INVESTIGATIONS OF

ORAL CANCER

BODH RAJ MAINALI.BDS 4th YEAR

ROLL NO 27

CONTENTS1. VITAL STAINING.2. BIOPSY

a) Definitionb) Objectivec) Indication and Contraindicationd) Classification and Types of Biopsye) Steps of Biopsy

3. INCISIONAL BIOPSY4. EXCISIONAL BIOPSY5. CORE BIOPSY6. FINE NEEDLE ASPIRATION CYTOLOGY.7. BRUSH BIOPSY

CONTENTS8. EXFOLIATIVE CYTOLOGY 9. VARIOUS IMAGING MODALITIES10.PLANE RADIOGRAPH11.COMPUTED TOMOGRAPHY AND MAGNETIC

RESONANCE IMAGING12.ULTRASONOGRAPHY13.ADVANCED DIAGNOSTIC TESTS

a) RAMAN SPECTROSCOPYb) LASER INDUCED FLUORESCENCEc) AUTO IMMUNOFLUORESCENCEd) HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY

VITAL STAINING(TOLUIDIN BLUE).

NIEBEL AND CHOMET FIRST REPORTED IN(1964).

toluidin blue (tolonium chloride) as a vital tissue stain to aid on early detection of oral precancerous and malignent lesions.

Toluidin is a basic metachromatic dye that stain for acidic tissue component and thus binds more readily to DNA.

Toluidin blue is also used as full adjunct to clinical examination and biopsy.

Toluidin blue based hyper chromatic dye , it provide deep blue staining that allows the identified lesions to be seen clearly under normal light

What is a Biopsy?Biopsy is derived from a Greek word

Bio – meaning LIFE and

Opsy – TO LOOK(Vision)

Biopsy is the removal of tissue from a living organism for the purpose of microscopic examination and diagnosis.

To confirm a diagnosis made on clinical findings.

To determine the treatment plan

Valuable self teaching diagnostic aid.

As a medical record

OBJECTIVES OF BIOPSY

INDICATION FOR BIOPSYPersistent hyperkeratosis changes in surface

tissue (ex: lips or oral mucosa)

Lesion that interfere with local function (ex :fibroma)

Any inflammatory lesion that does not respond to local treatment after 10 to 14 days (that is after removing local irritant)

INDICATION FOR BIOPSYBone lesions not specifically identified by

clinical and radiographic finding.

Any lesion persists for more than 2 weeks with no apparent etiology basis.

Any lesion that has the characteristics of malignancy .

WHEN IS ORAL BIOPSY NOT NEEDED?

There is no need to biopsy normal structures.

There is no need to biopsy for inflammatory or infectious lesions that respond to specific local treatments, as pericoronitis, gingivitis or periodontal abscesses.

No incisional biopsies should be performed on suspected angiomatous lesions.

CONTRA-INDICATIONS

Anticoagulant therapyOver-whelming sepsisSevere impaired lung functionUncontrolled bleeding.Uncooperative patientLocal infection near the site

CLASSIFICATION OF BIOPSY

According to the procedures applied, oral biopsies can be classified by:

a) Features of the lesion:

• Direct biopsy: when the lesion is located on the oral mucosa and can be easily accessed with a scalpel from the mucosal surface.

• Indirect biopsy: when the lesion is covered by an apparently normal oral mucosa.

B) AREA OF SURGICAL REMOVAL:

• INCISIONAL BIOPSY: consists of the removal of a representative sample of the lesion and normal adjacent tissue in order to make a diagnosis before treatment. definitive

• EXCISIONAL BIOPSY: is aimed at the complete surgical removal of the lesion for diagnostic and therapeutic purposes. This procedure is elective when the size and location of the lesion allows for a complete removal of the lesion and a wide margin of surrounding healthy tissue.

c) by the timing of the biopsy/ clinical timing of sampling:

• Pre-operative • Intra-operative • Post-operative

d) Purpose of the biopsy.Diagnostic BiopsyExperimental Biopsy

Types of BiopsySurgical biopsy- Incisional Biopsy ,Excisional

Biopsy and Punch Biopsy.Fine Needle Aspiration Cytology(FNAC) and CT

guided FNAC.Exfoliative Cytology.Brush Biopsy.Frozen Section Biopsy.Cone Biopsy.Core Needle Biopsy.Suction Assisted Core Needle Biopsy.Laser Biopsy.

STEPS OF BIOPSY

1.SELECTION OF AREA OF BIOPSY2.PREPARATION OF SURGICAL FIELD3.LOCAL ANASTHESIA4.INCISION5.HANDLING OF SPECIMEN6.SUTURING OF THE RESULTING WOUND

If a lesion is large or has different characteristics in various locations more than one area may need to be sampled

INCISIONAL BIOPSY

Incision should extend from the ulceration out onto clinically normal tissue

Grasp area to be removed with forceps and make an elliptical incision from the centre out onto clinically normal tissue: wound after removal of incised tissue: suturing completed

INCISIONAL BIOPSYIndications:

Size limitations Hazardous location of the lesionGreat suspicion of malignancy

Technique:Representative areas are biopsied in a wedge

fashion.Margins should extend into normal tissue on the

deep surface.Necrotic tissue should be avoided.A narrow deep specimen is better than a broad

shallow one.

DISADVANTAGES:1. Crush, splits and haemorrhage are the

artefacts most frequently found in incisional oral biopsies.

2. Theoretical seeding of cancer cells into the adjoining tissues.

EXCISIONAL BIOPSY

The entire lesion with 2 to 3mm of normal appearing tissue surrounding the lesion is excised if benign.

Excisional BiopsyAn excisional biposy implies the complete

removal of the lesion.Indications:

Should be employed with small lesions. Less than 1cm

The lesion on clinical exam appears benign.When complete excision with a margin of normal

tissue is possible without mutilation.

Excisional BiopsyTechnique:

The entire lesion with 2 to 3mm of normal appearing tissue surrounding the lesion is excised if benign.

FOR MUCOCELE LESIONS – CAREFUL EXCISIONAL BIOPSY

Punch Biopsy

Advantages :Ease of techniqueSutures may not be required if small

diameter punchMay produce a more satisfactory specimen in

bound down tissues (e.g. hard palate)

Drawbacks:May not be adequate for biopsy of deeper

pathologyMay be difficult to biopsy freely movable

tissues (e.g. soft palate, floor of mouth)

CORE BIOPSYFine needle biopsy has been established as

a safe procedure and is routinely performed under local anaesthesia. Many pathologists believe that for histologic study, core tissue is more useful than cytologic material

Core needle biopsy (CNB) has emerged as an important sampling method in the diagnosis of musculoskeletal tumours

FINE NEEDLE ASPIRATION CYTOLOGY

It is the “Technique of aspiration of cells/ fluid/ tissue fragments using a fine needle for examination under a microscope”

An 18 gauge needle on a 5 or 10 ml syringe is inserted into the area under investigation after anesthesia is obtained.

The syringe is aspirated and the needle redirected if necessary to find the fluid cavity.

ADVANTAGES

1. The technique is relatively painless, produces speedy results.

2. It is an inexpensive technique.3. It requires little equipment.4. The technique can be done as an out patient

or a chair side procedure.5. There is no problem with wound healing.6. The technique is readily repeatable

INDICATIONS1. Non palpable lesions, or area difficult to

biopsy but can be localized by CT, MRI, Ultrasound

2. To rule out vascular lesions prior to open surgery

3. In cases where Biopsy is contraindicated on medical background.

4. Used as a diagnostic screening test at community level for head and neck masses

5. Indicated for known tumors to assess effect of treatment.

6. Used to obtain tissue for specific studies.

FINE NEEDLE WITH ASPIRATION

FOR MAJOR SALIVARY GLAND/LYMPH

GLAND LESIONS FNAC MAY BE USEFUL

Brush BiopsyDiagnosis of oral epithelial dysplasia has

traditionally been based upon histopathological evaluation of a full thickness biopsy specimen from lesional tissue.

It has recently been proposed that cytological examination of “brush biopsy” samples is a non-invasive method of determining the presence of cellular atypia, and hence the likelihood of oral epithelial dysplasia.

EXFOLIATIVE CYTOLOGY

It is a quick and simple procedure, is an important alternative to biopsy in certain situations. In exfoliative cytology, cells shed from body surfaces, such as the inside of the mouth, are collected and examined. This technique is useful only for the examination of surface cells and often requires additional cytological analysis to confirm the results.

DANGERS OF BIOPSYSpreading of infectionHaemorrhageInfectionOperative trauma

For red & white lesions include both red & white area

For Vesiculobullous lesionsFluid is more representative. Intact vesicle or bulla should be biopsied.

ULCERS

Include margin, deep part of ulcer and site of maximal clinical activity. AVOID Superficial ulcers & necrotic tissue

VARIOUS RADIOLOGY INVESTIGATION

PLAIN RADIOGRAPHIC, EXAMINATION MAY INCLUDE THE USE OF OPG , OCCLUSAL AND INTRAORAL PERIORAL RADIOGRAPH

PARANASAL SINUS VIEW IS USE FULL FOR ASSESSING EXTENT OF BONE INVOLVEMENT IN THE MAXILLARY SINUS REGION.

PLAIN RADIOGRAPHS INTRAORAL PERRIAPICAL RADIOGRAPH ARE

USE FULL TO ASSESS FINER DETAILS SUCH SWITABLE BONE INVASITION, BONEY TRABECULAR ARCHITECTURE AND DISTRIBUTION OF LAMINA DURA.

OCCLUSAL RADIOGRAPHIC MAY HELP IN EVALUATION OF DESTRUCTION OF BONE IN THE ANTERIOR PORTION OF THE JAW WHICH CANNOT BE ASSESSED ADEQUATELY IN AN OPG.

COMPUTED TOMOGRAPHY AND MAGNETIC RESONANCE IMAGING

cross sectional imaging modalities such CT and MRI help in assesing the size and extend in of tumor three dimensions.

MRI imaging is considered more sensitive in evaluating the possibility of intracranial invasion.

typical radiographic feature of carcinoma invading bone include ill defined destrction(moth eaten appearance) of bone ragged borders

border may be thined out leading to pathological fracture.

lymph nodes which are not amenable for clinical examination(retro pharpharyngeal and parapharyngeal nodes) may be assessed by FNAC, CT and MRI

ULTRASONOGRAPHYIT IS A TECHNIQUE BASED ON SOUND

WAVE THAT ACQUIRE IMAGE IN REAL TIME WITH OUT THE USE OF IONIZING RADIATION.

DIAGNOSTIC ULTRADONOGRAPHY THE CLICAL APPLICATION OF ULTRASOUND USED VIBRATORY FREQUENCY IN THE RANGE OF 1 TO 20MZH.

THE ULTRASOUND SIGNAL TRANSMITTED IN TO A PATIENT IS ATTENUATED BYA COMBINATION OF ABSORPTION

REFLECTION REFRACTION AND DIFFUSION.

higher the frequence the sound wave the higher image resolution but les penitration of the sound through soft tissue.

tissue that do not produce signal such fluid filled cyst are said to be anechoic appear black.

tissue that produce a weak signal are hyper echoic. eg, where as tissue that produce intense signal such as ligament skin.

needle or catheters are hyper echoic and hyper bright.

USE ULTRASONOGRAPHY IN THE HEAD AND NECK REGION evaluation of neoplasms in the thyroid,

parathyroid, salivary gland or lymph node stone in the salivary gland or duct Sjogrens syndrome and vessels of the neck including carotid artery for the artherosclerotic plaque.

it is also used in fine needle aspiration in the neck .

more recent advances include three dimension imaging to allow multiplaner reformatting surface renderings eg, of afetal face and color dopplor sonography for evaluation of blood flow.

STAGING OF THE DISEASE

T , N , M Staging. Tx- primary tumour cannot be assessed T0- No evidence of primary tumorTis- carcinoma in situ. T1- ≤ 2cm in greatest dimension T2- 4cm < 2cm> in greatest dimension T3- > 4cm in greatest dimension

STAGING OF THE DISEASE T4a- Oral cavity: tumour invades through cortical bone, into deep(extrinsic) muscle of tongue, maxillary sinus or skin. Lips: cortical bone, inferior alveolar nerve, floor of mouth, skin i.e. chin or nose.

T4b- involves masticator space, pterygoid plates, skull base and/or encases internal carotid artery

STAGING OF THE DISEASEN stage: Nx- regional lymph nodes can not be assessed. N0- no regional lymph node metastasis. N1- metastasis in a single ipsilateral lymph node ≤ 3cm in greatest dimension. N2a- metastasis in a single ipsilateral LN > 3cm but < 6cm in greatest dimension.

STAGING OF THE DISEASE N2b- metastasis in multiple ipsilateral LNs, none > 6cm in greatest dimension. N2c- metastasis in B/L or C/L LNs, none > 6 cm. N3- metastasis in a LN > 6 cm in greatest dimension

M stage: Mx- cannot be assessed, M0- no distant metastasis, M1- distant metastasisi.

•Anatomy of Regional Lymphatics

The Head and Neck Service at Memorial Sloan Kettering Cancer Center has described a leveling system dividing the lymph nodes in the lateral aspect of the neck into feasibly reproducible

user-friendly method for description of regional cervical lymph nodes which establishes a common language between the clinician and the pathologist. The lymph nodes in the central compartment of the neck are assigned levels VI and VII.

Level I Submental group: The lymph nodes between the

anterior bellies of the digastric muscles and above the hyoid bone Submandibular group:

Lymph nodes in the triangular area bounded by the anterior and posterior bellies of the digastric muscle and the inferior border of the body of the mandible. The lymph nodes adjacent to the submandibular salivary gland and along the facial artery (facial nodes) are included in this group

Level II Upper jugular group: Lymph nodes around the upper portion of the

internal jugular vein and the upper part of the spinal accessory nerve, extending from the base of the skull up to the bifurcation of the carotid artery or the hyoid bone (clinical landmark). The posterior limit for this level is the posterior border of the sternocleidomastoid muscle and the anterior border is the lateral limit of the sternohyoid muscle.

Level III Mid-jugular group: Lymph nodes around the middle third of the

internal jugular vein from the inferior border of Level II up to the omohyoid muscle or the inferior border of cricoid cartilage (clinical landmark). The anterior and posterior borders are the same as those for Level II.

Level IV Lower jugular group: Lymph nodes around

the lower third of the internal jugular vein form the inferior border of Level III up to the clavicle. The anterior and posterior borders are the same as those for Levels II and III

Level V Posterior triangle group: Lymph nodes

around the lower portion of the spinal accessory nerve and along the transverse cervical vessels. It is bounded by the triangle formed by the clavicle, posterior border of the sternocleidomastoid muscle, and the anterior border of the trapezius muscle.

Level VI Central compartment group: Lymph nodes in

the prelaryngeal, pretracheal (Delphian), paratracheal, and trachoesophageal groove. The boundaries are: hyoid bone to suprasternal notch and between the medial borders of the carotid sheaths.

Level VII Superior mediastinal group: Lymph nodes in

the anterior superior mediastinum and tracheoesophageal grooves, extending from the suprasternal notch to the innominate artery.

ADVANCED DIAGNOSTIC TESTS

a) RAMAN SPECTROSCOPYb) LASER INDUCED FLUORESCENCEc) AUTO IMMUNOFLUORESCENCEd) HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY

Resonance Raman Spectroscopy

RAMAN SPECTROSCOPY.

Biological Applications of Raman SpectroscopyRaman spectroscopy has been applied widely for the study of biological systems. The advantages of his technique include the small sample requirement, the minimal sensitivity toward interference by water, the spectral detail, and the conformational and environmental sensitivity.

LASER INDUCED FLUORESCENCE(LIF).Fluorescent dyes (molecules) can absorb light at one

frequency and subsequently reemit (fluoresce) light at a different frequency. In experiments, the dyes are excited by laser light whose frequency closely matches the excitation frequency of the dye. For example, Fluorescein (maximum excitation at 490 nm) is best excited by an BlArgonIon,ue Green laser which predominantly emits wavelengths 488 (blue) and 514 (green) nm. Once excited, Fluorescein's maximum emission is at 520 nm. Because the fluoresced light is of a different frequency than the excitation light, the latter can be filtered out. Furthermore, only the dye that is exposed to the laser fluoresces, so specific planes within a flow field can be visualized.

Blue Dye Fluorescent Dye

AUTO IMMUNOFLUORESCENCE

development of tests to detect cancer markers in blood and body fluids is an active area of research. Some of the markers being evaluated include the detection of mutated APC, p53, and RAS in the stool of patients with colorectal carcinomas; the presence of mutated p53 and of hypermethylated genes in the sputum of patients with lung cancer and in the SALIVA OF PATIENTS WITH HEAD AND NECK CANCERS; and the detection of mutated p53 in the urine of patients with bladder cancer.

HPLC (High Performance Liquid Chromatography)HPLC-LIF for early detection of oral cancer detection of tumor markers in various forms of

malignancyTumor biomarkers could be used, not only for early

detection, but also for identification of pre-malignant lesions, prognosis of disease, monitoring of therapy, and early detection of recurrence or development of second primary cancer

high performance liquid chromatography- laser induced fluorescence (HPLC-LIF) technique:

to detect and record simultaneously spectra and chromatograms of physiological samples, which will enable the detection of multiple ‘markers’ in a single physiological sample in a short time

body fluids like saliva and serum of normal, premalignant and malignant subjects have substantially different protein profiles. By simultaneous recording of the chromatographic peaks and corresponding fluorescence spectra, it is possible to carry out unambiguous discrimination between normal, premalignant and malignant cases even when markers are present in femto/subfemtomole quantities, which should assist in early diagnosis of neoplasia

ADVANTAGES

1. technique can look at multiple markers in one run, thus improving the diagnostic capability

2. The time required for the test is also very short, being less than one hour after receiving the sample

3. No preprocessing or immuno reactions are involved

Reference

Textbook of oral medicine ,oral diagnosis and oral radiology .editors Ravikiranongole Praveen BN.2nd edition.

ROBBINS BASIC PATHOLOGY 8TH

EDITION,KUMARABBAS,FAUSTO, MITCHELL.

K. Venkatakrishna, V. B. Kartha*, Keerthilatha M. Pai†, C. Murali Krishna, O. Ravikiran†, Jacob Kurian§, Mohan Alexander† and G. Ullas Center for Laser Spectroscopy, †College of Dental Surgery, §Department of Surgical Oncology, Manipal Academy of Higher Education, Manipal 576 119, India

Reference