Presented by: Dr.

Kush Pathak

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Introduction Classification of Precancerous lesions and Conditions

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Detection techniques
Advanced diagnostic methods Conclusion References

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:
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Precancerous lesions Precancerous condition ( Pindborg 1980) The ability to diagnose premalignant state is critical to the battle

against oral cancer, so that it can be managed conservatively with
minimal surgical morbidity and 100% survival.

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 to

develop these methods for early diagnosis and treatment.

Precancerous lesion - (WHO 1978) is a morphologically altered tissue in which cancer is more likely to occur than in its apparently normal counterpart.

Clinical classification
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Leukoplakia Erythroplakia Proliferative Verrucous leukoplakia palatal keratosis with reverse smoking

Histological classification
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Squamous epithelial dysplasia Squamous cell carcinoma Solar keratosis Precancerous condition: (WHO 1978) is defined as a generalized

state associated with a significantly increased risk of cancer.

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Sideropenic dysphagia lichen planus Oral submucous fibrosis Syphilis Discoid lupus erythematosus Xeroderma pigmentosa Epidermolysis bullosa

Clinical examination Diagnostic adjuncts • Cytology • Brush cytology • Liquid cytology • Tissue fluorescence • Vital staining Diagnostic methods • Biopsy • Fine needle aspiration • Sentinel node biopsy • Imaging

Cytochemical and molecular methods
• Nucleolar organizing regions
• Loss of heterozygosity • Abnormal DNA segregation (DNA aneuploidy)

Advanced Molecular methods
Molecular methods
 Polymerase chain reaction  Filter hybridization  In situ hybridization

 Fluorescent in situ hybridization

 Comparative genomic hybridization  Flow cytometry

 Complemantry DNA (cDNA) microarrays

Immunologic methods  Immunohistochemistry  Immunofluorescency

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.

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.

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 packaged

and sent to an oral pathology laboratory for staining.

Cell slides are examined for benignancy or malignancy.

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 be suggestive of cancer, may represent precancerous lesions or carcinoma in situ. Biopsy is recommended in such cases • Class IV – (suggestive of cancer) few cells with malignant features or many cells with borderline. Biopsy is mandatory. • Class V – (positive of cancer) cells are obviously malignant. Biopsy is mandatory.

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

Introduced in 1999 as an alternative to conventional Exfoliative

cytology for suspicious epithelial lesions.
Procedure :

Transepithelial cell samples are obtained by twirling a patented spiral shaped 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:-

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Inadequate – incomplete transepithelial sample Negative – no epithelial abnormality Atypical – abnormal 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 of

persistent oral lesions is recommended.

Scuibba et al reported 100% sensitivity with 100% specificity for positive results and 92.9% specific results in 945 patients.


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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 sampling multiple areas of large lesions or in follow up patients. Disadvantages: Specimens have to be processed in commercial processing laboratory Not all of the harvested transepithelial cells are transferred to the glass slide Expensive

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.

Advantages:
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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.

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)

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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 the

epithelial cells to dehydrate slightly, increasing the prominence of
their nuclei.

The vizilite light stick is activated by bending until the inner

capsule breaks.

The examiner shakes until it glows (activated chemiluminescence) then inserts the light stick into the hollow end of the retractor.

After dimming the lights, the oral cavity is examined using vizilite. Under blue whit illumination normal epithelium appears light bluish in color, whereas abnormal epithelium appears distinctly white.

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

Velscope: is an alternating current powered portable, reusable

light source and uses filters to block the reflected blue light to allow the visualization of the natural tissue fluorescence.

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 by naked 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.

The clinician is then able to differentiate different fluorescence response of normal 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% . The

authors stated that this device has a potential as an adjunct to
convention white light screening.

Advantage:
• •

Vizilite is simple to use VELscope is portable, multiuse device and simple to use Vizilite plus is costly and light stick can only be activated once VELscope unit is expensive and its durability has not been proven

Disadvantage:
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Toludine blue or tolonium chloride. In 1964, Nibel amd Chomet first reported on the use the use of toludine 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 basic metachromatic stain that binds to the DNA. Although not cancer specific, it stains mitochondrial DNA, altered DNA in Premalignant/malignant lesions and cells with increased amounts of DNA.

The practitioners swab the blue dye onto a suspicious oral lesion and 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 exhibit sensitivity that ranged from 86% to 100% with a specificity ranging from 63% to 100%. Recent studies have also shown that TB positivity is higher in OPL that show loss of heterozygosity at chromosome regions associated with development of squamous cell carcinoma (3p,17p).

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

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

It is the removal of tissue from the living organism for the purposes of microscopic examination and diagnosis. The different methods of biopsy are:

Excision biopsy: The goal of excision biopsy is to obtain the entire abnormality for histopathologic examination and to provide definitive treatment by the total removal of the lesion. Adequate deep and lateral margins of normal appearing tissue must be excised to ensure that no remnants of the lesion remain as a potential source of recurrence. It is reserved for clinically benign and precancerous mucosal lesions that are less than 2 cm in diameter.

Incisional Biopsy – Most suspicious lesions of the oral cavity are diagnosed through an incision biopsy, where a portion of the abnormal surface tissue is removed for histopathologic examination.

The tissue samples should include the most clinically suspicious portion of the lesion, including areas of erythroplakia, speckled leukoplakia, surface granularity or ulceration.
Application of TB stain may be useful in highlighting suspicious areas.

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 epithelial dysplasia which is assigned to histopathological changes associated with an increases risk of malignant development. The individual cellular changes are referred to as atypia.

The criteria used for diagnosing epithelial dysplasia (Pindborg et al 1997) are:
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Loss of polarity of basal cells

The presence of more than one layer of cells having a basaloid
appearance Increased nuclear cytoplasmic ratio


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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

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Cellular and nuclear pleomorphism Nuclear hyperchromatism Enlarged nuclei Loss of intercellular adherence Keratinization of single cell or cell groups in the prickle cell layer

Brandwein Gensler et al 2005 proposed a histologic risk assessment system based on:
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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 and positron emission tomography.

CT scan with intravenous contrast is the most common imaging modality used in the assessment of deep tissue extension of tumors of 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

MRI is superior in providing soft tissue details has multiplanar imaging capability and can better demonstrate intracranial extension 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 bone involvement can not be assessed as bone does not transmit sound.

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.

Procedure
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Preoperative lymphoscintigraphy Intraoperative lymphatic mapping Excision of sentinel lymph nodes Pathologic evaluation of sentinel lymph nodes

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.

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 non histonic proteins that can be visualized by silver staining techniques. Number and size of AgNORs correlate positively with cellular proliferation. Mean AgNOR counts differ significantly between nondysplastic clinical leukoplakia with a sensitivity of 75 %. Limitation with the technique includes the time and effort required to perform the study manually as well as staining variability and counting subjectivity.

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 progression

risk 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)

Accordingly a staging system of oral PML was proposed by Zang and Rosin with both pathologic and genetic findings.
Stage (cancer risk) 1 2 Pathology P % genetic G pattern P1+G1 P2+G1,P1+G2,P2+G2

3

P3+G1,P3+G2, ANY P with G3

P1 no or mild dyslasia, P2 moderate dysplasia, P3 severe dysplasia G1 low risk, G2 intermediate, G3 high risk

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 the cellular and clinical level. Carcinogenesis does not result from a single area of DNA damage but is a multistep process that requires an accumulation of several DNA alterations collectively resulting in an uncontrolled neoplastic growth. Recent studies show that loss of allelic balance or LOH in precancerous lesions is found in the 3p and 9p regions.

For a given lesion the risk for progression to cancer can be classified into
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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 based on the microscopic study of cells and tissues and remains the standard 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

Immunologic methods – • Immunohistochemistry • Immunofluorescency

DNA & RNA extraction: Currently all pathologic materials are suitable for nucleotide extraction. The steps involved in DNA extraction are:  Lysis of the cell usually performed with a detergent such as sodium dodecy sulfate.  Proteniase K is used to both release the DNA from the chromatin and to destroy DNAases. DNAases can also be destroyed 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 functions to remove any excess proteinase.

 After this extraction step, the DNA is concentrated by ethanol precipitation 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 by measurement of its optical density of the solution at a wavelength of 260nm.  The sample is then ready for further processing or it can be stored for long periods of time. If stored for a period up to 1 year, a 4 degree C refrigerator is sufficient, but for longer period a – 20 degree C freezing is required.

The steps involved in RNA extraction are  RNA extraction is complicated by the need to remove ubiquitously 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 of – 70 degree C.

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.

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.

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The initial labeling step is performed before hybridization. The various methods of labeling are  Nick translation  Random hexamer priming  End labeling  Polymerase chain reaction

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For autoradiography – radioactive 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.

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.

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

3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.
4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction (Taq Polymerase) 5) Mg++ ions - cofactor of the enzyme 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

PCR tube

THERMOCYCLER

Denature (heat to 95oC)

Lower temperature to 56oC Anneal with primers

Increase temperature to 72oC DNA polymerase + dNTPs


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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

Basic Research
• Mutation screening • Drug discovery • Classification of organisms • Genotyping • Molecular Archaeology • Molecular Epidemiology • Molecular Ecology • Bioinformatics • Genomic cloning • Site-directed mutagenesis • Gene expression studies

Applied Research
• Genetic matching • Detection of pathogens • Pre-natal diagnosis • DNA fingerprinting • Gene therapy

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2.
3. 4. 5.

6.
7. 8. 9.

Reverse transcription PCR, Nested PCR, In situ PCR, Competitive PCR, Single cell PCR, Immuno PCR, Inverse PCR, Anchored PCR, Asymmetrical PCR

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).

• •

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).

Whatman 3mm paper is the world’s most widely used blotting paper. This acceptance and usage is due to the high quality, purity and consistency that are relied upon by researchers doing Southern, Northern and Western transfers.

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

Filter Paper

Gel Matrix

Nitrocellulose membrane

Another Filter paper

Cut two pieces of filter paper and a piece of nitrocellulose membrane 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 membrane against 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 and the gel.

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.

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Gene discovery Mapping Evolution Development studies Diagnostics and forensics.

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.

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.

2. Sample’s are loaded on gel and the RNA samples are separated according to their size on an agarose gel. The resulting gel following after the electrophoresis run.

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

4. The membrane is placed in a dish containing hybridization buffer with a labeled probe. 5. The membrane is washed to remove unbound probe.

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

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

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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)

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.

Is a similar technique used to identify and locate proteins based on their ability to bind to specific antibodies. Western Blot gives information on the: size of protein & expression amount of protein.

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

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 primary antibody - Ab1) is added to the nitrocellulose sheet and reacts with the antigen.

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

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

Ab2 is radioactively labeled, or is covalently linked to a reporter enzyme, which allows to visualize the protein-Ab1-Ab2 complex.

The confirmatory HIV test employs a Western blot to detect antiHIV 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.

Is a technique used to examine DNA and RNA in their normal topographic surroundings. In situ hybridization presents a unique set of problems as the sequence to be detected will be at a lower concentration, be masked because of associated protein, or protected within a cell or cellular structure. Therefore, in order to probe the tissue or cells of interest one has to increase the permeability of the cell and the visibility of the nucleotide sequence to the probe without destroying the structural integrity of the cell or tissue.

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

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Oligonucleotide probes Single stranded DNA probes. Double stranded DNA probes RNA probes (cRNA probes or riboprobes)

A process which vividly paints chromosomes or portions of chromosomes with fluorescent molecules. Identifies chromosomal abnormalities.

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

of

Used to identify the presence and location of a region of DNA or RNA within morphologically preserved chromosome preparations, fixed cells or tissue sections.

Advantage:

Less labor-intensive method for confirming the presence of a DNA
segment within an entire genome than other conventional methods like Southern blotting.

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Denature the chromosomes Denature the probe Hybridization Fluorescence staining Examine slides or store in the dark

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 unexplained mental retardation and miscarriages. FISH can be used in the study of transgenic animals.

In 1986, Hector Battifora first introduced the so-called ‘sausage’ or Multi Tumor Tissue Block, where up to 100 separate tissues were processed together in one single paraffin wax block. Also known as DNA Chip. Allows simultaneous measurement of the level of transcription for every gene in a genome (gene expression). Microarray detects mRNA, or rather the more stable cDNA

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Start with individual genes, e.g. the ~6,200 genes of the yeast genome. Amplify all of them using polymerase chain reaction (PCR). “Spot” them on a medium, e.g. an ordinary glass microscope slide.

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Each spot is about 100 µm in diameter.
Spotting is done by a robot. Complex and potentially expensive task.

Ngai Lab arrayer , UC Berkeley

Print-tip head

Building the chip:
MASSIVE PCR PREPARING SLIDES PCR PURIFICATION AND PREPARATION PRINTING

RNA preparation:
CELL CULTURE AND HARVEST

Hybing the chip:

POST PROCESSING

ARRAY HYBRIDIZATION RNA ISOLATION

cDNA PRODUCTION

PROBE LABELING

DATA ANALYSIS

Pins collect cDNA from wells

384 well plate
Print-tip group 1 Contains cDNA probes

Glass Slide
Array of bound cDNA probes

cDNA clones
Spotted in duplicate

4x4 blocks = 16 print-tip groups

Print-tip group 6

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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;

Flow cytometry is a technology that simultaneously measures and then

analyzes 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 particle’s 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.

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Cytometry Localization of antigen is possible Poor enumeration of cell subtypes Limiting number of simultaneous measurements

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Flow Cytometry Cannot tell you where antigen is. Can analyze many cells in a short time frame. Can look at numerous parameters at once.

Instrument Overview
Fluidic system

Computer LASER SOURCE Detector Optical filters

Interrogation Point

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 machine’s detection system. This process is managed by the fluidics system.

Hydrodynamic Focusing

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.

Central Core

Sheath fluid

Single flow

After hydrodynamic focusing, each particle passes through beams of light. The laser are the most commonly used light sources in modern flow cytometry. It gives information about the particle’s by forward scattering Channel and Side scattering channel. The detectors used in flow cytometry is Photomultiplier Tube(PMTs).

As a cell passes through the laser, it will refract or scatter light at all angles.

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 the detector.

Small cells produce a small amount of forward scatter and large cells produce a large amount of forward scatter, the magnitude of the voltage pulse recorded for each cell is proportional to the cell size.

Side Scatter

The side scatter channel (SSC) provides information about the

granular content within a particle.

This side-scattered light is focused through a lens system and is collected by a separate detector, usually located 90 degrees from the laser’s path. The signals collected by the side-scatter detector

The fluorescent light travels along this path, it is directed through a series of filters and mirrors, so that particular wavelength ranges are delivered to the appropriate detectors.

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 (5–10 volts) tobe plotted graphically.

 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

 Neoplasia  Infectious disease  Hereditary disease

 Identity determination

The practice of pathology is currently undergoing significant change, in large part due to advances in the analysis of DNA, RNA, and proteins in tissues. These advances have permitted improved biologic insights into many developmental, 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 the future, these methods will increasingly enter into the day-to-day diagnosis and management of patients. The pathologist will continue to play a fundamental role in diagnosis and will likely be in a pivotal position to guide the implementation and interpretation of these tests as they move from the research laboratory into diagnostic pathology.

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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 cancerous lesion: clinic of north America 2006

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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

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Review of oral premalignant lesions: Crit Rev Biol Med 2003
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