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WO2023137340A1 - Système de carottage et d'analyse de tissu - Google Patents

Système de carottage et d'analyse de tissu Download PDF

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Publication number
WO2023137340A1
WO2023137340A1 PCT/US2023/060501 US2023060501W WO2023137340A1 WO 2023137340 A1 WO2023137340 A1 WO 2023137340A1 US 2023060501 W US2023060501 W US 2023060501W WO 2023137340 A1 WO2023137340 A1 WO 2023137340A1
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WO
WIPO (PCT)
Prior art keywords
ffpe
tissue
heatmap
block
interest
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2023/060501
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English (en)
Inventor
Leif Honda
Phil CESTARO
Jon Wetzel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Trimetis Life Sciences LLC
Original Assignee
Trimetis Life Sciences LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Trimetis Life Sciences LLC filed Critical Trimetis Life Sciences LLC
Priority claimed from US18/153,253 external-priority patent/US20230221221A1/en
Publication of WO2023137340A1 publication Critical patent/WO2023137340A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2873—Cutting or cleaving

Definitions

  • the present disclosure relates to high-throughput method for analyzing tissue samples of nucleic acid material disposed on a Formalin Fixed Paraffin Embedded (“FFPE”) block is disclosed, and will provide analyses without undue delays.
  • FFPE Formalin Fixed Paraffin Embedded
  • a high-throughput method for analyzing tissue samples of nucleic acid material disposed on a Formalin Fixed Paraffin Embedded (FFPE) block involves generating, with a computer, a heatmap of locations of interest for the FFPE block and then obtaining core samples from each location of interest in the heatmap by sterilizing a hollow punch tip, punching a respective location of interest with the hollow punch tip to pick up a respective core sample, and depositing the respective core sample to a respective receptacle.
  • FFPE Formalin Fixed Paraffin Embedded
  • the presently disclosed method further includes treating the core samples deposited in the receptacles with an appropriate reagent to extract the nucleic acid material, then isolating the extracted nucleic acid material, and analyzing the isolated nucleic acid material.
  • the presently disclosed method utilizes the TriMetis Computer-assisted
  • TCAP Treatment Agents for identifying various characteristics in the Formalin Fixed Paraffin Embedded (“FFPE”) tissue blocks.
  • the presently disclosed method can also analyze tissue samples presented in forms or fixates other than FFPE blocks, such as, without limitation, fresh tissue, preserved tissue using fixatives other than formalin, such as, Weigners, Greenfix, UPM, CyMol, Bouin and Hollande.
  • the FFPE block may be a slide, a substrate or other sample holders.
  • Bar-coded digital Images are utilized to identify pathological and morphological features of the tumor including the location of tumor, presence and count of tumor nuclei, and the concentration of tumor nuclei and other features.
  • the TCAP system From these images and Ai output, the TCAP system generates a heatmap that identifies the region of interest.
  • the heatmap is set by the end-user to the desired selection criteria.
  • the result of the criteria identifies the locations of interest, or the target core.
  • the presently disclosed system can utilize images of both stained and unstained tissue slides or blocks, identify the high density tumor locations, and then collect (extract) the identified tumor materials for downstream DNA or other testing and analysis.
  • FIG. 1 illustrates an exemplary method according to various embodiments
  • Figs. 2A thru 21 are representative of the presently disclosed system according to one embodiment of the present disclosure.
  • the method involves generating, with a computer, a heatmap of locations of interest for the FFPE block and then obtaining core samples from each location of interest in the heatmap.
  • the samples are obtained by sterilizing a hollow punch tip, punching a respective location of interest with the hollow punch tip to pick up a respective core sample, and depositing the respective core sample to a respective receptacle.
  • Further steps include treating the core samples deposited in the receptacles with an appropriate reagent to extract the nucleic acid material, then isolating the extracted nucleic acid material, and analyzing the isolated nucleic acid material.
  • the tissue samples analyzed by the method can include human or animal tissue, and can be studied for the presence of carcinogenic tissue, a tumor, and a necrotic tissue, to name a few examples.
  • One way the depositing step can be performed is by ejecting the respective core sample with a plunger, which can be a mechanical plunger or a pneumatic impulse.
  • a plunger which can be a mechanical plunger or a pneumatic impulse.
  • the presently disclosed method can be conducted with unstained FFPE blocks, or in some embodiments the FFPE block can be stained with a stain selected from one or more of Hematoxylin and Eosin (H&E), Immunohistochemistry (THC), Fluorescence In-situ Hybridization (FISH), Chromogenic In-situ Hybridization (CISH), Spectral Imaging, Confocal Microscopy and other simulated staining techniques.
  • H&E Hematoxylin and Eosin
  • THC Immunohistochemistry
  • FISH Fluorescence In-situ Hybridization
  • CISH Chromogenic In-situ Hybridization
  • Spectral Imaging Confocal Microscopy and other simulated staining techniques.
  • the presently disclosed method can utilize information to generate a heatmap with the heatmap being generated by the computer using a machine learning system trained to identify a carcinogenic tissue, a tumor, or a necrotic tissue on the FFPE block.
  • tracing of the FFPE blocks, the heatmap, and the receptacles can be carried out by unique identifiers, such as barcodes, QR codes, or other similar machine readable identifiers.
  • the heatmap can include an X and Y coordinate for each of the locations of interest on the FFPE block.
  • the heatmap can analyze for, and provide, X, Y, and Z coordinates, or three dimensional images, for the locations of interest on the tissue sample.
  • the Z coordinate may be set to zero.
  • the presently disclosed method can also analyze tissue samples presented in other forms or fixates than FFPE blocks, such as, without limitation, fresh tissue, or preserved tissue using fixatives other than formalin, such as, Weigners fixative, Greenfix, UPM, CyMol, Bouin and Hollande.
  • fixatives other than formalin such as, Weigners fixative, Greenfix, UPM, CyMol, Bouin and Hollande.
  • the tissue sample to be analyzed can be embedded in a nonparaffin based substrate, or cryogenically preserved.
  • a system to analyze tissue samples of nucleic acid material disposed on a Formalin Fixed Paraffin Embedded (FFPE) block including a computer to generate a heatmap of locations of interest for the FFPE block, an automated punching system to obtain core samples from each location of interest in the heatmap by sterilizing a hollow punch tip, punching a respective location of interest with the hollow punch tip to pick up a respective core sample, and depositing the respective core sample to a respective receptacle is taught.
  • FFPE Formalin Fixed Paraffin Embedded
  • the presently disclosed system further includes a nucleic acid extractor and a nucleic acid analyzer.
  • the nucleic acid extractor can treat the core samples deposited in the receptacles with an appropriate reagent to extract the nucleic acid material, and isolate the extracted nucleic acid material.
  • the nucleic acid analyzer such as a genome sequencer, can be used for analyzing the isolated nucleic acid material.
  • the receptacles receiving the core samples may include solutions of the appropriate reagents and/or solvents to initiate the nucleic acid extraction or, in some instances, preserve the core sample for subsequent analysis and characterization.
  • the tissue samples analyzed by this system can include, for example, human or animal tissue, and can be studied for the presence of carcinogenic tissue, a tumor, and a necrotic tissue.
  • One way the depositing step can be performed is by ejecting the respective core sample with a plunger, which can be a mechanical plunger or a pneumatic impulse.
  • a plunger which can be a mechanical plunger or a pneumatic impulse.
  • the presently disclosed system can utilize unstained FFPE blocks, or in some embodiments the FFPE block can be stained with a stain selected from one or more of Hematoxylin and Eosin (H&E), Immunohistochemistry (THC), Fluorescence In-situ Hybridization (FISH), Chromogenic In-situ Hybridization (CISH), Spectral Imaging, Confocal Microscopy and other simulated staining techniques.
  • H&E Hematoxylin and Eosin
  • THC Immunohistochemistry
  • FISH Fluorescence In-situ Hybridization
  • CISH Chromogenic In-situ Hybridization
  • Spectral Imaging Confocal Microscopy and other simulated staining techniques.
  • the presently disclosed system and method can utilize a heatmap the generation of which is performed by a computer using a machine learning system trained to identify a carcinogenic tissue, a tumor, or a necrotic tissue on the FFPE block.
  • the machine learning system is further described in pending patent application No. 17/449,727.
  • the heatmap can comprise X, Y, and Z coordinates for each of the locations of interest on the FFPE block.
  • Unique identifiers such as bar codes or QR codes, can be used to trace and identify the FFPE blocks, the heatmap, and the receptacles.
  • the system also records any or all of the punch locations of each core sample, the tissue obtained, treatment of the core samples, and results obtained.
  • Corresponding bar-coded FFPE blocks can be loaded into the instrument.
  • the FFPE blocks are moved to slots for punching.
  • the heatmap is checked for orientation and associated with coordinates on a xyz plane. These coordinates are sent to a robotic arm that selects a sterile hollow coring tip.
  • the hollow punch tip is brought to locations of interest as directed by the heatmap. Then the robotic arm depresses and cores with the hollow punch tip the FFPE block.
  • the core is removed and carried to the corresponding tray with bar-coded tubes for downstream processing.
  • a used hollow punch tip can be returned to discard into a waste bin.
  • the robot tip holder can also be dipped in a disinfectant bath to clean any remanent DNA. This procedure removes any possibility for cross-contamination.
  • the robot head then moves to another tray to pick up a new hollow punch tip for the next core.
  • Post coring trays can be located on a conveyor belt.
  • the trays can hold arrays of 24, 48, 96, etc. of test tubes or other suitable sample holders.
  • the arrays are arranged such that the core sample held in the array can be traced back to the FFPE block, heatmap, and coring position. Once a tray is full, it can be moved to a dock where the tray remains until a technician removes it for testing, or if configured appropriately the tray will move to the next processing step, for example, dissociation for genomic testing.
  • One purpose of the presently disclosed system is to streamline and expedite the process of accessing the highest concentration of tissue for downstream molecular, genomic and DNA testing, which is currently a manual process.
  • the presently disclosed system removes several time-consuming and wasteful manual sample preparation steps including cutting multiple unstained slides, manual estimation by a highly trained pathologist to circle with a marker the highest concentration of DNA on an H&E (“Haemotoxylin and Eosin”) stained slide, technician micro dissecting or scraping slide for material of interest, and discarding unstained slide waste.
  • H&E Haemotoxylin and Eosin
  • FIG. 1 illustrates an exemplary method for a high-throughput method for analyzing tissue samples of nucleic acid material disposed on a Formalin Fixed Paraffin Embedded (FFPE) block according to various embodiments.
  • FFPE Formalin Fixed Paraffin Embedded
  • a method 100 for analyzing tissue samples of nucleic acid material disposed on a Formalin Fixed Paraffin Embedded (FFPE) block may include operation 102 to generate, with a computer, a heatmap of locations of interest for the FFPE block. This operation can be followed by operation 110 to obtain core samples from each location of interest in the heatmap.
  • This operation 110 can include three sequential methods 112, 114 and 116, to sterilize a hollow punch tip, move the hollow punch tip to respective location of interest, and then punch the respective location of interest with the hollow punch.
  • Operation 120 of depositing the respective core sample to a respective receptacle follows obtaining the core sample, and may include operation 122 to move the hollow punch tip to location of respective receptacle, and then operation 124 of ejecting the respective core sample with a plunger.
  • the plunger can be replaced with a pneumatic impulse driver.
  • FIG. 2A shows a scanned H&E image that can be analyzed to generate, with a computer, a heatmap of locations of interest for the FFPE block as shown in Fig. 2B.
  • High- throughput FFPE blocks are shown in Fig. 2C.
  • the blocks are then punched in an automated tissue puncher shown in Fig. 2D.
  • a multi-tissue, multi-arm robotic system as shown in Fig. 2E can be utilized in some embodiments of the presently disclosed system to further increase processing rates.
  • Fig. 2A shows a scanned H&E image that can be analyzed to generate, with a computer, a heatmap of locations of interest for the FFPE block as shown in Fig. 2B.
  • High- throughput FFPE blocks are shown in Fig. 2C.
  • the blocks are then punched in an automated tissue puncher shown in Fig. 2D.
  • a multi-tissue, multi-arm robotic system as shown in Fig. 2E can be utilized in some embodiments of the
  • FIG. 2F shows the process of punching a core sample from a donor block and then depositing same into the recipient block, in some embodiments of the presently disclosed system core samples are not inserted into recipient blocks but rather are deposited into designated receptacles for subsequent nucleic acid analysis.
  • Fig. 2G illustrates an array of test tubes as possible receptacles for the core samples.
  • An example of a nucleic acid extractor is presented in Fig. 2H.
  • the nucleic acid extracted by the extractor of Fig. 2H can be subsequently genome sequenced as shown in Fig. 21. Not all of the illustrated aspects of Figs. 2A-2I may be present in all embodiments of the presently disclosed system.
  • the presently disclosed system can increase throughput by adding processing 24x7x365, each instrument can increase production superior to current human manual processes, multiple instruments can function simultaneously for higher volume, and higher accuracy of quantifiable DNA identification equals higher success rate.
  • the system can be superior to the current human gated manual processes.
  • the output of the TCAP system allows for the identification of the area of the tumor tissue that has the highest concentration of nuclei, which in turn is an indication of the DNA available for subsequent immunohistochemistry and genomic sequencing.
  • tissue test regime that would benefit from the increased accuracy and quantification that results from the TCAP in combination with the presently disclosed system can utilize these teachings.
  • the presently disclosed system can be applied to veterinary applications in addition to human.
  • the identification, selection, and picking of tissue elements can provide faster processing for downstream processes such as tissue dissociation or cellular isolation.
  • TMA systems still utilize a manual process for identifying and punching FFPE blocks for Tissue Microarrays.
  • the presently disclosed system can allow for manual punching of the FFPE but differs on many other levels as the known systems are limited to individual punches.
  • the machine learning identification of locations of interest on the tissue sample provided by the TCAP process leads to higher throughput.
  • the known systems are also not designed for high-throughput analyses, nor for placement of samples into tissue dissociation cartridges.
  • known systems do not utilize Ai or machine learning to handle the identification of the region of interest with the highest tumor nuclei density, nor are known systems capable or designed to handle multiple tissue assays in a production workflow.
  • the present disclosure will be of interest to molecular genomic profiling entities, pathology equipment manufacturers and pathology diagnostic entities, and addresses the slow manual process of extracting tumor tissue (or any other tissues of concern) and identification of areas of interest.
  • the presently disclosed system can be utilized in the production for throughput of a molecular testing or diagnostic laboratory. Grunkin et al. provide background information in their U.S. Patents Nos. 9,697,582 and 10,209,165.
  • the presently disclosed system can be interfaced with various intermediate networks, wireless or wired communications equipment, and the internet and the like to allow for interaction and/or control by parties located physical away from the system.
  • the present teachings may be a system, a method, and/or a computer program product at any possible technical detail level of integration
  • the computer program product may include a computer readable storage medium (or media) having computer readable program instructions thereon for causing a processor to carry out aspects of the present invention
  • the computer readable storage medium can be a tangible device that can retain and store instructions for use by an instruction execution device.
  • the computer readable storage medium may be, for example, but is not limited to, an electronic storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination of the foregoing.
  • a non-exhaustive list of more specific examples of the computer readable storage medium includes the following: a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a static random access memory (SRAM), a portable compact disc read-only memory (CD-ROM), a digital versatile disk (DVD), a memory stick, a floppy disk, a mechanically encoded device such as punch-cards or raised structures in a groove having instructions recorded thereon, and any suitable combination of the foregoing.
  • RAM random access memory
  • ROM read-only memory
  • EPROM or Flash memory erasable programmable read-only memory
  • SRAM static random access memory
  • CD-ROM compact disc read-only memory
  • DVD digital versatile disk
  • memory stick a floppy disk
  • a mechanically encoded device such as punch-cards or raised structures in a groove having instructions recorded thereon
  • a computer readable storage medium is not to be construed as being transitory signals per se, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through a waveguide or other transmission media (e.g., light pulses passing through a fiber-optic cable), or electrical signals transmitted through a wire.
  • Computer readable program instructions described herein can be downloaded to respective computing/processing devices from a computer readable storage medium or to an external computer or external storage device via a network, for example, the Internet, a local area network, a wide area network and/or a wireless network.
  • the network may comprise copper transmission cables, optical transmission fibers, wireless transmission, routers, firewalls, switches, gateway computers and/or edge servers.
  • a network adapter card or network interface in each computing/processing device receives computer readable program instructions from the network and forwards the computer readable program instructions for storage in a computer readable storage medium within the respective computing/processing device.
  • Computer readable program instructions for carrying out operations of the present invention may be assembler instructions, instruction-set-architecture (ISA) instructions, machine instructions, machine dependent instructions, microcode, firmware instructions, state-setting data, or either source code or object code written in any combination of one or more programming languages, including an object oriented programming language such as SMALLTALK, C++ or the like, and conventional procedural programming languages, such as the "C" programming language or similar programming languages.
  • the computer readable program instructions may execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer or server.
  • the remote computer may be connected to the user's computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider).
  • electronic circuitry including, for example, programmable logic circuitry, field-programmable gate arrays (FPGA), or programmable logic arrays (PLA) may execute the computer readable program instructions by utilizing state information of the computer readable program instructions to personalize the electronic circuitry, in order to perform aspects of the present invention.
  • These computer readable program instructions may be provided to a processor of a general-purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions/acts specified in the flowchart and/or block diagram block or blocks.
  • These computer readable program instructions may also be stored in a computer readable storage medium that can direct a computer, a programmable data processing apparatus, and/or other devices to function in a particular manner, such that the computer readable storage medium having instructions stored therein comprises an article of manufacture including instructions which implement aspects of the function/act specified in the flowchart and/or block diagram block or blocks.
  • the computer readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device to cause a series of operational steps to be performed on the computer, other programmable apparatus or other device to produce a computer implemented process, such that the instructions which execute on the computer, other programmable apparatus, or other device implement the functions/acts specified in the flowchart and/or block diagram block or blocks.
  • each block in the flowchart or block diagrams may represent a module, segment, or portion of instructions, which comprises one or more executable instructions for implementing the specified logical function(s).
  • the functions noted in the block may occur out of the order noted in the figures.
  • two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved.

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé à haut débit d'analyse de multiples échantillons de tissu pour un matériau d'acide nucléique. Le procédé consiste à fournir des blocs FFPE contenant des échantillons de tissu, une carte thermique d'emplacements d'intérêt pour chaque bloc FFPE contenant un échantillon, et un dispositif de carottage ayant une pointe de poinçon creuse. Le bloc FFPE contenant un échantillon est poinçonné avec la pointe de poinçon creuse à un emplacement déterminé par la carte thermique afin d'obtenir un échantillon de carotte qui est ensuite éjecté hors de la pointe de poinçon creuse vers un réceptacle désigné. La pointe de poinçon creuse est stérilisée après éjection de l'échantillon de carotte. Ces étapes sont répétées selon les besoins pour obtenir un nombre souhaité et des échantillons de carotte typés à partir des blocs FFPE contenant un échantillon. Les échantillons de carotte individuels sont ensuite traités dans les réceptacles individuels désignés avec un réactif approprié pour extraire un matériau d'acide nucléique qui est ensuite isolé et analysé.
PCT/US2023/060501 2022-01-11 2023-01-11 Système de carottage et d'analyse de tissu Ceased WO2023137340A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202263266673P 2022-01-11 2022-01-11
US63/266,673 2022-01-11
US18/153,253 2023-01-11
US18/153,253 US20230221221A1 (en) 2022-01-11 2023-01-11 Tissue coring and analysis system

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Citations (5)

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US9697582B2 (en) 2006-11-16 2017-07-04 Visiopharm A/S Methods for obtaining and analyzing images
US10209165B2 (en) 2014-03-10 2019-02-19 Visiopharm A/S Assessment of staining quality
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Patent Citations (5)

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US9697582B2 (en) 2006-11-16 2017-07-04 Visiopharm A/S Methods for obtaining and analyzing images
US10866170B2 (en) * 2011-01-24 2020-12-15 Roche Molecular Systems, Inc Devices, systems, and methods for extracting a material from a material sample
US10209165B2 (en) 2014-03-10 2019-02-19 Visiopharm A/S Assessment of staining quality
US20190390252A1 (en) * 2018-06-21 2019-12-26 Genomic Health, Inc. Systems and methods for pre-analytical substrate processing
US20220108442A1 (en) 2020-10-02 2022-04-07 Leif E. Honda Identifying Morphologic, Histopathologic, and Pathologic Features with a Neural Network

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GHOSHAL BIRAJA ET AL: "DeepHistoClass: A Novel Strategy for Confident Classification of Immunohistochemistry Images Using Deep Learning", MOLECULAR & CELLULAR PROTEOMICS, vol. 20, 1 January 2021 (2021-01-01), US, pages 100140, XP055869422, ISSN: 1535-9476, DOI: 10.1016/j.mcpro.2021.100140 *
VITI FEDERICA ET AL: "Semi-automatic identification of punching areas for tissue microarray building: the tubular breast cancer pilot study", BMC BIOINFORMATICS, BIOMED CENTRAL , LONDON, GB, vol. 11, no. 1, 18 November 2010 (2010-11-18), pages 566, XP021085840, ISSN: 1471-2105, DOI: 10.1186/1471-2105-11-566 *

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