Diagnostics of Precancerous and Cancerous Lesions

8/2/2019 Diagnostics of Precancerous and Cancerous Lesions

1/133

Presented by:Dr. Kush Pathak


2/133

Introduction

Classification of Precancerous lesions and

Conditions Detection techniques

Advanced diagnostic methods

Conclusion References


3/133

Oral cancer is sometimes preceded by clinically visible lesions

which are noncancerous to begin with and which have therefore

been termed precancerous or premalignant. WHO divided this

premalignant state into two:

Precancerous lesions

Precancerous condition ( Pindborg 1980)

The ability to diagnose premalignant state is critical to the battleagainst oral cancer, so that it can be managed conservatively with

minimal surgical morbidity and 100% survival.


4/133

The most important and widely used method of evaluating the

malignant potential of oral precancerous lesions and conditions is

by a conventional microscopical study of epithelial dysplasia.

Advances in molecular diagnosis suggest that genetic markers of

precancerous changes are likely detectable before clinical and

histopathologic changes can be identified making it necessary todevelop these methods for early diagnosis and treatment.


5/133

Precancerous lesion - (WHO 1978) is a morphologically alteredtissue in which cancer is more likely to occur than in its apparently

normal counterpart.

Clinical classification Leukoplakia

Erythroplakia Proliferative Verrucous leukoplakia

palatal keratosis with reverse smoking


6/133

Histological classification Squamous epithelial dysplasia

Squamous cell carcinoma

Solar keratosis

Precancerous condition: (WHO 1978) is defined as a generalizedstate associated with a significantly increased risk of cancer.


7/133

Sideropenic dysphagia

lichen planus

Oral submucous fibrosis

Syphilis

Discoid lupus erythematosus

Xeroderma pigmentosa

Epidermolysis bullosa


8/133

Clinical examination Diagnostic adjuncts

Cytology

Brush cytology

Liquid cytology

Tissue fluorescence

Vital staining Diagnostic methods

Biopsy Fine needle aspiration

Sentinel node biopsy

Imaging


9/133

Cytochemical and molecular methods Nucleolar organizing regions

Abnormal DNA segregation (DNA aneuploidy)

Loss of heterozygosity

Advanced Molecular methods Molecular methods

Polymerase chain reaction

Filter hybridization

In situ hybridization

Fluorescent in situ hybridization


10/133

Comparative genomic hybridization

Complemantry DNA (cDNA) microarrays

Flow cytometry

Immunologic methods Immunohistochemistry

Immunofluorescency


11/133

Should include a thorough head and neck & intraoral examination,

with examination of the lymph nodes and visual examination,

palpation of the oral mucosa.

The location, size, border, color and surface characteristics of the

lesion should be noted.


12/133

A variety of aids to the diagnosis of oral precancerous and

cancerous lesions have been developed in past few years. Increased

use of these adjunctive tools by dentists would likely improve early

cancer detection. However proper case selection and correct

performance of the test itself is critical to the sensitivity and

specificity of its result.


13/133

This is the study of cells which exfoliate or abrade from the body

surface.

Procedure:- Involves stroking the lesion gently but firmly with a wet wooden

tongue blade or cotton tip applicator.

The collected cells are smeared on a frosted slide and immediately

fixed with alcohol ether. After drying the glass slide is packagedand sent to an oral pathology laboratory for staining.

Cell slides are examined for benignancy or malignancy.


14/133


15/133

The Cytological smear falls in one of the five classes

Class I (normal) indicates that only normal cells were seen Class II (atypical) indicates the presence of minor atypia but no

evidence of malignant changes

Class III (indeterminate) cells display wider atypia that may besuggestive of cancer, may represent precancerous lesions orcarcinoma in situ. Biopsy is recommended in such cases

Class IV (suggestive of cancer) few cells with malignantfeatures or many cells with borderline. Biopsy is mandatory.

Class V (positive of cancer) cells are obviously malignant.Biopsy is mandatory.


16/133

Advantage: Quick, simple, painless and bloodless procedure Helps as a check against false negative biopsies Especially helpful in follow-up detection of recurrent carcinoma in

previously treated cases Screening lesion where biopsy is not warranted

Disadvantages: Presence and extent of invasion can not be assessed

Scarcity of viable surface cells Biopsy is indicated in all clinically suspicious lesions even on

negative result Has high false negative results


17/133

Introduced in 1999 as an alternative to conventional Exfoliative

cytology for suspicious epithelial lesions.

Procedure :

Transepithelial cell samples are obtained by twirling a patented spiralshaped stiff nylon bristle brush on the lesion.

Collected cells are transferred to a bar coded, clear glass slide and a

supplied pouch of alcohol fixative is immediately poured over the

slide.

The specimen is mailed. Results of brush cytology specimens are

classified into one of four categories:-


18/133


19/133

Inadequateincomplete transepithelial sample Negativeno epithelial abnormality Atypicalabnormal epithelial changes Positive definitive cellular evidence of epithelial dysplasia or

carcinoma

For atypical and positive results follow up scalpel biopsy is

recommended. In case of a negative result clinical follow up ofpersistent oral lesions is recommended.

Scuibba et al reported 100% sensitivity with 100% specificity for

positive results and 92.9% specific results in 945 patients.


20/133

Advantages: Full thickness sample Easy to use Useful alternative in patients who refuse a scalpel biopsy

In combination with vital staining may be useful for samplingmultiple areas of large lesions or in follow up patients.Disadvantages:

Specimens have to be processed in commercial processinglaboratory

Not all of the harvested transepithelial cells are transferred to theglass slide

Expensive


21/133

Provides a clearer and more attractive presentation.

Procedure: Transepithelial cells are harvested with a nylon bristle brush that is

then immersed and twirled in a liquid preservative container; the

brush can be disposed of or included in the specimen.

The liquid container is sent to the oral pathology laboratory where

a patented machine filters the harvested cells from debris and lays

the cells in monolayer on a glass slide, followed by staining and

examination.


22/133


23/133

Advantages: Better representation of collected lesional cells

Easier interpretation since there is a monolayer of cells with

elimination of blood and debris

False positive and false negative results lessened.


24/133

Used in combination with conventional visual oral mucosal

examination by healthcare providers to improve identification,

evaluation and monitoring of oral mucosal abnormalities. Several

different products designed for this technique have been marketed

including:

Vizilite, now available as Vizilite plus or vizilite with Tblue

marking system. MIcrolux DL

VELscope (visually enhanced lesion scope)


25/133

Vizilite Is a single use product that consists of an acetic acid rinse, retractor

and light stick. The patient rinses with vizilite acetic acid solution

and expectorates.

Acetic acid wash helps to remove surface debris and causes theepithelial cells to dehydrate slightly, increasing the prominence of

their nuclei.

The vizilite light stick is activated by bending until the innercapsule breaks.

The examiner shakes until it glows (activated chemiluminescence)

then inserts the light stick into the hollow end of the retractor.


26/133

After dimming the lights, the oral cavity is examined using vizilite.Under blue whit illumination normal epithelium appears lightbluish in color, whereas abnormal epithelium appears distinctlywhite.

Vizilite plus: Consists of the same device packaged together with atolonium chloride solution. The stain is intended for use as amarking dye to help highlight lesions identified with light source.

Velscope: is an alternating current powered portable, reusablelight source and uses filters to block the reflected blue light to allowthe visualization of the natural tissue fluorescence.


27/133

It is intended to be used as an adjunct to traditional oral

examination to enhance the visualization of oral mucosal

abnormalities that may not be apparent bynaked eye.

It is intended to be used by surgeons to identify diseased tissue

around clinically apparent lesion and thus aid in determining the

appropriate margin for surgical excision.


28/133

The clinician is then able to differentiate different fluorescence

response ofnormal and abnormal tissue. Healthy tissue appears as

bright apple green glow while suspicious regions are identified by a

loss of fluorescence which thus appears dark.

In a study by Lane et al(2006) using histology as gold standard, the

device achieved a sensitivity of 98% and a specificity of 100% . Theauthors stated that this device has a potential as an adjunct to

convention white light screening.


29/133


30/133

Advantage: Vizilite is simple to use

VELscope is portable, multiuse device and simple to use

Disadvantage: Vizilite plus is costly and light stick can only be activated once

VELscope unit is expensive and its durability has not been proven


31/133

Toludine blue or tolonium chloride.

In 1964, Nibel amd Chomet first reported on the use the use oftoludine blue as a vital tissue stain to aid in the early detection of

oral precancerous and malignant lesions.

The chemical name of the dye used is toludine chloride. It is a basicmetachromatic stain that binds to the DNA.

Although not cancer specific, it stains mitochondrial DNA, alteredDNA in Premalignant/malignant lesions and cells with increasedamounts of DNA.


32/133

The practitioners swab the blue dye onto a suspicious oral lesionand based on its retention and changes in blue tint, determine with

greater reliability whether to proceed to biopsy.

Studies from 1964 to 1992, show that toludine blue exhibitsensitivity that ranged from 86% to 100% with a specificity rangingfrom 63% to 100%.

Recent studies have also shown that TB positivity is higher in OPL

that show loss of heterozygosity at chromosome regions associatedwith development of squamous cell carcinoma (3p,17p).


33/133


34/133

Despite the growing number of adjuncts available to assist in theclinical detection of lesions with uncertain biologic potential,surgical biopsy remains by far the most popular method ofobtaining a final tissue diagnosis.

Once a diagnosis is established, additional studies (includingimaging modalities) may be needed to determine the stage ofdiseases and to guide the treatment plan development.


35/133

It is the removal of tissue from the living organism for the purposesof microscopic examination and diagnosis. The different methods ofbiopsy are:Excision biopsy: The goal of excision biopsy is to obtain theentire abnormality for histopathologic examination and toprovide definitive treatment by the total removal of the lesion.Adequate deep and lateral margins of normal appearing tissuemust be excised to ensure that no remnants of the lesionremain as a potential source of recurrence. It is reserved forclinically benign and precancerous mucosal lesions that areless than 2 cm in diameter.


36/133


37/133

Incisional Biopsy Most suspicious lesions of the oral cavity arediagnosed through an incision biopsy, where a portion of theabnormal surface tissue is removed for histopathologicexamination.

The tissue samples should include the most clinically suspiciousportion of the lesion, including areas of erythroplakia, speckledleukoplakia, surface granularity or ulceration.

Application of TB stain may be useful in highlighting suspiciousareas.


38/133


39/133

Punch biopsy - Punch is a soft tissue sampling instrument having a circular cutting

edge of varying diameter.

Deep biopsies in the areas like palate can be relatively simple to

obtain. But the disadvantages are the difficulty in controlling the sample

depth and necessary subsequent use of scalpel or scissors.

The tissue sample should be studied histopatholgically for epithelialdysplasia which is assigned to histopathological changes associatedwith an increases risk of malignant development. The individualcellular changes are referred to as atypia.


40/133


41/133

The criteria used for diagnosing epithelial dysplasia (Pindborg et al1997) are:

Loss of polarity of basal cells

The presence of more than one layer of cells having a basaloid

appearance

Increased nuclear cytoplasmic ratio Drop shaped rete ridges

Irregular epithelial stratification

Increase number of mitotic figures

Mitotic figures that are abnormal in form

The presence of mitotic figures in the superficial half of the

epithelium


42/133

Cellular and nuclear pleomorphism

Nuclear hyperchromatism

Enlarged nuclei

Loss of intercellular adherence

Keratinization of single cell or cell groups in the prickle cell layer


43/133

Brandwein Gensler et al 2005 proposed a histologic risk assessmentsystem based on:

Perineural invasion greater than 1mm involving nerves

Lymphocytic response Worst pattern of invasion at interface

Imaging Evaluation of deep tissue involvement of oral cancer,presence of cervical lymphadenopathy and future evaluation of

primary tumor often requires use of several imaging modalities.

Imaging options include panoramic radiography, CT, MRI andpositron emission tomography.


44/133

CT scan with intravenous contrast is the most common imagingmodalityused in the assessment ofdeep tissue extension of tumorsof oral cavity.

Advantage: Clearly demonstrates bone changes and tumor invasion More sensitive than MRI for lymphadenopathy Lower cost and good soft tissue contrast Better tolerated than MRI by patients

Disadvantage: Radiation dose Beam hardening artifact attributable to dental amalgam


45/133

MRI is superior in providing soft tissue details has multiplanarimaging capability and can better demonstrate intracranialextension of the tumor.

Disadvantage: Bony details are not clear Imaging times are longer Can not be used in patients who can not easily lie still or are

claustrophobic

Ultrasound can help in evaluation of neck nodes. However boneinvolvement can not be assessed as bone does not transmit sound.


46/133

Sentinel node biopsy This procedure is intended to identify micrometastatic disease

within a sentinel node considered most likely to drain the tumor

bed and receive initial metastatic deposits from the primary

malignancy.

Represents a less invasive means of providing staging information

for the patient with oral cancer. So that not all the patients have to

undergo lymphadenopathy.


47/133


48/133

Procedure Preoperative lymphoscintigraphy

Intraoperative lymphatic mapping

Excision of sentinel lymph nodes

Pathologic evaluation of sentinel lymph nodes


49/133

Recent advances in molecular studies show that alterations at DNA

level precede microscopic morphologic changes in pre cancer and

cancer.

Evaluation of these markers may help in early characterization of

oral epithelial dysplasia and squamous cell carcinoma.


50/133

Nucleolar organizing regions: These are loops of ribosomal DNA located on the short arms of

chromosome 13,14,15,21 and 22 & are associated with acidic nonhistonic proteins that can be visualized by silver stainingtechniques.

Number and size of AgNORs correlate positively with cellularproliferation.

Mean AgNOR counts differ significantly between nondysplasticclinical leukoplakia with a sensitivity of 75 %.

Limitation with the technique includes the time and effort requiredto perform the study manually as well as staining variability andcounting subjectivity.


51/133

DNA aneuploidy - (abnormal DNA segregation)

Abnormal chromosomal segregation resulting in aneuploidy can be

a marker for neoplastic transformation. Studies have reported an

association of ploidy status in dysplastic lesions with progressionrisk of oral pre cancer.

Lowest risk was associated with diploid lesions, tetreploid lesions

had intermediate risk and aneuploid lesions show greater risk.

(Lipmann SM, Hong WK 2001)


52/133

Accordingly a staging system of oral PML was proposed by Zangand Rosin with both pathologic and genetic findings.

P1 no or mild dyslasia, P2 moderate dysplasia, P3 severe dysplasiaG1 low risk, G2 intermediate, G3 high risk

Stage (cancer risk) Pathology P % genetic Gpattern

1 P1+G1

2 P2+G1,P1+G2,P2+G2

3 P3+G1,P3+G2, ANY P withG3


53/133

DNA alteration (loss of heterozygosity) The progression of oral epithelium from a benign to malignant

process begins at genetic level and is ultimately expressed at thecellular and clinical level.

Carcinogenesis does not result from a single area of DNA damage

but is a multistep process that requires an accumulation ofseveralDNA alterations collectively resulting in an uncontrolled neoplastic

growth.

Recent studies show that loss of allelic balance or LOH inprecancerous lesions is found in the 3p and 9p regions.


54/133

For a given lesion the risk for progression to cancer can be

classified into

Low risk if no LOH is found.

Intermediate if LOH at 3p & 9p is found.

High if LOH at 3p & 9p is found along with additional genetic

damages.

The diagnosis of cancer and many other diseases is fundamentally


55/133

The diagnosis of cancer and many other diseases is fundamentallybased on the microscopic study of cells and tissues and remains thestandard by which all other diagnostic tests are measured.

The methods include

Molecular methods Polymerase chain reaction Filter hybridization

In situ hybridization

Fluorescent in situ hybridization

Comparative genomic hybridization

Complemantry DNA (cDNA) microarrays

Flow cytometry


56/133

Immunologic methods Immunohistochemistry

Immunofluorescency


57/133

DNA & RNA extraction: Currently all pathologic materials aresuitable for nucleotide extraction.

The steps involved in DNA extraction are:

Lysis of the cell usually performed with a detergent such assodium dodecy sulfate.

Proteniase K is used to both release the DNA from thechromatin and to destroy DNAases. DNAases can also bedestroyed through their heat sensitivity(65 degree for 15 min)

or with high concentration of EDTA. Cellular macromolecules are removed by extraction of the

sample in phenol or chloroform or both, which also functionsto remove any excess proteinase.


58/133

After this extraction step, the DNA is concentrated byethanolprecipitation in the presence of acetate salts.

The DNA is then brought into the solution with a buffer or

distilled water and its concentration is assessed bymeasurement of its optical density of the solution at awavelength of 260nm.

The sample is then ready for further processing or it can bestored for long periods of time. If stored for a period up to 1

year, a 4 degree C refrigerator is sufficient, but for longerperiod a 20 degree C freezing is required.


59/133

The steps involved in RNA extraction are

RNA extraction is complicated by the need to removeubiquitously present RNAases which may be endogenous or

exogenous, or both Guanidium isothiocyanate (GITC) is the most effective reagent

for RNA extraction.

RNA needs to store at much lower temperature of70 degreeC.


60/133


61/133

A probe refers to a stretch of nucleotides that is used to detect a

specific region of DNA or RNA.

Those derived from native DNA are known as genomic probes and

may include both the exons and introns.

A probe derived from RNA is a cDNA probe and recognizes only

exons.

Oligonucleotide probes are formed in the laboratory and are much

shorter that the other two.


62/133

Probes need to be further manipulated by the incorporation of a

label, allowing for the detection of the specific binding of the probe

to the target.

The initial labeling step is performed before hybridization.

The various methods of labeling are

Nick translation

Random hexamer priming End labeling

Polymerase chain reaction


63/133

For autoradiographyradioactive labeled probes.

Nonradioisotope labeling (digoxigenin or biotin) - Offers safe work

environment, faster signal detection and are easier to use than

radiolabeled probes.

Digoxigenin labeled probes - detected with alkaline phosphatase

conjugated, anti digoxygenin antibody.

Biotin labeled probes - detected by use of a streptavidin alkaline

phosphatase conjugate system.

A third method for visualization is through chemiluminescence, which

can be detected either luminometrically or on photographic film.


64/133

PCR is an exponentially progressing synthesis of the defined target

DNA sequences in vitro.

Was introduced in 1983 by Kary mullis for which he received

Nobel prize in 1993 in chemistry.

Through this, a single piece of DNA can be used to generate infinite

supply of identical copies for a variety of research, clinical, forensic

and commercial purposes.


65/133

1) Target DNA - contains the sequence to be amplified.2) Pair of Primers - oligonucleotides that define the sequenceto be amplified.

4) Thermostable DNA Polymerase - enzyme that catalyzes thereaction (Taq Polymerase)

5) Mg++

ions - cofactor of the enzyme

6) Buffer solution maintains pH and ionic strength of thereaction solution suitable for the activity of the enzyme

3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.


66/133

THERMOCYCLERPCR tube


67/133

Denature (heat to 95oC)

Lower temperature to 56oCAnneal with primers

Increase temperature to 72oC

DNA polymerase + dNTPs


68/133


69/133

Classification of organisms Genotyping Molecular archaeology Mutagenesis

Mutation detection Sequencing Cancer research Detection of pathogens DNA fingerprinting

Drug discovery Genetic matching Genetic engineering Pre-natal diagnosis


70/133

Basic Research Applied Research

Genetic matching Detection of pathogens Pre-natal diagnosis DNA fingerprinting

Gene therapy

Mutation screening Drug discovery Classification of organisms Genotyping

Molecular Archaeology Molecular Epidemiology Molecular Ecology Bioinformatics Genomic cloning Site-directed mutagenesis

Gene expression studies


71/133

1. Reverse transcription PCR,2. Nested PCR,3. In situ PCR,4. Competitive PCR,5.

Single cell PCR,6. Immuno PCR,7. Inverse PCR,8. Anchored PCR,9. Asymmetrical PCR


72/133

Refers to the pairing of complementary RNA or DNA strands to

produce a double stranded nucleic acid.

All hybridization methods use a radio labeled DNA or RNA probe

that binds to the target DNA or RNA of interest, permitting

visualization.

The target molecule can be either immobilized in a membrane

(blotting) or examined in tissue sections (in situ).


73/133

This method was first described by Southern in 1975.

This involves the transfer or blotting of DNA fragments onto a

membrane.

Southern blotting is a technique which allows the detection of a

specific DNA sequence (gene or other) in a large, complex sample

of DNA (e.g. cellular DNA).


74/133

Whatman 3mm paper is the worlds most widely used blottingpaper. This acceptance and usage is due to the high quality, purityand consistency that are relied upon by researchers doing Southern,Northern and Western transfers.

A medium thickness paper (0.34 mm) is used extensively inelectrophoresis for lifting of sequencing gels.


75/133

Filter Paper

Another Filter paper

Gel Matrix

Nitrocellulose membrane


76/133

Cut two pieces of filter paper and a piece of nitrocellulosemembrane to an appropriate size, and soak them in transfer buffer.

Place a piece of buffer soaked filter paper over the gel.

Flip the gel over, Place the buffer soaked nitrocellulose membraneagainst the exposed gel.

Place the second piece of buffer soaked filter paper on the

nitrocellulose membrane (covering it)

Check to see that there are no bubbles between the membrane andthe gel.


77/133

1. The mixture of molecules is separated.

2. The molecules are immobilized on a matrix.

3. The probe is added to the matrix to bind to the molecules.

4. Any unbound probes are then removed.

5. The place where the probe is connected corresponds to the

location of the immobilized target molecule.


78/133


79/133


80/133


81/133

Gene discovery

Mapping

Evolution

Development studies

Diagnostics and forensics.


82/133

Is an RNA blotting technique which was developed in 1977 by

Alwine et al.

It was named after the Southern blotting technique.

Northern blot analysis allows a direct comparison of the messenger

RNA abundance between samples on a single membrane.


83/133

1. RNA is isolated from several biological samples (e.g. various tissues,various developmental stages of same tissue etc.)RNA is more susceptible to degradation than DNA.


84/133

2. Samples are loaded on gel and the RNA samples are separatedaccording to their size on an agarose gel.

The resulting gel following after the electrophoresis run.


85/133

3. The gel is then blotted on a nylon membrane or a nitrocellulosefilter paper by creating the sandwich arrangement.


86/133

4. The membrane is placed in a dish containing hybridization bufferwith a labeled probe.

5. The membrane is washed to remove unbound probe.


87/133

6. The labeled probe is detected via autoradiography or via achemiluminescence reaction (if a chemically labeled probe isused). In both cases this results in the formation of a dark band onan X-ray film.


88/133

A standard for the direct study of gene expression at the level of mRNA

(messenger RNA transcripts).

Detection of mRNA transcript size

Study RNA degradation Study RNA splicing - can detect alternatively spliced transcripts

Study RNA half-life

Study IRES (internal ribosomal entry site) - to remove possibility of RNA

digestion vs. 2nd cistron translation.

Often used to confirm and check transgenic / knockout mice (animals)


89/133

Often radioactivity is used. This prevents ease of performing it, use and

disposal.

The whole process of northern blotting takes a long time usually, from

sample preparation through to detection.

If RNA samples are even slightly degraded by RNases, the quality of the

data and quantization of expression is quite negatively affected.

The standard northern blot method is relatively less sensitive than nuclease

protection assays and RT-PCR. Detection with multiple probes is a problem. Often, the membranes must

be stripped before hybridization and detection with a second probe.


90/133

Is a similar technique used to identify and locate proteins based ontheir ability to bind to specific antibodies.

Western Blot gives information on the: size of protein & expressionamount of protein.


91/133

Steps :1. A protein sample is subjected to electrophoresis on anSDS-polyacrylamide gel.2. Electro blotting transfers the separated proteins from thegel to the surface of a nitrocellulose membrane.


92/133

3. The blot is incubated with a generic protein (such as milk proteins)which binds to any remaining sticky places on the nitrocellulose.

4. An antibody that is specific for the protein of interest (the primaryantibody - Ab1) is added to the nitrocellulose sheet and reacts withthe antigen.

Only the band containing the protein of interest binds theantibody, forming a layer of antibody molecules .


93/133

5. Following several rinses for removal of non-specifically bound Ab1,the Ab1-antigen complex on the nitrocellulose sheet is incubatedwith a second antibody (Ab2), which specifically recognizes the Fcdomain of the primary antibody and binds it.

Ab2 is radioactively labeled, or is covalentlylinked to a reporter enzyme, which allows tovisualize the protein-Ab1-Ab2 complex.


94/133

The confirmatory HIV test employs a Western blot to detect anti-

HIV antibody in a human serum sample.

A Western blot is also used as the definitive test for Bovine

spongiform encephalopathy (BSE, commonly referred to as 'mad

cow disease').

Some forms of Lyme disease testing employ Western blotting.


95/133

Is a technique used to examine DNA and RNA in their normaltopographic surroundings.

In situ hybridization presents a unique set of problems as thesequence to be detected will be at a lower concentration, be maskedbecause of associated protein, or protected within a cell or cellularstructure.

Therefore, in order to probe the tissue or cells of interest one has to

increase the permeability of the cell and the visibility of thenucleotide sequence to the probe without destroying the structuralintegrity of the cell or tissue.


96/133

a) Frozen sections - Fresh tissue is snap frozen (rapidly put into a -80

freezer) and then frozen embedded in a special support medium for

thin cryosectioning. The sections are lightly and rapidly fixed in 4%

paraformaldehyde just prior to processing for hybridization.b) Paraffin embedded sections - Sections are fixed in formalin as one

would normally fix tissues for histology and then embedded in wax

(paraffin sections) before being sectioned

c) Cells in suspension - Cells can be cytospun onto glass slides and

fixed with methanol


97/133

Oligonucleotide probes

Single stranded DNA probes.

Double stranded DNA probes

RNA probes (cRNA probes or riboprobes)


98/133

A process which vividly paints chromosomes or portions ofchromosomes with fluorescent molecules.

Identifies chromosomal abnormalities.

Aids in gene mapping, toxicological studies, analysis ofchromosome structural aberrations, and ploidy determination.

Used to identify the presence and location of a region of DNA or

RNA within morphologically preserved chromosome preparations,fixed cells or tissue sections.


99/133

Advantage:

Less labor-intensive method for confirming the presence of a DNA

segment within an entire genome than other conventional methods

like Southern blotting.


100/133

Denature the chromosomes

Denature the probe

Hybridization

Fluorescence staining

Examine slides or store in the dark


101/133


102/133

Detection of high concentrations of base pairs.

Also used in germ cell or prenatal diagnosis of conditions such as

aneuploidies.

Telomeric probes define the terminal boundaries of chromosomes. Used in research of chromosomal rearrangements and deletions

related to cell aging or other genetic abnormalities.

Application in cytogenetics - can detect submicroscopic deletions

and cryptic translocations of genes associated with unexplainedmental retardation and miscarriages.

FISH can be used in the study of transgenic animals.


103/133

In 1986, Hector Battifora first introduced the so-called sausage orMulti Tumor Tissue Block, where up to 100 separate tissues wereprocessed together in one single paraffin wax block.

Also known as DNA Chip.

Allows simultaneous measurement of the level of transcription forevery gene in a genome (gene expression).

Microarray detects mRNA, or rather the more stable cDNA


104/133


105/133

Start with individual genes, e.g. the ~6,200 genes of the yeast

genome.

Amplifyall of them using polymerase chain reaction (PCR).

Spot them on a medium, e.g. an ordinary glass microscope

slide.

Each spot is about 100 m in diameter. Spotting is done by a robot.

Complex and potentially expensive task.


106/133


107/133

Ngai Lab arrayer , UC Berkeley Print-tip head


108/133

Building the chip:

MASSIVE PCR PCR PURIFICATIONAND PREPARATION

PREPARING

SLIDES PRINTING

RNA preparation:CELL CULTURE

AND HARVEST

RNA ISOLATION

cDNA PRODUCTION

Hybing the chip:

ARRAY HYBRIDIZATION

PROBE LABELING DATA ANALYSIS

POST PROCESSING


109/133


110/133

Pins collect cDNAfrom wells


111/133

384 wellplate

Contains cDNAprobes

Glass Slide

Array of bound cDNA probes

4x4 blocks = 16 print-tip group

Print-tip

group 6

cDNA clonesSpotted in duplicate

Print-tipgroup 1

from wells


112/133

Measuring transcript abundance (cDNA arrays);

Genotyping;

Estimating DNA copy number (CGH);

Determining identity by descent (GMS);

Measuring mRNA decay rates;

Identifying protein binding sites;

Determining sub-cellular localization of gene products;


113/133

Flow cytometry is a technology that simultaneouslymeasures and thenanalyzes multiple physical characteristics of single particles, usually

cells, as they flow in a fluid stream through a beam of light. The

properties measured include a particles relative size, relative

granularity or internal complexity, and relative fluorescence intensity.

These characteristics are determined using an optical-to-electronic

coupling system that records how the cell or particle scatters incident

laser light and emits fluorescence.


114/133

Cytometry Localization of antigen is

possible Poor enumeration of cell

subtypes

Limiting number ofsimultaneous measurements

Flow Cytometry Cannot tell you where

antigen is. Can analyze many cells in a

short time frame.

Can look at numerousparameters at once.

Instrument Overview


115/133

LASER SOURCE

Fluidic system

DetectorOptical filters

Computer


116/133

Interrogation Point


117/133

When a solution is injected into a flow cytometer the particles are

randomly distributed in three-dimensional space.

The sample must therefore be ordered into a stream of single

particles. That can be interrogated by the machines detection

system. This process is managed by the fluidics system.

Hydrodynamic Focusing


118/133

Here we see how the sample is

transported through the interrogation

point. For accurate data collection, it is

important that particles or cells are

passed through the laser beam one at

a time.

Sheath fluidCentral Core

Single flow

y y g


119/133

After hydrodynamic focusing, each particle passes through beamsof light.

The laser are the most commonly used light sources in modern flow

cytometry.

It gives information about the particles by forward scatteringChannel and Side scattering channel.

The detectors used in flow cytometry is PhotomultiplierTube(PMTs).


120/133

As a cell passes through the laser, itwill refract or scatter light at all

angles.


121/133

In most cytometers, a blocking bar

(called an obscuration bar) is placed in

front of the forward scatter detector.

The obscuration bar prevents any of

the intense laser light from reaching

the detector. As a cell crosses the

laser, light is scattered around the

obscuration bar and is collected by thedetector.


122/133

Small cells produce a smallamount of forward scatter andlarge cells produce a largeamount of forward scatter, themagnitude of the voltage pulserecorded for each cell isproportional to the cell size.

Side Scatter


123/133

The side scatter channel (SSC) provides information about the

granular content within a particle.


124/133

This side-scattered light is focusedthrough a lens system and is collected bya separate detector, usually located 90

degrees from the lasers path.The signals collected by the side-scatterdetector


125/133

The fluorescent light travelsalong this path, it is directedthrough a series of filters andmirrors, so that particularwavelength ranges aredelivered to the appropriatedetectors.


126/133


127/133

When light hits a photo detector a small current (a few

microamperes) is generated.

Its associated voltage has an amplitude proportional to the total

number of light photons received by the detector.

This voltage is then amplified by a series of linear or logarithmic

amplifiers, and by analog to digital convertors (ADCs), into

electrical signals large enough (510 volts) tobe plottedgraphically.


128/133

Support a diagnosis of malignancy when the morphologic

changes are equivocal

Subclassify lesions of borderline malignancy

Provide prognostic information independent of stage and

grade

Monitor response to therapy

Establish development of tumor relapse

Establish the origin of synchronus or metachronus tmors


129/133

Neoplasia

Infectious disease

Hereditary disease

Identity determination


130/133

The practice of pathology is currently undergoing significant change, inlarge part due to advances in the analysis of DNA, RNA, and proteins intissues.

These advances have permitted improved biologic insights into manydevelopmental, inflammatory, metabolic, infectious, and neoplastic

diseases. Moreover, molecular analysis has also led to improvements in the

accuracy of disease diagnosis and classification. It is likely that, in thefuture, these methods will increasingly enter into the day-to-day diagnosisand management of patients.

The pathologist will continue to play a fundamental role in diagnosis andwill likely be in a pivotal position to guide the implementation andinterpretation of these tests as they move from the research laboratory intodiagnostic pathology.


131/133

ABC of clinical genetics by Helen M Kingston

Textbook of surgical pathology by Ackerman

Advanced diagnostic methods in oral and maxillofacial pathology:

molecular methods: journal of oral surg, oral med,oral path: dec 2001

Textbook of diagnostic molecular pathology

www.molecularstation.com

Textbook of oral pathology by Shafer

Advances in detection and diagnosis of oral precancerous and cancerouslesion: clinic of north America 2006
http://www.molecularstation.com/http://www.molecularstation.com/http://www.molecularstation.com/http://www.molecularstation.com/http://www.molecularstation.com/http://www.molecularstation.com/


132/133

Application of cytology and molecular biology in diagonising malignant

oral lesions: molecular cancer 2006

Role of oral health care specialists in the diagnostic purpose of oral cancer:M Akhar; AEGIS series

Review of oral premalignant lesions: Crit Rev Biol Med 2003

Nomenclature precancer: Journal of oral pathology medicine 2007

http://teachline.ls.huji.ac.il/72320/methods-tutorial/western/western-

blot.html

http://www.bio.davidson.edu/COURSES/GENOMICS/method/westernblot.html


133/133

http://www.bio.davidson.edu/COURSES/GENOMICS/method/Southernblot.html

http://www.med.unc.edu/pmbb/MBT2000/Northern%20Blotting.htm\

http://www.bme.gatech.edu/vcl/WesternBlotting/Background/Introduction.

htm

Documents

Diagnostics of Precancerous and Cancerous Lesions