwent evaluation of their primary specimen after systemic infusion of panitumumab-IRDye800 that we have shown to be sensitive and specific for squamous cell cancer (14,15). Primary surgical specimens underwent closed-field imaging of the deep margin immediately after surgical removal. Patient characteristics are summarized in Table 1 . All 18 patients could undergo successful imaging and peak intensity analysis, but six of the patients were excluded because pathological reconstruction of the specimen could not be performed in a manner where the individual histological sections could be correlated precisely ( ⁇ 1 mm) to the corresponding PIA on the fluorescence imaging of the primary specimen. For the remaining 12 patients, a total of 80 PIAs and corresponding ANSs were isolated whereby a total of 400 tumor depth measurements were performed.
  • the margin distance (the measurement in mm between the invasive tumor edge and the cut surface of the specimen) correlates directly with the peak fluorescence region.
  • tumor was always closer to the deep margin in the first region of peak intensity on the specimen compared to the second which harbored tumor closer in 83% compared to the averaged third/fourth fluorescence peak.
  • FIG. 3 shows decreasing peak fluorescence intensity corresponds with increasing tumor depth on histological section for each patient.
  • the average increase of tumor depth between the second and the third/fourth PIA was 1 6mm. In only two (2/12) cases was the tumor depth that correlated to the second PIA was found to be larger than that of the third/fourth PIA tumor distance. However, in both of these cases the second PIA and third/fourth PIA were not substantially different: the PIAs were found to correlate with a tumor thickness that was within the margin of error in measurement (5.8 ⁇ 0.1 mm versus 5.6 ⁇ 0.4mm; and 1 1 .4 ⁇ 0.6mm versus 10.2 ⁇ 0.6mm).
  • Frozen section itself is considered highly accurate (>90%;), but its application in head and neck surgery results in 10-20 frozen sections per case and as mentioned, has not impacted success rates despite wide availability (12,19).
  • the current study has shown that identifying fluorescence peak-intensity can be performed which could direct intraoperative margin sampling for frozen section analysis.
  • Retrospective data analysis on patient specimens suggests that intraoperative ex vivo fluorescence imaging of the resected specimen can direct the surgeon to the closest involved margin on the deep surface.
  • the success of this ex vivo specimen mapping method relies on several key optical principles.
  • the NIR fluorescent signal will not travel much further than 6mm through tissue. This allows the signal to become relatively constant after the margin exceeds 5mm, an important number clinically. Detection of a truly positive number is not as common as a close margin (1 -5mm) which can be successfully identified using this technique. Other surgical imaging methods dependent on cancer on the surface of the specimen are less helpful.
  • the optical signal will intensify as the thickness of the surgical margin decreases. The region with the peak fluorescence intensity may or may not be positive, however it is most likely the spot where tumor tissue is closest to the surface. It objectively delineates the most likely suspicious region and therefore transcends the need to identify positive margins.
  • Hinni ML Ferlito A
  • Brandwein-Gensler MS Takes RP, Silver CE, Westra WH, et al. Surgical margins in head and neck cancer: a contemporary review. Head Neck. 2013 Sep;35(9):1362-70.

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Abstract

L'invention concerne des méthodes, des kits et des systèmes d'identification de zones suspectes dans des échantillons chirurgicaux contenant potentiellement des cellules cancéreuses. Les méthodes utilisent l'imagerie par fluorescence d'un échantillon chirurgical avec un agent de contraste fluorescent se localisant sélectivement sur des cellules cancéreuses afin d'identifier des régions de pic d'intensité de fluorescence correspondant à des emplacements dans l'échantillon, la distance entre la tumeur et la bordure de marge étant la plus proche. Les méthodes de l'invention permettent l'identification de sections d'un échantillon de tumeur devant être sélectionnées pour un examen histopathologique supplémentaire. De plus, lesdites méthodes aident à fournir un guidage intraopératoire permettant à un chirurgien de prendre une action corrective pendant des procédures chirurgicales.
PCT/US2020/015436 2019-01-28 2020-01-28 Identification rapide de marges chirurgicales proches sur des échantillons chirurgicaux Ceased WO2020160006A1 (fr)

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CN117373545A (zh) * 2023-12-07 2024-01-09 广州金墁利医药科技有限公司 肠癌放化疗预测评分模型、应用、系统及其构建方法
WO2024060432A1 (fr) * 2022-09-23 2024-03-28 上海纳米技术及应用国家工程研究中心有限公司 Kit de test de cancer gastrique proche infrarouge et son procédé de préparation

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080015448A1 (en) * 2006-06-22 2008-01-17 Keely Patricia J Stromal collagen in the diagnosis and characterization of breast cancer
US20130066199A1 (en) * 2010-04-15 2013-03-14 Cedars-Sinai Medical Center Tumor margin detection method based on nuclear morphometry and tissue topology
US20150030542A1 (en) * 2013-07-26 2015-01-29 Sunil Singhal Methods for medical imaging
US20170059487A1 (en) * 2015-02-23 2017-03-02 Li-Cor, Inc. Fluorescence Biopsy Specimen Imager and Methods
US20170340755A1 (en) * 2014-12-19 2017-11-30 Bracco Imaging S.P.A. Intra-operative imaging

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080015448A1 (en) * 2006-06-22 2008-01-17 Keely Patricia J Stromal collagen in the diagnosis and characterization of breast cancer
US20130066199A1 (en) * 2010-04-15 2013-03-14 Cedars-Sinai Medical Center Tumor margin detection method based on nuclear morphometry and tissue topology
US20150030542A1 (en) * 2013-07-26 2015-01-29 Sunil Singhal Methods for medical imaging
US20170340755A1 (en) * 2014-12-19 2017-11-30 Bracco Imaging S.P.A. Intra-operative imaging
US20170059487A1 (en) * 2015-02-23 2017-03-02 Li-Cor, Inc. Fluorescence Biopsy Specimen Imager and Methods

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024060432A1 (fr) * 2022-09-23 2024-03-28 上海纳米技术及应用国家工程研究中心有限公司 Kit de test de cancer gastrique proche infrarouge et son procédé de préparation
CN117373545A (zh) * 2023-12-07 2024-01-09 广州金墁利医药科技有限公司 肠癌放化疗预测评分模型、应用、系统及其构建方法
CN117373545B (zh) * 2023-12-07 2024-05-10 广州金墁利医药科技有限公司 肠癌放化疗预测评分的系统及模型的应用和构建方法

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