EP1135680A1 - Systeme a haut debit pour l'evaluation de l'utilite clinique de cibles moleculaires dans des echantillons de tissus - Google Patents

Systeme a haut debit pour l'evaluation de l'utilite clinique de cibles moleculaires dans des echantillons de tissus

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Publication number
EP1135680A1
EP1135680A1 EP00965348A EP00965348A EP1135680A1 EP 1135680 A1 EP1135680 A1 EP 1135680A1 EP 00965348 A EP00965348 A EP 00965348A EP 00965348 A EP00965348 A EP 00965348A EP 1135680 A1 EP1135680 A1 EP 1135680A1
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EP
European Patent Office
Prior art keywords
tissue
stain
target
tissue samples
target molecule
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.)
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Application number
EP00965348A
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German (de)
English (en)
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EP1135680A4 (fr
Inventor
Jonathan Cohen
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Ventana Medical Systems Inc
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Jonathan Cohen
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Filing date
Publication date
Application filed by Jonathan Cohen filed Critical Jonathan Cohen
Publication of EP1135680A1 publication Critical patent/EP1135680A1/fr
Publication of EP1135680A4 publication Critical patent/EP1135680A4/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention is directed to a system and process for evaluating the potential clinical utility of molecular targets in tissue and, more particularly, to such a process that is fully automated so that large numbers of unique targets from multiple sources can be tested on large quantities of different tissue samples mounted on microscope slides.
  • HERCEPTIN ® Genetech, S. San Francisco, CA
  • HERCEPTIN ® a monoclonal antibody that targets metastatic breast cancer cells that overexpress the HER-2 oncogene.
  • HERCEPTIN ® works by binding to the HER-2 growth factor receptors present in excessive amounts on the surface of the cancer cells.
  • the drug is indicated only for patients whose turners have either amplification (i.e. extra copies) of the HER-2 gene as determined by in-situ by hybridization (ISH) or protein overexpression as determined by immunohistochemistry (IHC).
  • HER-2 status has also been found to predict patient response to a variety of conventional therapeutic agents such as doxorubican.
  • HER-2 and most other cancer markers are acquired (i.e. somatic) aberrations present only in the tumor. These markers are best identified in intact cells (in-situ) using ISH or IHC in order to allow the pathologist or scientist to study the histopathologic architecture or morphology of the tissue specimen under the microscope at the same time that the nucleic acid or proteins are being assayed.
  • ISH can be used to look for the presence of a genetic abnormality or condition such as amplification of cancer causing genes specifically in cells that, when viewed under a microscope, morphologically appear to be malignant.
  • target molecules sought to be detected are RNA sequences ISH permits researchers to learn where, and in what tissues, a particular gene is over or under expressed.
  • Immunohistochemistry allows the detection of target antigens (usually proteins) within tissues.
  • Antibodies to specific antigens are used to probe tissues to detect the target in specific cells of the tissue being assayed.
  • the level of protein expression and subcellular location of the target can also be elevated.
  • IHC can ascertain whether or not the targets are expressed in the cell nucleus, cytoplasm or on the cell membrane in a semi-quantitative manner.
  • tissue microarray In order to permit parallel in-situ detection of molecular targets in large numbers of tumor samples a "tissue microarray" was recently developed by U.S. government scientists as part of the Human Genome Project. (Kononen, J. et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nature Med. 7, 844-847 (1998)). As described elsewhere herein up to 1000 circular tissue biopsies from different tumors can be distributed in a grid format on a standard microscope slide. This allows nucleic acid probes or antibodies to be applied to hundreds of different samples at one time.
  • ISH and IHC remain highly specialized techniques that are complex, time consuming, and vary from lab to lab.
  • IHC requires a series of treatment steps conducted on a tissue section or cells (e.g. blood or bone marrow) mounted on a glass slide to highlight by selective staining certain morphological features indicative of disease states.
  • Typical steps include pretreatment of the tissue section to remove the paraffin and reduce non-specific binding, retrieval of antigens masked by cross-linking of the proteins from the chemical fixatives, antibody treatment and incubation, enzyme labeled secondary antibody treatment and incubation, substrate reaction with the enzyme to produce a fluorophore or chromophore highlighting areas of the tissue section having epitopes binding with the antibody, counter staining, and the like.
  • Most of these steps are separated by multiple rinse steps to remove unreacted residual reagent from the prior step. Incubations can be conducted at elevated temperatures, usually around 3 7°C, and the tissue must be continuously protected from dehydration. Lack of uniformity among different labs in their use of reagents and reaction conditions often yields to results that differ improperly.
  • ISH analysis which relies upon the specific binding affinity of probes with unique or repetitive nucleotide sequences from the cells of tissue samples or infectious agents, requires a similar series of process steps with many different reagents and is further complicated by varying temperature requirements. As with antibodies each probe must be individually optimized for reactivity in tissue. The variables include probe length and labeling, probe concentration, hybridization conditions, stringency washes and detection methodology.
  • the present invention is directed to apparatus and methods which can be employed in a high-throughput manner to establish the clinical utility of a molecular target by screening for the target in large quantities of tissues from different patients, organs, diseases, or disease stages. For each tissue sample the quantity or location of target is determined and compared to other samples from different organs or from patients in different disease states. For example, determining that amplification or overexpression of a particular gene is more frequent in tumors from patients with a recurrent form of cancer may create a prognostic marker used in planning treatment strategies as well as a target for designing new drugs that interact with the gene or its product.
  • the inventive apparatus and method are particularly adapted for providing services to various sources of potential target molecules, in particular pharmaceutical and biotechnology companies seeking ISH/IHC markers to help identify patients that would benefit from their drug as well as validation of their drug targets using in-situ techniques.
  • the apparatus generally comprises (i) a tissue microarray having hundreds of small tissue samples upon which reagents may be applied, (ii) an automated staining instrument for applying reagents to the tissue samples and automatically carrying out most of the steps required for ISH and IHC, and (iii) an imaging instrument to allow the user to readily ascertain the presence and/or quantity of target in each of the samples.
  • a key advantage of the present invention is the economies of scale and efficiencies created by performing essentially the same services using the same instrumentation for multiple clients having multiple targets to test or validate.
  • Another advantage of the present invention is the speed and high-throughput achieved through the use of the combination of tissue microarrays together with the automated staining instrumentation.
  • Still another advantage of the present invention is that it allows accurate comparison of results from multiple different tissue samples each having been treated in precisely the same manner. Yet another advantage of the present invention is that each sample can receive an individualized staining or treatment protocol even when such protocols require different temperature parameters and other conditions.
  • Still another advantage of the present invention is that it allows the temperature of the entire surface of the slide to which the tissue is mounted to be carefully controlled (i.e. within plus or minus two degrees Celsius of the desired temperature).
  • Such precision is particularly necessary for DNA denaturation and probe hybridization in ISH and related processes such as in-situ PCR. Furthermore, since the heating according to the present invention is made uniform, this narrow temperature range is maintained throughout the surface of the slide so that the tissue is evenly heated regardless of its position on the slide. This feature is particularly advantageous when heating tissue microarrays as it allows each of the plurality of tissue samples to be heated uniformly. This improves accuracy when comparing reactions in different tissue samples placed at opposite ends of the slide. Still another advantage of the present invention is the negation of human error and increase in productivity resulting from automation.
  • Yet another advantage of the present invention is that the same staining protocol (reagents, times, temperatures, etc.) developed for evaluating or validating a target in a research setting can be subsequently employed a clinical (patient care) setting for disease prognosis or treatment selection.
  • FIG. 1 is a schematic illustration of the method according to the present invention.
  • FIGS. 2a-2c are perspective views of a tissue microarray according to the present invention and the instruments employed in creating a microarray from tissue blocks.
  • FIG. 3 is a perspective view of the staining instrument according to the present invention.
  • FIG. 4 is a perspective view of the staining instrument according to the present invention shown in conjunction with a computer and other instruments with which it operates.
  • FIG. 5 is a perspective of the staining instrument according to the present invention shown with a reagent dispenser.
  • FIG. 6 is an enlarged perspective view of a thermal platform according to the present invention shown supporting a tissue microarray slide.
  • FIG. 1 a schematic illustration of a preferred embodiment of the system and apparatus according to the present invention which is designated generally by reference numeral 10.
  • System 10 generally comprises tissue microarray apparams 12 for constructing arrays of hundreds of minute tissue samples mounted on a single glass microscope slide, staining apparatus 14 for automatically conducting most of the steps required for ISH/IHC, and imaging apparams 16 to allow the results of the ISH/IHC staining to be visualized and analyzed by the user.
  • System 10 preferably has access to one or more tissue banks 18 (a-c) having thousands of preserved surgical samples catalogued by organ type, disease, and patient history.
  • system 10 is adapted to serve multiple sources of different targets 20 such as pharmaceutical companies and the like who each supply the system with one or more molecular targets 22 (DNA, RNA, or protein) and receive data 24 regarding the clinical relevance of the targets based on screening of the tissue samples assayed.
  • targets 20 such as pharmaceutical companies and the like who each supply the system with one or more molecular targets 22 (DNA, RNA, or protein) and receive data 24 regarding the clinical relevance of the targets based on screening of the tissue samples assayed.
  • “Clinical Utility” means usefulness of a target for (i) designing or prescribing a drug or therapy that interacts with the target, or (ii) determining which patients would be most likely to benefit from a particular drug or therapy.
  • tissue means tissue from different patients, organs, diseases, and/or disease stages.
  • High-Throughput means the capability to treat more than about 20,000 different tissue samples in one day.
  • Sources means companies or similar entities that provide the system according to the present invention with at least one target, receive services from the system, and are separately controlled from the company that uses the system.
  • Target and “Targeted molecules” means detectable molecules found in cells including without limitation nucleic acids, proteins, antigens, carbohydrates, lipids, and small molecules.
  • tissue means any collection of cells that can be mounted on a standard glass microscope slide including, without limitation, sections of organs, tumor sections, bodily fluids, smears, frozen sections, cytology preps, and cell lines.
  • tissue Array and "Tissue Microarray” means a glass microscope slide or similar solid surface having a plurality of different tissue samples thereupon.
  • “Screen” means determining the presence, absence, quantity, location, and/or other characteristics of a target in a tissue sample. "Stain” means any biological or chemical substance which, when applied to targeted molecules in tissue, renders the molecules detectable under a microscope. Stains include without limitation detectable nucleic acid probes, antibodies, and dyes.
  • Treating shall mean application of a stain to a tissue as well as other processes associated with such application including, without limitation, heating, cooling, washing, rinsing, drying, evaporation inhibition, deparaffinization, cell conditioning, mixing, incubating, and/or evaporation.
  • tissue microarray apparatus 12 In order to permit parallel in-situ detection of molecular targets in large numbers of tumor or other tissue samples a tissue microarray apparatus 12 is provided as illustrated in FIG. 2 (a-c). Developed at the National Human Genome Research Instimte at the NIH (Bethesda, MD), apparams 12 and the uses thereof to make a tissue microarray 15 having as many as 1000 cylindrical tissue biopsies distributed in a single array is described in Kononen, J. et al. "Tissue microarrays for high-throughput molecular profiling of tumor specimens. " Nature Med. 7, 844-847 (1998) which is incorporated herein in its entirety. Materials needed to construct the array (FIG.
  • H&E hemotxylin & eosin
  • a bar code label 17 is preferably applied to tissue microarray 15 for identification by the automated staining instrumentation 14.
  • machine readable indicia or media including optically readable letters or symbols, magnetic strips, and the like.
  • automated staining instrumentation 14 is provided to significantly reduce human labor and the costs and error rate associated therewith, and to introduce uniformity.
  • the preferred apparams utilized is the DISCOVERYTM automated staining instrument available from Ventana Medical Systems, Inc. (Tucson, Arizona). This instrument is a microprocessor controlled system including a revolving carousel supporting radially positioned slides. A stepper motor rotates the carousel placing each slide under one of a series of reagent dispensers positioned above the slides. Bar codes on the slides and reagent dispensers permits the computer controlled positioning of the dispensers and slides so that different reagent treatments can be performed for each of the various tissue samples by appropriate programming of the computer.
  • a unique feature of the DISCOVERYTM instrument that distinguishes it from other instruments known in the art, and renders it particularly suitable for inclusion in system 10 is that each slide rests on a separate heating platform allowing the temperature of each sample to be precisely controlled independent of the other samples.
  • This feature is particularly advantageous when performing in-situ hybridization experiments which is nucleic acid based and requires higher and more precise temperature control.
  • the temperature In order to denature the DNA double helix of both the target sample and the probe so as to render them single stranded, the temperature must be raised above the melting point of the duplex, usually about 65°- 105 °C. At the same time it is imperative that the sample not be overheated past 100° C as doing so destroys cell morphology making it difficult to view under a microscope.
  • Precise temperature control is also required in ISH to effect probe hybridization and washing at the desired stringency. The selected temperature must be low enough to enable hybridization between probe and template, but high enough to prevent mismatched hybrids from forming.
  • instrumentation 14 comprises automated staining instrument 32, a host computer 33 preferably a dedicated personal computer, bulk fluid containers 34, waste container 36 and related equipment.
  • a host computer 33 preferably a dedicated personal computer
  • bulk fluid containers 34 preferably a waste container 36
  • waste container 36 preferably a waste container 36
  • instrumentation 14 is found in U.S. Patent Application Serial No. 09/259,240 to Richards et al. which corresponds to PCT International Application No. PCT/US99/04181 , both filed on February 26, 1999 which are incorporated herein in their entirety.
  • Instrument 32 is a computer controlled, bar code driven, staining instrument that automatically applies chemical and biological reagents to tissue or cells mounted or affixed to standard glass microscope slides or tissue microarrays.
  • reagent dispensers Up to 20 slides are mounted in a circular arrangement to a carousel which rotates, as directed by the computer, placing each slide under one of a series of reagent dispensers positioned above the slides.
  • Each slide receives the selected reagents (e.g. DNA probe) and is washed, mixed and/or heated in an optimum sequence and for the required period of time.
  • Tissue sections so stained or treated are then removed from the apparams by the user to be viewed under a microscope or other imaging instrument 16 by a scientist or pathologist who analyzes the stained sample to determine the presence, location, and quantity of target present.
  • the computer controlled automation permits use of the apparatus in a "walk-away" manner, i.e. with little manual labor.
  • Individualized slide temperature control is accomplished by the heating system according to the present invention that has thermal platforms radially mounted to the carousel for heating the slides and sensing the temperature of each.
  • a printed circuit board also mounted to the slide carousel, individually monitors and controls each thermal platform separately.
  • Information and power pass between the rotating carousel and the fixed apparatus via a slip ring assembly. This information includes the upper and lower temperature parameters needed for heating each of the 20 slides for the appropriate time period.
  • instrument 32 comprises a housing 40 having a lid 42 hingedly mounted thereto.
  • a slide carousel 44 is mounted within housing 40 for rotation in the direction of arrow A.
  • a plurality of thermal platforms 50 are mounted radially about the perimeter of carousel 44 upon which standard glass slides with tissue samples (standard or microarray) may be placed. It is a key feature of instrument 32 that the temperature of each slide may be individually controlled and is constant across the top surface of the slide.
  • wash dispense nozzles Liquid CoverslipTM dispense nozzle, fluid knife, wash volume adjust nozzle, bar code reader mirror, and air vortex mixers as shown and described in U.S. Patent Application Serial No 09/259,240 supra.
  • reagent carousel 52 Rotatably mounted atop lid 42 is a reagent carousel 52.
  • Dispensers 54 are removably mounted to a reagent tray 56 (FIG. 5) which, in turn, is adapted to engage carousel 52.
  • Reagents may include any chemical or biological material conventionally applied to slides including nucleic acid probes or primers, polymerase. primary and secondary antibodies, digestion enzymes, pre-fixatives, post-fixatives, readout chemistry, and counterstains.
  • dispensers 54 are preferably modified so that the reagent is dispensed evenly over all of the minute tissue samples in microarray 15. This could be accomplished by adjusting the orifice of the dispenser to widen the angle of spray or by moving the dispenser to drop reagent on two or more different locations of the microarray.
  • Reagent dispensers 54 are preferably bar code labeled 58 for identification by the computer. For each slide, a single reagent is applied and then incubated for a precise period of time in a temperature-controlled environment. Mixing of the reagents is accomplished by compressed air jets (not shown) aimed along the edge of the slide thus causing rotation of the reagent. After the appropriate incubation, the reagent is washed off the slide using nozzles. Then the remaining wash buffer volume is adjusted using the volume adjust nozzle. Liquid CoverslipTM solution, to inhibit evaporation, is then applied to the slide via nozzle. The air knife divides the pool of Liquid CoverslipTM followed by the application of the next reagent. These steps are repeated as the carousels turn until the protocol is completed.
  • Instrument 14 permits the automatic staining or treating of multiple tissue samples mounted on microscope slides so that each sample can receive an individualized staining or treatment protocol even when such protocols require different temperature parameters.
  • samples requiring de -waxing e.g. tumor sections
  • samples requiring de -waxing can be automatically processed at the same time as other samples (e.g. smears) that do not require this preliminary step.
  • paraffinization Prior to staining, complete removal of the paraffin, a hydrophobic substance, is also required so that it does not interfere with antibody or probe binding.
  • Deparaffinization is normally achieved by the use of two or three successive clearing reagents that are paraffin solvents such as xylene, xylene substitutes or toluene which may be toxic, flammable and pose environmental hazards. Safer and faster methods to deparaffinize the slides would be advantageous and may be used.
  • some of the treatment parameters may have to be changed from those employed when treating conventional tissue section. These changes may include, for example, more time for cell conditioning to restore the three dimensional structure of the tissue. Additionally the reagent concentrations may need to be increased when staining microarrays due to the quantity of target that is present from that typically present with conventional sections.
  • thermal platforms 50 are radially mounted to carousel 44 for heating the slides and monitoring the temperature thereof.
  • Each thermal platform 50 comprise a plate 52 made of brass or a similar material that conducts heat upon which a glass microscope slide may rest.
  • a control electronics printed circuit board (not shown) is also mounted to the slide carousel for monitoring the sensors and controlling the heaters. Information and power are transferred from the fixed instrument platform to the rotating carousel via a slip ring assembly. This information includes the temperature parameters needed for heating the slides (upper and lower) communicated from a microprocessor (after having been downloaded from computer 32) to control electronics as described in the aforementioned application to Richards et al.
  • the thermal platform heats the slide. Likewise, if the slide temperature is found to be above the upper limit, heating is stopped. A power supply of sufficient capacity to provide about eight watts per heater is provided to meet the requisite rate of temperature rise (a/k/a "ramp up").
  • Heating the slide surface uniformly is a key goal of the present invention since tissue specimens in a microarray are mounted at different positions on the slide. This poses a challenge for conduction heating, even when done manually, since traditional hot plates often generate patchy "hotspots" making it hard to know where to place the slide on the plate. If cells in different tissue samples are not heating evenly the slide cannot be reliably interpreted. For example if the temperature on one side of the slide is not high enough to denature the probe or tissue DNA this may lead to false negatives for samples positions at that side.
  • the resistive heater (not shown) is bonded to the bottom side of plate 52.
  • the brass plate has sufficient conductivity to smooth out any local non-uniformity caused by the fact that the heating traces are not continuous over the surface, but rather are adjacent lines separated by a space where no heat is generated. The separation in on the order of .015 inch, so this non-uniformity in the heat source is not felt at the side of the plate which abuts the slide.
  • Uniformity of temperature of the slide should also be achieved by modifying the heating traces so that they produce heat in a non-uniform manner. More heat is dissipated to the surrounding air at the edges of the plate than in the center. To compensate for this phenomenon the heater flux in the center of the slide should be adjusted in the manner described in the application to Richards, et al. infra at page 14. As a result the temperature differential between samples positioned anywhere on the slide should be within plus or minus two degrees Celsius.
  • staining instrument 14 is substantially the same as the instrument described in USSN 09/259,240 except for the aforementioned modifications to dispenser 54 and the detection chemistries in order to be used in conjunction with tissue microarray 15.
  • Equipment 16 for viewing and interpreting the assays performed on the tissue array section are preferably provided as part of system 10.
  • Equipment 16 may comprise a standard microscope (fluorescent or brightfield) or a digital imaging system such as those employed in imaging DNA arrays.
  • a digital imaging system would likely comprise an electronic camera, a frame grabber, and image processing software installed on a personal computer (PC).
  • PC personal computer
  • a variety of low-cost electronic cameras based on charge-coupled device (CCD) technology are commercially available.
  • a frame grabber is a printed circuit board that can be added to a PC as an interface between the camera and the software.
  • the image processing software uses picture data stored in the frame grabber and is commercially available. Automated readers and optical scanners may also be employed to assist in interpreting the stained slide.
  • tissue banks 18 consisting of standard blocks of paraffin embedded tissue as has been utilized for decades.
  • the blocks should be readily accessible by disease type and stage, patient history at the time the tissue was excised, and preferably the post-surgical clinical follow-up. It is also desirable that the blocks identify the method of fixation (e.g. formalin) as well as the results any diagnostic tests performed.
  • Such banks exists at most hospitals having a surgical pathology department. For example, an extensive 100 year old tissue bank exists at the Johns Hopkins University Medical Center (Baltimore, Maryland).
  • Sources 20 of target molecules for system 10 would include pharmaceutical and biotechnology companies, research institutes, and university laboratories that have identified novel targets believed to be associated with a particular disease or disorder including genes, gene fragments, mRNA sequences, or antigens. Typically they have an idea or prediction of the targets' biological function from profiling the expression pattern of clinical samples using one or more of the aforementioned technologies such as SAGE or DNA microarrays.
  • tissue banks 18 and select between 30 and 1000 blocks representing different patient populations and disease states.
  • the selected blocks are used as donor blocks 26.
  • the types of tissue samples selected would depend largely on the diseases for which new in situ assays would be deemed useful in medical practice. This would include cancer, arthritis, and skin disorders such as psoriasis and eczema. This might also include tissues from patients diagnosed Chron's disease, type I diabetes, and certain other autoimmune disorders. Additionally, tissue infected with a pathogen (e.g. Human Papaloma Virus) would be used for developing in situ assays for selecting treatments to various infectious diseases.
  • a pathogen e.g. Human Papaloma Virus
  • a H&E stained section 30 [FIG. 2(a)] is made from each block to define representative tumor regions.
  • Tissue cylinders with a diameter 0.6mm are punched from tumor areas of each "donor" tissue block and brought into a recipient paraffin block using steel tube 25.
  • Five- ⁇ m sections of the resulting multitumor tissue microarray block are transferred to glass slides using the paraffin sectioning aid system [adhesive coated slides (PSA-CS4x), adhesive tape, and UV lamp; Intermedics, Inc. hackensack, NJ] supporting the cohesion of 0.6 mm array elements.
  • PSA-CS4x paraffin sectioning aid system
  • nucleic acid probe is constructed that will preferentially hybridize with the target.
  • the techniques of probe construction are described by Herrington and McGee, Diagnostic Molecular Pathology, IRL Press (1992).
  • the target is a protein
  • a detectable antibody is raised that will preferentially bind to the target.
  • the techniques of raising antibodies are described in Lidell and Cryer, A Practical Guide to Monoclonal Antibodies (1991).
  • Sections cut from the array allow parallel detection of DNA (fluorescense in situ hybridization, FISH), RNA (mRNA ISH) or protein (immunohistochemistry, IHC) targets in each of the hundreds of specimens in the array.
  • staining instrument 14 is employed to carry out the staining protocols in an automated manner.
  • manual staining of the microarray may first be employed followed by automatic staining of conventional samples with instrumentation 14 to confirm the results of the array.
  • Staining instrument 14 may be used to perform in-situ hybridization (ISH), in- situ, PCR, immunohistochemistry (IHC), Special Stains, as well as a variety of chemical (non-biological) tissue staining techniques on an array or conventional tissue specimens. Moreover, two or more of the above techniques may be employed during a single run despite their differing temperamre requirements due to the inventive heating system herein.
  • ISH in-situ hybridization
  • IHC immunohistochemistry
  • Special Stains as well as a variety of chemical (non-biological) tissue staining techniques on an array or conventional tissue specimens.
  • two or more of the above techniques may be employed during a single run despite their differing temperamre requirements due to the inventive heating system herein.
  • In-situ hybridization is clearly a technique that may be advantageously employed with the instrument according to the present invention, either alone or in combination with other techniques, since many of the steps in ISH must be carefully temperamre controlled for a precise period of time. The precise amount of heat for a specific period of time is necessary to sufficiently denature the DNA so that subsequent hybridization may occur without over-heating to the point where cell morphology is degraded. Different specimens may require different times and temperamres for denaturation depending on the tissue type and how it was prepared and fixed. The steps of denaturation, hybridization, and posthybridization washes each have unique temperamre and time requirements that depend on the particulars of the probe and tissue being tested.
  • DNA probes require and are typically hybridized at between 30°- 65 °C while RNA probes are typically hybridized at higher temperamres with the time for hybridization varying from 30 min. to 24 hours depending on target copy number, probe size and specimen type.
  • Standard denaturation for cytogenetic preparations is performed at about 72 °C for 2 min. while for tissue sections the conditions may vary from 55 °C to 95 °C from 2 to 30 min.
  • Post hybridization wash temperamres may vary from about 65 °C to 100°C for 2 min. to 15 min. Salt concentration may vary from O.lx to 2x SSC.
  • Probe detection temperamres may vary from ambient to 42 °C for 2 min. to 30 min.
  • the low mass of the plate 60 and heater 64 enables the rapid heating and cooling of the slide and consequently the tissue on the slide (i.e. from 37°C to 95°C in 180 seconds).
  • the increased rapidity of heating and cooling increases the efficiency of in situ hybridization. Concomitantly the background is decreased and the quality of the resulting test is vastly improved.
  • Hybridization or denaturation of DNA is absolutely essential to an ISH tissue staining process and requires that temperamres in the range of 92-100 degrees C be quickly reached, precisely controlled and maintained.
  • the thermal platform brings treated tissue on microscope slides to the required temperamre range in less than 180 seconds with an accuracy of plus or minus 2 degrees C. Rapid loss of temperamre in hybridized tissue is essential to successful staining and diagnosis.
  • a fan or other rapid cooling feature may be added to bring the required temperamre to 37 degrees C in less than 420 seconds.
  • ISH includes both fluorescent detection (FISH) and non-fluorescent detection (e.g. brightfield detection).
  • Apparatus 10 may also be employed for the simultaneous application of ISH and
  • Sections embedded in paraffin require as an early step deparaffinization of the embedded tissue.
  • Using the thermal platform eliminates the use of harsh chemicals such as xylene, through the use of precisely controlled heating of individual slides allowing the paraffin embedded in the tissue to melt out and float in aqueous solution where it can be rinsed away.
  • Paraffin being less dense than water, once liquified rises through the aqueous buffer on the tissue sample and floats on top of this fluid.
  • the liquid paraffin can then be removed from the microscope slide and away from the tissue sample by passing a fluid stream, either liquid or gaseous, over the liquid paraffin. Details of this procedure are set forth in U.S. Patent Application Serial No. 60/099,018 filed September 3, 1998 which is incorporated herein.
  • a similar technique may be employed to remove embedding materials other than paraffin such as plastics although the addition of etching reagents may be required.
  • Heating the tissue with thermal platforms 50 in an appropriate aqueous solution has been found to improve the accessibility of the stain to the target molecule in the cell (protein, nucleic acid, carbohydrate, lipid, or small molecule). Lack of accessibility may be caused by cross-linking of the molecules by aldehydes used in preserving the tissue or by other changes in the confirmation caused by fixatives. Cross-linking of antigens causes a loss of antigenicity due to the chemical modification of antigenic proteins.
  • This process of improving accessibility of the stain (biological or chemical) to the molecular target is referred to herein as "cell conditioning. " For protein targets the preferred conditioning solution is citrate buffer, the preferred temperature is up to about 95 degrees C, and the preferred time of heating is about one hour.
  • the preferred conditioning solution is citrate buffer and the preferred temperamre is up to about 100 degrees C for about 42 minutes. Heating the tissue sample by the thermal platform decreases the degree of cross-linking in aldehyde treated tissue such that the modified antigen reverts to a form recognizable by a corresponding antibody thereby enhancing the staining.
  • the preferred conditioning solution is citrate buffer and the preferred temperamre is up to about 75 degrees C for about one hour. Many alternatives to citrate buffer may be employed as cell conditioning solution. A list of such solutions appears in Analytical Morphology, Gu, ed. , Eaton Publishing Co. (1997) at pp. 1-40. The solutions should generally have known molarity, pH, and composition. Sodium dodecyl sulfate (SDS), ethylene glycol are preferably added to the conditioning solution.
  • SDS sodium dodecyl sulfate
  • Typical In-Situ Hybridization (ISH), In-Situ PCR, Immunohistochemical (IHC), Histochemical (HC), or Enzymehistochemical (EHC) methods as carried out with the apparams of this invention includes the following steps are described in the patent application of Richards et al. infra.
  • the stained slides would be scored and analyzed by a pathologist or pathology support personnel using techniques known in the art.
  • the results would be preferably be correlated by a biostatistician to arrive at clinical utility of the target in tissue. For example, it might be determined that overexpression of the gene target is a particular tumor type correlates with extended survival in patients treated with a drug designed to block expression of the gene target. A useful in situ assay could then be developed for use in selecting patients to receive the drug.
  • System 10 should be capable of screening large volumes of tissue samples in a high-throughput manner. If both tissue microarray 12 and automated staining instrumentation 14 are used at least one run and perhaps two runs of twenty slides, each supporting up to 1000 minute tissue samples may be treated in one day. Thus between 20,000 and 40,000 different samples may be screened per day using system 10.

Abstract

L'invention concerne une méthode visant à établir l'utilité clinique d'une cible moléculaire, caractérisée en ce qu'elle consiste à mettre en contact une grande quantité de différents échantillons de tissus, en un grand débit, avec une tache liant spécifiquement une molécule cible in situ, puis à déterminer dans quelle mesure la tache a lié la molécule cible dans les échantillons de tissus. A cet effet, l'appareil pouvant être employé comprend (I) une micro-matrice de tissus comportant des centaines de petits échantillons de tissus sur lesquels des réactifs peuvent être appliqués; (ii) un instrument de coloration automatisé pour l'application des réactifs sur les échantillons de tissus et pour effectuer la plupart des étapes requises pour l'hybridisation et l'immunohistochimie in situ, et (iii) des moyens d'imagerie permettant à l'utilisateur de constater facilement la présence et/ou la quantité d'une cible dans chacun des échantillons.
EP00965348A 1999-09-24 2000-09-22 Systeme a haut debit pour l'evaluation de l'utilite clinique de cibles moleculaires dans des echantillons de tissus Withdrawn EP1135680A4 (fr)

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US15566599P 1999-09-24 1999-09-24
US155665P 1999-09-24
PCT/US2000/026113 WO2001022086A1 (fr) 1999-09-24 2000-09-22 Systeme a haut debit pour l'evaluation de l'utilite clinique de cibles moleculaires dans des echantillons de tissus

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