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Diagnostic Approaches Of Oral Diseases And Screening For Oral Cancer

Systematic Approach To Assessment Of Oral Mucosal Lesion

• 1) History Of Current Illness –

             onset, location, intensity, frequency, duration

             aggravating &or  relieving variables

             better,  unchanged  or  worse over time

• 2) Medical, Tobacco  &or  Alcohol  history –

              medical conditions

              medications  &  allergies

              tobacco & alcohol  ( type, frequency, duration )

• 3) Clinical  examination

             extra oral

             intra oral

             lesion inspection

• 4) Differential Diagnosis
• 5) Diagnostic Tests
• 6) Definitive Diagnosis
• 7) Suggested Management

Diagnostics Aids

• Haemogram, biochemical analysis, bacteriological examination, Immunostaining, Molecular Biology Tests,
• Imaging
• X-rays
• CAT Scan
• Magnetic Resonance Imaging (MRI)
• Functional & Metabolic Imaging—        
• a) Positron  Emission Tomography        
• b) Magnetic Resonance Spectroscopy
• Ultrasonography , less commonly used for oral lesions
• Biopsy , Exfoliative Cytology
• Screening  Devices
• Photography,   Referrals.

HAEMATOLOGY

• Essential for diagnosis of Blood Dyscrasias  like anaemias, Leukaemia, Multiple Myeloma, Leukopenia, Haemophilia, Purpura etc.
• Useful in diagnosis of other conditions  like  infections, sore tongue, recurrent apthae  which are associated with anaemia.
• Completely  filled request form  with  sufficient clinical details is essential for pathologist to check whether appropriate tests are ordered.

MICROBIOLOGY

• It  is  unfortunate that  this aspect of investigation  is  not  explored as it  should have  been  utilized. Pus, plaque, aspirate or scrapping  should  be  send for  Direct  Gram  Staining  of Gram +ve , Gram –ve bacilli & cocci, Actinomycetes associated ray-fungus,  ZN staining  for Mycobacteria   Or for

• PAS staining for Candida  
• PAP smears for Virally  infected cells.
• Especially  important  in   acute  ulcerative  gingivitis, candidiasis, herpetic  lesions, measles,  or  for diagnosis  of  various  vesiculo-bullous  lesions.
• Viral  culture  is  rarely  required.
• Rapid  diagnosis can  also  be  achieved  by  ELISA.

IMMUNOSTAINING

• Has revolutionized  histological  diagnosis  & made  complex  techniques  of  electron  microscope redundant.
• Principle- There is highly  specific  binding between Antibody & Antigen to  stain  specific  molecules  within  the  tissues.
• Two  basic  methods are  used  either Direct  or  Indirect ,  & the final  product  is  either  labeled  with a  Fluoroscein dye or an enzyme  such  as peroxidase.
• Important in diagnosis Pemphigus,  Pemphigoid,  Multiple Myeloma, or other undifferentiated malgnancies, identification of Viral infections like HPV, EBV, HSV  

MOLECULAR  BIOLOGY  TESTS

• Useful  particularly  in  screening  for  &  identification  of  Genetic  abnormalities  like  Down’s syndrome.
• Rapid  diagnosis of bacteria ( Mycobacteria), Viruses HPV, HIV
• Diagnosis of malignancies having  genetic  abnormalities like  chromosomal  translocation in  Leukaemia.
• Diagnosis  of some  forms  of  Lymphoma which  cannot  be  categorized  by  routine  biological  methods.
• Tests are  like  PCR, In-Situ-Hybridization.
• PCR  is  especially  important  for  Mycobacterial infections since  these  method  is  rapid, more sensitive, can  differentiate  different  types of MB with  high  degree of   precision.
• PCR  is  ideal  to  investigate enlarged lymph  nodes in neck &  recently  is being  used  to  detect micrometastases  of tumors.

PHOTOGRAPHY   

• Photography   or  computerized   video  imaging   is  an  valuable  adjunct  to  clinical  record. It  is 
• 1) useful  to  monitor lesions  which  vary  in  the  course  of  long  follow-up like  leukoplakia.
• 2) useful  to  explain  to  patients  about  their  condition 
• 3) To  show  patients  the  effect  of   treatment.

OTHER  CLINICAL  TESTS

• like  urine  analysis  in  diabetes  is  essential  in  case  of  repeated  candidal or periodontal infections
• Autoimmune  conditions  require  rigorous  tests to  detect  systemic  involvement
• Bence Jones Protein  in  Multiple  Myeloma
• Paul Bunnel Test in Infectious Mononucleosis

TEMPERATURE

Temperature  recording of patient  is  very  essential  to  differentiate  facial      inflammatory  edema  from  cellulitis.  It  indicates  systemic effects  of  infections &  the  need  for  aggressive  therapy.

REFERRALS

to concerned medical faculty is very  essential for  further  investigations  especially  when  oral cause  is  eliminated,  &  also  to  exclude any severe systemic illness  &  refer the patient  back to dentist

       IMAGING

• Imaging is useful for diagnosis of all tumors that are not accessible  to clinical evaluation or endoscopy.
• Sites in which imaging plays a key role for diagnosis are  brain, lung, breast, mediastinum, abdominal organs, bones, etc.
• The choice  of  imaging  system to be selected , for a given pt.  depends upon its physical capabilities.
• Imaging techniques allow differentiation of benign from malignant lesions, & assist in accurate staging of disease

Conventional XG  provides the highest  spatial  resolution  & is suited best for     barium studies of GIT,  breast & bone  diseases.

CAT SCAN

• CT scan has higher density discrimination than conventional  XG, &  is  best suited for distinguishing  differences between soft tissues, fluid, fat & other structures.
• Recent innovation include Spiral (helical) CT, Multiphase Imaging, Multi detector scanning.
• Spiral CT is currently  preferred for detecting Pulmonary & liver lesions prior to surgery & for surgical planning of Pancreatic & renal cancer treatment

      Magnetic Resonance Imaging

• Benefits- superb soft tissue contrast, multiplanar  & 3D acquisition, freedom from ionizing radiation & bony artifacts, ability to acquire biological & physiological information.
• MRI is choice for evaluating tumors of brain, HN, spine, breast, liver & adrenal glands.

Positron Emission Tomography

• PET creates images that represent metabolic activity of underlying tissue processes like glucose, oxygen,  amino acid metabolism.
• Commonly used radiotracer for tumor imaging  is glucose analogue 2[F-18] fluoro 2deoxyDglucose (18-FDG)
• FDG enters cells & is phosphorylated to FDG-6- phosphate, which becomes trapped within malignant cells with high glucose metabolism.
• PET is the most accurate non- invasive technique for detecting & staging Lung CA, detecting intrahepatic metastases, identifying metastatic deposits in nodes less than 1 cm dia. 

Magnetic Resonance Spectroscopy

• MRS is powerful , non-invasive method for studying tumor biochemistry & physiology.
• It measures signals from chemical compounds within tissues.
• 31 P MRS provides information on tissue energetics & pH while I H MRS provides information on cell membrane synthesis  & degradation, reflecting cellular proliferation & necrosis.

• MRS can provide diagnostic information on tumor grade & are also used to monitor tumor response to therapy.

ULTRASOUND

• Ultrasound has major advantage that ionizing radiation is not used,  & is often used for evaluating thyroid, parotid, testicles, liver, kidneys, & pelvic organs.
• It has limited scope in Oral cancer diagnosis

BIOPSY

It is the procedure of  removal of living tissue for the purpose of microscopic  examination

     Methods for obtaining the material—

      1) surgical scalpel biopsy

      2) surgical removal by Cautery or High        Frequency  Cutting Knife

      3) punch biopsy

      4) FNAC

      5) FNAB

      6) Exfoliative Cytology             

Point’s to be considered before biopsy

1) Why is biopsy been taken ? e.g to confirm a mucosal disease such as Lichen Planus,  cyst etc.

    Or  to rule out malignancy- then take it from the edge of the lesion

  2) Is the biopsy incisional or excisional ? For excisional biopsies a margin of surrounding normal tissue will be required.

Provisional  clinical diagnosis is especially important in guiding the technique to be used & tissue handling 

For  lesion  such  as  Mucocele, Lichen Planus, Fibroepithelial  Polyp, Pyogenic Granuloma,  Leukoplakia, epulis  incisional  biopsy  can  be  performed on OPD  basis  But  for  suspected  lesions of  Malignancies, salivary Gland  tumors, Pemphigus,  Granulomatous Diseases  Referrals to Hospitals is essential.

• Periapical surgical material  should  always be sent for histopathological  examination. It  is mostly inflammatory ,rarely it can be odontogenic or non- odontogenic cyst, odontogenic tumor.
• Even rarely it can be bone lesion like giant cell granuloma, multiple myeloma, langerhans cell histiocyosis, or even more rarely malignant metastatic deposits or SCC
• Occasionally dental hard tissues are sent  to rule out abnormality of dentine or enamel
• Vascular small lesion is excised , no incision biopsy
• Large Vascular lesions are ablated with Laser or Cryosurgery (no tissue remains for histologic exam.)
• Sjogren’s syndrome – confirmation incisional biopsy from lower lip to include at least 5 minor SAL. Glands

Limitations of Biopsy

• 1) invasive procedure, has surgical implications
• 2) technique is limited to professionals
• 3) psychological implications to patient’s
• 4) for large lesions , site selection is difficult
• 5) has limited sensitivity because of subjective                interpretation of examining pathologist

Adjuncts to Biopsy

• All the limitations of biopsy  & the need for early diagnosis of oral cancer  have underlined the importance of discovering & developing new diagnostic methods  & improving existing ones
• Detecting presence of innocuous lesions &  Selecting the site  for biopsy  can be done by adjuncts like—
•  Supravital staining with Toluidine Blue  or
•  By use of Exfoliative Cytology  

Supra-vital Staining

• Toluidine Blue is a vital  tissue dye which exhibits differential uptake into tissues, resulting in metabolically active areas of lesions being stained a deep blue.
• T-Blue is effective in differentially staining the nucleus  in precancerous & cancerous lesions
• Helps to delineate the margin of the lesion
• Positive correlation between degree of dye uptake & presence of chromosomal abnormalities
• Adjunctive use of T- Blue helps-
• Identify abnormal lesions
• Facilitates biopsy site selection
• Eventual definitive diagnosis

Exfoliative Cytology

• When epithelium becomes seat of pathology, cells loose their cohesiveness & deeper cells shed along with superficial cells .
• These exfoliated cells are collected  & studied Quantitatively or Qualitatively.
•  Collection of cells is done by  different physical  systems  like—

    -Scraping the mucosal surface

    -by rinsing the oral cavity

    -taking saliva samples from pt’s

    -by using Brush Biopsy

• Exfoliative Cytology was introduced by George N Papanicolau in 1941
• It is a Quick, simple, painless, bloodless procedure
• Helps check against false negative  biopsies
• Valuable for screening of lesions whose clinical appearance does not warrant a biopsy
• Apart from Cancers, can be applied to other disease like  Candidiasis, EBV related Hairy Leukoplakia, HSV infection, HZ,  Pemphigus, Benign Familial Pemphigus, Keratosis Follicularis, HBID, White Sponge Nevus, Pernicious anaemia, Sickle Cell Anaemia
• Application in Forensic Odontology

Oral Brush Biopsy CDX (scan lab)

• Simple
• Highly sensitive
• Risk free for screening for cancer
• Aids in clinical examination
• Full trans-epithelial cellular sample is obtained
• Smears are evaluated with image analysis adapted  specifically to detect oral epithelial abnormalities including cancer
• Can  be  applied  to  Precancerous & cancerous  lesions & other diseases as well 

 Disadvantages of brush biopsy

• False negative results, no diagnosis, so difficult to determine appropriate treatment or anticipate whether an additional procedure is  necessary.
• Adds time & cost to the diagnosis of oral lesions without added benefit to the patient
• Positive  results  have to be confirmed by routine biopsy
• The need for 2 procedures delays the diagnosis & this delay may be potentially hazardous

Applications of Cytology  technique

 Apart  from  screening  of lesions, Cytology techniques can  also  be  used for other valuable purposes  like  evaluating   Response to Radiation therapy, Molecular  analysis,  Immunohistochemistry

Response to Radiation therapy

•  useful to study radiation response of oral  tumour,  and  the  changes are like--
• cellular enlargement, cytoplasmic granulation, multinucleation, micronucleation
• Micronucleation is accepted as reliable indicator to monitor the effectiveness of therapy
• Apoptosis - % of apoptotic cells  is useful to monitor pt’s reaction to chemotherapy
• DNA Ploidy analysis to predict the aggressiveness & prognosis of cancers.

Molecular AnalysisGene Alterations—

•     Most of the oral cavity carcinogens like chemicals (tobacco), physical (radiation), infections (HPV, candida) are mutagenic agents
•  They  can cause changes in gene & chromosome  structure  by point mutations , deletions, insertions  & rearrangements.  Some of  these changes are spontaneous. 
• These genetic alterations  occuring during carcinogenesis can be used  as targets  for detecting  tumor cells in clinical samples.
• Point Mutations in  Tumor Suppressor Gene - P 53  is frequently used genetic alteration in Clinical  &  Oral  Cytology as specific marker in  Oral SCC.
• Aberrations  in the  3p, 9p, & 17p chromosomal sites  is implicated as high- risk predictor

Immunohistochemistry

• Cytokeratin Expression Profile in smears from oral cavity are used to provide information on cell differentiation status
• Certain Cytokeratins like K8, k 19 are useful if not definitive indicator of malignancy

Summary Of Oral Cytology

• Cytologic studies are easy, simple,  rapid, non-aggressive  & relatively painless. It is accepted well by the pt’s  &  are suitable for  routine application in -
• Screening Programmes
• Early detection of suspect lesions
• Pre & Post Operative  monitoring of confirmed malignant lesions

Screening For Oral Cancer

• Unaided visual examination
• Palpation
• Radiographs
• Supplemental screening techniques-
•   1) use of special wavelength lights
•   2) Chemiluminescence
•   3) Use of dyes for selective staining
•   4)Laser induced Fluorescence Emission  after exposure to photosensitizer 5-Aminolevulinic Acid
•  Screening devices available at present
  • Microlux DL (Ad Dent)
  • Velscpoe  (LED)  (Dental  INC)
  • Vizilite & Vizilite Plus (Zila Pharmaceuticals)

Microlux /DL

• It is a hand held device, that uses light emitting diodes (LEDs) as illumination source.
• Prior to exposing the mucosa to the light, the pt. rinses with 1% acetic acid for 30-60 seconds.
• Upon illumination, the abnormal tissue will appear white (aceto-white

VELSCOPE

• VEL-Scope is hand held device that emits a blue light to fluoresce the mucosa.
• No pre-rinse is required .
• The amount  of fluorescence depends on the health of  mucosa.
• When exposed to blue light , normal mucosa emits a pale green autofluorescence, while abnormal tissue appears dark green to black.
• Inflammed mucosa results in loss of fluorescence  (false +ve)
• VELScope is used to-  
•  ---delineate the extent of visible lesion                
• ---identify  lesions  that are  difficult to appreciate by unaided visual     examination. 

Vizilite  & Vizilite Plus

• Vizilite & Vizilite Plus is a hand held device that emits Chemiluminescent light.
• The pt. rinses with 1% acetic acid for 30-60 sec. The device then illuminates the oral cavity. Abnormal areas appear white ( aceto-white)
• The light increases both the sharpness &  brightness of lesions. 

CONCLUSION

• Although Biopsy still remains the gold standard for diagnosis, recent advances
• -in the field of molecular biology 
• -advent of  new screening devices
• -newer diagnostic technologies  has  widened the horizon of diagnostic  approaches .
• It won’t be too long before these technologies reach down to the level of use from research  to   well being of the patient’s

About the Author

Dr Mrs Priya Shirish Joshi, MDS, Professor & HOD , Postgraduate-Teacher, Dept Of Oral Pathology & Microbiology, vasantdada Patil Dental College & Hos[ital, Kavalupur, Sangli, Maharashtra, India.

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