Diagnostic Molecular Pathology Dr. med. vet. Matti Kiupel, BS, MS, PhD, DACVP Diagnostic Center for Population and Animal Health College of Veterinary Medicine, Michigan State University 4125 Beaumont Road Room 152A, Lansing, MI 48848, USA Tel.: ** 517 432 2670; Fax: ** 517 432 6557; E-mail: [email protected]

Visualizing a Target • • •

Fluorescent Antibody Staining Immunohistochemistry In -situ Hybdridization In-situ – Fluoresecent – Chromogenic

Ferret Coronavirus

Fluorescent Antibody Staining • Well established, widely used technique – Primary antibody to detect target – Either direct or indirect labeling • Diagnostic tool for fresh/frozen material • Identification of specific infectious organisms • Commonly used on cell cultures • Antigen: protein, glyco glyco-- or lipoprotein, carbohydrate

Advantages and Disadvantages of Fluorescent Antibody Staining • • • • • • • • • • •

Used on fresh/frozen samples Not useful for formalin fixed material Fast, relatively cheap Easy to detect, highly sensitive Signal can be quantitated Architecture difficult to evaluate Not a good tool for herd surveillance Requires expensive specialized equipment Fluorescent signals will commonly fade Autofluorescence Success depends on antibody (mono (mono-- vs. polyclonal)

Immunohistochemistry • Well established, widely used technique – Primary antibody to detect target – Detection system – Visualizing reagent • Antigen: protein, glyco glyco-- or lipoprotein, carbohydrate • Diagnostic tool for routine formalin fixed material – Identification of specific infectious organisms – Identification of cell types and tumor entities – Determination of primary site of tumor – Determination of tumor malignancy

Advantages and Disadvantages of IHC • • • • •

• • • •

Routinely available, relatively inexpensive Rapid (48 hours), automated systems Can study antigens: protein levels and activities differ from those of RNA Connects visualized target with microscopic lesion Mainly used on fixed tissues: – free of infectious agents so no human health risk – Good cell morphology preservation – Anchored antigens (no displacement like unfixed) Standardization is very difficult (antigen retrieval) Difficult to quantitate Must have well -trained pathologists and lab well-trained personnel (standardization & interpretation) Success depends on antibody (mono (mono-- vs. polyclonal)

Double Staining

Avian Poxvirus and ILT

IHC vs ISH

West Nile virus

IHC vs ISH

Feline Herpes virus

In -situ Hybridization In-situ •• •• •• ••

•• •• ••

Detects Detects nucleic nucleic acids acids (DNA, (DNA, RNA, RNA, mRNA) mRNA) –– Gene Gene amplification, amplification, deletion, deletion, chromosome chromosome translocation, translocation, aneuploidy aneuploidy Visualize Visualize gene gene expression expression patterns patterns Can ne Can provide provide spatial spatial and and temporal temporal information information on on understanding understanding ge gene function function Sensitivity Sensitivity depends depends on on detection detection system system –– Radioactive semiquantitative) Radioactive labeling labeling ((semiquantitative) 35S] •• [[35 -uridine triphosphatase S]-uridine triphosphatase •• Less -accurate cellular Less-accurate cellular localization localization –– Biotin Biotin labeled labeled probes probes –– decreased decreased sensitivity, sensitivity, background background –– Digoxigenin Digoxigenin labeled labeled probes probes Higher Higher sensitivity sensitivity with with increased increased probe probe conc./time conc./time Reduce -specific binding Reduce non non-specific binding with with 1000mM 1000mM dithiothreitol dithiothreitol Probes: Probes: –– Purified Purified DNA DNA (labeled (labeled with with nick nick translation translation or or random random priming) priming) •• Lower Lower sensitivity, sensitivity, strands strands bind bind to to each each other other –– Oligoprobes Oligoprobes through through DNA DNA synthesis, synthesis, lower lower labeling labeling efficiency efficiency –– Riboprobes Riboprobes through through genetic genetic cloning, cloning, RNA RNA probes probes –– Selection Selection of of sense sense vs. vs. antisense antisense –– Use Use HPLC HPLC purification purification of of labeled labeled probes probes

In -situ Hybridization In-situ

Criteria to Determining the Type of Probe Criteria of Choice

Probes Double Stranded DNA

Single Oligonucleotide Stranded DNA

RNA

Availability

++

++

+++

+

Storage

+++

+++

+++

+

Stability

+++

+++

+++

+

Specific activity

++

++

++

+++

Manipulation

+++

+++

+++

+

Efficiency

+

++

++

+++

Controls

+

+

++

+++

In -situ Hybridization In-situ • Fixation (preventing detachment) • Pretreatment ((Permeabilization, Permeabilization, Deproteinization Deproteinization,, Acetylation Acetylation,, Dehydration) • Denaturation (breaking the double strand) • Hybridization • Post -hybridization stringency washes Post-hybridization • Detection system

Porcine Circovirus

Criteria

Signal

Background

Sensitivity

Specificity

Probe (vs. oligo)

cDNA

►

►

▼

►

Ribo

▲

▼

▲

►

Homology

3h

►

▲

▲

▼

time

▼

▼

▼

▼

▲

▲

▲

▼

▼

Advantages and Disadvantages of ISH • Independent of antibody, unnecessary to have target/antigen available • Highly sensitive and specific (target sequence!!!) • Connects visualized target with microscopic lesion • RNA easily degrades in tissues • Fixation protects RNA, but cross -linking masks target cross-linking • Labor intensive, slow, difficult to automate • Standardization is very difficult (antigen retrieval) • Can be quantitated quantitated,, except radioactive probes • Does not detect post -translational changes post-translational • Protein overexpression can be related to cell proliferation (different pathways with same result) • Must have well -trained pathologists and lab personnel well-trained (standardization & interpretation)

FISH versus CISH • FISH: – Requires expensive specialized equipment – Fluorescent signals will commonly fade – Results are normally recorded with camera, time consuming and expensive – Direct detection, thereby faster – Easily used with many color systems – Autofluorescence • CISH: – Can be interpreted on regular microscope – Lower ratio of signal to background staining – HPLC purified probes with sensitive sensitive detection system have overcome most of these problems – Restricted to 11-2 -2 colors colors – Combines target detection with morphology – Can screen a section on low magnification – Permanent labeling and can be archieved

IHC vs IFA vs ISH

Porcine Circovirus

IHC vs IFA vs ISH Cost

Speed

Sensitivity

Specificity Morphology

Target

IFA

low

fastest

variable

antibody dependent

no

protein

IHC

low

fast

variable

antibody dependent

yes

protein

FISH

high

slower

high

sequence dependent

no

nucleic acid

CISH

high slowest

high

sequence dependent

yes

nucleic acid

Eastern Equine Encephalitis

How else can we go after a Target?

PCR and In -situ PCR In-situ

Combining Tools

1

2

3

~ 450 bp

Negative

Bat

Combining Tools

Combining Tools

Laser Capture Microscopy

Laser Capture Microscopy

• Powerful imaging tool that weds imaging to molecular methods and utilizes shared pathology informatics networks (SPIN)

Laser Capture Microscopy Fixation and slide preparation are the key

RNA – Isolation from Tissue Vacuum systems with UV -sensitive UV-sensitive tape transfer systems are an option

On On approach approach is is to to change change from from aa fixation fixation model model to to aa frozen frozen section section process process ….. …..

Laser Capture Microscopy Pulmonary Fibrosis Epithelium

Smooth Muscle

Laser Capture Microscopy L

M

N

NC

L= 100bp Ladder N= Normal M= Mutation NC= Negative Control

Canine Mast Cell Tumors

Genes Associated with the Progression of Canine MCTs • 60mer long oligonucleotide microarray • 851 genes associated with – Cancer, Inflammation, Stem cells, Osteogenesis • Internal controls – Positive: • 5 housekeeping genes – Negative: • Empty • Nonsense, scrambled

What have the following done to boost awareness and hope? Glevec Her2neu P450 – Roche cyp2d6 “poor metabolizer” “extensive metabolizer” It is our hope … and not only ours … but of the science community and the public as well … that we can develop individualized diagnostic, prognostic and therapeutic procedures.

New Paradigm for Analysis Fresh Frozen Tissues - OCT Embedded tissue Histopathology DNA

Whole Whole tissue tissue LCMD LCMD

Image RNA

Whole Whole tissue tissue LCMD LCMD

Image

Whole Whole tissue tissue

Image

Protein

LCMD LCMD

Anatomical Anatomical Pathology: Pathology: Past Past and and Present Present Mast Mast Cell Cell Tumor Tumor Prognosis Prognosis

So what do we propose ?

Histopathology is qualitative and excellent at assigning a case to a “class” .. But it does not indicate the appropriate treatment with a great deal of assurance or indicate an “individual” prognosis.

Anatomical Anatomical Pathology: Pathology: Past Past and and Present Present Mast Mast Cell Cell Tumor Tumor Prognosis Prognosis L

M

N

NC

L= 100bp Ladder N= Normal M= Mutation NC= Negative Control

Anatomical Anatomical Pathology: Pathology: Past Past and and Present Present Feline Feline Lymphoma Lymphoma Diagnosis Diagnosis

So what do we propose ?

Inflammatory bowel disease and intestinal lymphoma (most are T-cell) commonly present morphological identical, especially in absence of muscularis involvement or endoscopic biopsies that don’t allow for more detailed evaluation

Anatomical Anatomical Pathology: Pathology: Past Past and and Present Present Feline Feline Lymphoma Lymphoma Diagnosis Diagnosis PCR

Feline TCRG V-N-J alignment CDR3 region

5’ primer

3’ V segment

3’ primer

N region

J segment

How do We Quantitate Reactions?

Tissue Mirco -Arrays Mirco-Arrays • Tissue microarray work station • Stainless needles cores in mobile arm • Digital precision devise • 0.6 mm core samples

Tissue Mirco -Arrays Mirco-Arrays 0.6 mm

1.5 mm 1.5 1.5 mm mm

0.6 0.6 mm mm

Tissue Array Technology: Paraffin or Frozen

Quantitative ELISA -like IHC (QUELI) ELISA-like

Confocal Microscopy • Used to increase micrograph contrast and/or to reconstruct three-dimensional images • Uses point illumination and a spatial pinhole to eliminate out-of-focus light or flares in specimens that are thicker than the focal plane • Only the light within the focal plane can be detected • 2D or 3D imaging requires scanning over a regular raster • Three types of confocal microscopes: • Confocal laser scanning microscope • Spinning-disk (Nipkow disk) confocal microscope • Programmable Array Microscopes (PAM)

Confocal Laser Scanning Microscopy

Confocal Laser Scanning Microscopy

Porcine Circovirus

Image Analysis

Image Analysis

Image Analysis

Image Analysis

Analysis of Cell Proliferation M

The intermediate step

G0

Ki-67

G2 G1

PCNA increasing

S

Mitotic Index G1

BrdU

Analysis of Cell Proliferation

Quantification and Spatial Recognition

Units are “Lymphocytes per 100 pixels”

Nuance Multispectral Imaging

STEP STEP 1: 1: RGB RGB image image

STEP STEP 2: 2: Image Image Cube Cube

Shown ylin counter Shown is is the the traditional traditional image image of of IHC IHC // DAB DAB (step (step 1) 1) and and hematox hematoxylin counter-stain n step stain with with the the spectral spectral separation separation of of the the DAB DAB from from hematoxylin hematoxylin iin step 2. 2.

STEP STEP 3: 3: TIFF TIFF representation representation of of the the image image cube cube

STEP -fluorescent STEP 4: 4: Pseudo Pseudo-fluorescent image image of of the the cube cube

Brown -color selection Brown has has been been changed changed to to red red by by pseudo pseudo-color selection for for ease ease of of visualization te quantification. visualization and and aa pseudo pseudo fluorescent fluorescent image image created created to to facilita facilitate quantification. STEP STEP 5: 5: Quantification: Quantification: From -fluorescent image, From the the Pseudo Pseudo-fluorescent image, the the following following may may be be quantified: quantified: --Number Number of of cells cells --Intensity Intensity per per cell cell --Intensity Intensity per per number number of of cells cells --Intensity Intensity per per unit unit area area

Pathology the Future: Needs • Monoclonal antibodies against proteins for diagnosis • Gene targeted probes and antibodies for directed “surgical targeting and killing” • Tissue -array slides for Tissue-array controls • Micro -array and serum -array Micro-array serum-array “chips” • New instrumentation for molecular imaging • Scientists, technologists and informed public