DISSERTATION
on
“EARLY DIAGNOSIS OF ORAL CANCER BY
EXFOLIATIVE CYTOLOGY USING METHYLENE BLUE AND CORRELATION WITH BIOPSY AND SPECIAL STAINS IN
GMKMC, SALEM”
submitted in partial fulfillment of requirements for
MD DEGREE EXAMINATION BRANCH-III PATHOLOGY
Reg No. 201713754
THE TAMIL NADU DR. M.G.R. MEDICAL UNIVERSITY CHENNAI
GOVERNMENT MOHAN KUMARAMANGALAM MEDICAL COLLEGE
SALEM
MAY 2020
CERTIFICATE
This is to certify that this dissertation entitled as “ EARLY DIAGNOSIS OF ORAL CANCER BY EXFOLIATIVE CYTOLOGY USING METHYLENE BLUE AND CORRELATION WITH BIOPSY AND SPECIAL STAINS IN GMKMC, SALEM ”, is a bonafide work done by Dr.V.KOKILA., Post Graduate Student, Department of Pathology, Govt Mohan Kumaramangalam Medical College, Salem, in partial fulfillment of the university rules and regulations for the award of MD DEGREE in PATHOLOGY BRANCH-III, during the academic period from January 2018 to June 2019.
Dr.K. THIRUMAL BABU M.D., D.M., Dr.M.THENMOZHI, M.D.,
DEAN, PROFESSOR AND HEAD,
GMKMC, DEPARTMENT OF PATHOLOGY,
SALEM. GMKMC, SALEM
CERTIFICATE BY THE DISSERTATION GUIDE
This is to certify that this dissertation entitled as “EARLY DIAGNOSIS OF ORAL CANCER BY EXFOLIATIVE CYTOLOGY USING METHYLENE BLUE AND CORRELATION WITH BIOPSY AND SPECIAL STAINS IN GMKMC, SALEM”, is a bonafide work done by Dr.V.KOKILA, Post Graduate Student, Department of Pathology, Govt Mohan Kumaramangalam Medical College, Salem, under my supervision and guidance, in partial fulfillment of the university rules and regulations for the award of MD DEGREE in PATHOLOGY BRANCH-III, during the academic period from January 2018 to June 2019.
.
Dr.K.KASTHURI THILAGAM, MD., ASSOCIATE PROFESSOR,
DEPARTMENT OF PATHOLOGY, GMKMC, SALEM.
DECLARATION
I solemnly declare that the dissertation titled “EARLY DIAGNOSIS OF ORAL CANCER BY EXFOLIATIVE CYTOLOGY USING METHYLENE BLUE AND CORRELATION WITH BIOPSY AND SPECIAL STAINS IN GMKMC, SALEM”
was done by me at Govt. Mohan Kumaramangalam Medical College, Salem, during the period of January 2018 to June 2019 under the guidance and supervision of Dr.K.KASTHURI THILAGAM, MD, to be submitted to The Tamil Nadu Dr. M.G.R.
Medical University towards the partial fulfillment of requirements for the award of MD DEGREE in PATHOLOGY BRANCH-III to be held in May 2020.
Place : Salem Date :
Dr.V.KOKILA, MD PATHOLOGY, Post Graduate Student, Department of Pathology,
GMKMC, SALEM.
ACKNOWLEDGEMENT
To begin with, I thank the Almighty in making this project a successful one.
I express my deep gratitude to Dr.K.THIRUMAL BABU, M.D., D.M, Dean, Govt.
Mohan Kumaramangalam Medical College, Salem, for permitting me to undertake this study.
I express my sincere gratitude to Dr.M.THENMOZHI,M.D, Professor and Head, Department of Pathology, GMKMC, Salem, for having rendered her valuable support and encouragement.
I am extremely grateful to my guide Dr.K.KASTHURI THILAGAM, M.D, Associate Professor, Department of pathology, GMKMC, Salem, for his valuable guidance, suggestions, constant encouragement and support during this endurable work.
I also sincerely thank all faculties of Department of Pathology, GMKMC, Salem, for their support and encouragement.
I thank my fellow postgraduates, lab technicians and all the staffs of my department for their constant cooperation.
I extend my sincere thanks to Departments of Dental Surgery and surgery, GMKMCH, Salem, for their cooperation.
It would not be complete without mentioning my family and friends, I express my gratitude for their moral support while pursuing this study.
CERTIFICATE
This is to certify that this dissertation work titled “EARLY DIAGNOSIS OF ORAL CANCER BY EXFOLIATIVE CYTOLOGY USING METHYLENE BLUE AND CORRELATION WITH BIOPSY AND SPECIAL STAINS IN GMKMC, SALEM” of the candidate Dr. V.KOKILA with the registration number 201713754 for the award of M.D DEGREE in the branch of BRANCH-III PATHOLOGY. I personally verified the urkund.com website for the purpose of plagiarism Check. I found that the uploaded thesis file contains from Introduction to Conclusion pages and results show 13 percentage of plagiarism in the dissertation.
Guide and supervisor with seal.
EARLY DIAGNOSIS OF ORAL CANCER BY
EXFOLIATIVE CYTOLOGY USING METHYLENE BLUE AND CORRELATION WITH BIOPSY AND SPECIAL STAINS IN
GMKMC, SALEM.
CONTENTS
S.No. TOPICS PAGE No.
1. INTRODUCTION 1
2. AIMS AND OBJECTIVES 4
3. REVIEW OF LITERATURE 5
4. MATERIALS AND METHODS 45
5. OBSERVATION AND RESULTS 50
6. DISCUSSION 61
7. SUMMARY 75
8. CONCLUSION 77
COLOUR PLATES
BIBLIOGRAPHY
ANNEXURE
MASTER CHART
LIST OF TABLES
S.No. TITLE PAGE
No.
1. Correlation of brush cytology in relation to histopathology 49
2. Distribution in relation to age group 50
3. Sex distribution 51
4. Distribution in relation to adverse habits 53 5. Distribution in relation to site of lesion 54 6. Distribution in relation to clinical diagnosis 55 7. Distribution in relation to brush cytology diagnosis 56 8. Distribution in relation to HPE diagnosis 57 9. Correlation of brush cytology with histopathology 58 10. Brush cytology diagnosis in relation to histopathology diagnosis 60
11. Comparison of sex distribution 62
12. Comparison of adverse habits associated with oral lesions 63
13. Comparison of site of oral lesions 64
14. Comparison of brush cytology diagnosis 66 15. Comparison of statistical parameters of brush cytology with
other studies 67
LIST OF CHARTS
S.No. TITLE PAGE
No.
1. Distribution in relation to age group 51
2. Sex distribution 52
3. Distribution in relation to adverse habits 53 4. Distribution in relation to site of lesion 54 5. Distribution in relation to clinical diagnosis 55 6. Distribution in relation to brush cytology diagnosis 56 7. Distribution in relation to HPE diagnosis 58
8. Comparison of sex distribution 62
9. Comparison of adverse habits associated with oral lesions 64
10. Comparison of site of oral lesions 65
11. Comparison of brush cytology diagnosis 66 12. Comparison of sensitivity and specificity of brush cytology 68 13. Comparison of PPV and NPV of brush cytology 68
ABBREVIATIONS
AJCC - American Joint Committee on Cancer
CEA - Carcino Embryonic Antigen
CK - Cytokeratin
DNA - Deoxyribo nucleic acid
EFGR - Epidermal growth factor receptor
HPV - Human papilloma virus
HHV - Human herpes virus
HIV - Human immunodeficiency virus
OC - Oral cancer
OED - Oral epithelial dysplasia
OPMDs - Oral potentially malignant disorders OSCC - Oral squamous cell carcinoma
STAT3 - Signal transducer and activator of transcription 3 VEGFR - Vascular endothelial growth factor receptor
WHO - World health organization
1
INTRODUCTION
Oral cancer is the most common cancer and constitutes a major health problem in developing countries, representing the leading cause of death. Although representing 2-4% of the malignancies in the West, oral squamous cell carcinoma (OSSC) accounts for almost 40% of all cancers in the Indian subcontinent1. Worldwide oral cancer is accounting for sixth most common cancer. The GLOBOCAN project done by International Agency for Research in Cancer (IARC) estimated 3, 00,373 new cases in 2012, with a global age specific standardized incidence rate of 4.0 cases per 100000 populations per year. And also the project estimated that global mortality rate of 1.9 deaths per 1, 00,000 populations per year.
High incidence of oral cancer is seen in southern Asia (e.g.) India, Sri Lanka, Taiwan, China and Pakistan with age specific standardized incidence rates of > 10 cases per 1,00,000 population per year in parts of India and Pakistan. Incidence is also found high in Western and Eastern Europe, Latin America and the Caribbean and Melanesia.
Worldwide, the incidence of oral cancer is higher among Males (5.5 cases per 100 000 population per year) than females (2.5 cases per 1, 00,000). However the reported male- to-female ratio in India and Thailand are 1:2 and 1: 1.56, respectively. Most oral cancers occur in patients with age group of 50-70 years. As smoking and tobacco chewing rates decline, the incidence of oral cancer is decreasing in some countries. In few countries the incidence of oral cancer is increasing among younger patients2.
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Exposure to tobacco, alcohol consumption, genetic susceptibility and radiation, are the main predisposing factors associated with oral cancers. Other etiological factors are malnutrition, viruses, syphilis and traumatic irritation. In India the high incidence of oral cancer is associated with tobacco chewing and smoking habits3.
Oral squamous cell carcinoma (OSCC) is accounting for 92–95% of all oral cancers. In the occurrence of oral cancer, initially there will be a precursor premalignant lesion subsequently it developed into well established cancer. The premalignant lesions are morphologically altered tissue having greater risk for malignant transformation. In oral potentially malignant disorders the presence of epithelial dysplasia is a very important predictor of malignant development. In oral cavity lesions the most common OPMDs are leukoplakia, erythroplakia, lichen planus and actinic keratosis and they have increased risk for malignant transformation. These lesions are often asymptomatic and subtle, requiring a high index of suspicion, especially if it is associated with tobacco chewing or smoking.
Early diagnosis and treatment of OPMDs leads to prevention of malignant transformation. When a suspected oral lesion is present, the options for the clinician include an observation period of some defined time, vital staining like methylene blue, toluidine blue, etc., to assist in selection of most appropriate site for biopsy, various fluorescent and chemiluminescent helps to visualize the extent of the lesion, oral exfoliative cytology and scalpel biopsy to evaluate the lesion. Vital stains are very much
3
useful in choosing biopsy sites, to mark the margin of the lesion and to screen the patients who have asymptomatic suspicious oral lesions.
Although surgical biopsy followed by histopathology is considered as gold standard for diagnosing the oral lesions, it may not be possible to carry out biopsy in every patient because some patients may be medically compromised and a few patients with asymptomatic lesion may refuse to do biopsy.
Exfoliative cytological study of oral cells is a less painful, non-invasive technique that is well accepted by the patient, and it is an attractive option for the early diagnosis of potentially malignant and malignant lesions of oral mucosa. There are different techniques to obtain the oral cavity cells by scraping the surface of the mucosa, rinsing the oral cavity or by taking a sample of saliva from the patients. The use of brush yields cells from deeper layers of the epithelium.
The use of brush cytology without computer-assisted system analysis using ordinary nylon toothbrush is less expensive and also used in resource challenged areas and could be a risk free method of diagnosing oral premalignant and malignant lesions. In this present study, brush cytology of potentially malignant and malignant lesions of oral cavity was performed using a conventional tooth brush and subjected to cytological examination. Brush cytology results were compared with that of conventional punch biopsy results4.
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AIMS AND OBJECTIVES
• To evaluate the clinical profile of the patients with oral mucosal lesions.
• To evaluate the diagnostic correlation between brush cytology and histopathology of oral mucosal lesions.
• To evaluate the sensitivity, specificity and accuracy of brush cytology in the detection of oral premalignant and malignant lesions in comparison to histopathology.
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REVIEW OF LITERATURE
Anatomy
The oral cavity is divided into vestibule and oral cavity proper. The vestibule is the space bounded internally by the teeth and gums and externally by the cheeks and lips.
The oral cavity proper is bounded superiorly by soft and hard palate, inferiorly by the tongue, anteriorly by the teeth and posteriorly by the palatoglossal arches. Posteriorly the cavity communicates with the oropharynx. The palatine tonsils are situated between the palatoglossal and palatopharyngeal arches. The retromolar trigone is a triangular space which is situated behind the last molar teeth and it represents the posterior most region of the vestibule of the mouth.
Figure 1 Anatomy of oral cavity63
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Based on the location of the oral lesions, the oral cavity is subdivided into various sites as 1) Lip, 2) Buccal mucosa, 3) Tongue, 4) Soft palate, 5) Gingiva, 6) Floor of the mouth, 7) Hard palate, 8) Retromolar trigone, 9)Base of the tongue, 10) Tonsillar area and 11) Pharyngeal wall5.
Functions i) Mastication ii) Taste sensation iii) Speech
iv) Aids in swallowing by providing saliva for lubrication v) Immunity
vi) Digestion Embryology
The lining epithelium of the inner surface of the lips, floor of the mouth, gingiva, cheeks and hard palate is derived from embryonic ectoderm, and the epithelium covering the tongue is derived from both ectoderm and endoderm6-8.
Histology
The oral mucosa is divided into three groups based on the lining epithelium, connective tissue and its function.
1. Masticatory (hard palate and gingival), 2. Specialized (dorsal surface of the tongue)
3. Lining (buccal mucosa, ventral surface of the tongue, soft palate, intra-oral surfaces of the lips and alveolar mucosa) 6.
7
In the oral cavity lining, approximately 25% is lined by keratinized stratified squamous epithelium similar to that of the epidermis lining the skin in regions where subject to mechanical forces (masticatory mucosa of the gingiva and hard palate), 60% is covered by non keratinized squamous lining in the areas, where more flexibility occurs to accommodate speech, chewing or swallowing (floor of the mouth, buccal regions, esophagus, etc), and the remaining 15% of the oral cavity is lined by specialized mucosa (dorsum of the tongue) which can be represented as a mosaic of non-keratinized and keratinized epithelium7.
In the keratinized areas the oral epithelium has various layers of stratified squamous epithelium: basal, spinous, granular and corneal layer. In the non keratinized areas it has layers of basal, spinous, intermediate and superficial layer. The oral epithelium is in direct contact with an underlying, dense connective tissue called lamina propria containing structural fibers, blood vessels, fibroblasts and minor salivary glands, along with other cell types 7-12. Its histological structure mainly involves undulations of epithelium (rete ridges) protruding downwards into the lamina propria, with corresponding upward projections of lamina propria (dermal papillae) and thus provides increased surface contact, which prevent separation of the oral epithelium from the underlying lamina propria during mastication13.
The squamous epithelium covering the oral mucosa relies on epithelial stem cells for tissue renewal 14. It is unanimously accepted that normal tissue stem cells constitute a lifelong reservoir of cells with active mechanisms for self renewal. Cell division in oral
8
mucosa epithelial cells takes place in the basal layer which contains the stem cells compartment from which the oral mucosa is being regenerated8.
After dividing, the committed cells undergo differentiation that leads to the expression of structural keratin proteins as cells move superficially, and eventually fall off the surface. In the oral epithelium, it takes 14–24 days for a stem cell to divide and the progeny to traverse through the entire thickness of the epithelium (turnover time)9.
ORAL CANCER
Most common cancer occurring in the oral cavity is squamous cell carcinoma.
Adenocarcinoma from minor salivary glands and other malignant neoplasm are very rare in this site. Tobacco and alcohol consumption appears to be major determinants of OSCC.
Etiology and Major risk Factors
Many risk factors or causative agents for OC have been established. Chemical factors like tobacco, betel nut chewing and alcohol, biological factors like human papillomavirus (HPV), syphilis, chronic candidiasis , viruses, oro-dental factors and dietary deficiencies are significantly associated with OC. Figure 2 represents the etiological factors for occurrence of oral cancer.
Chemical Factors Tobacco
There are many evidences showing that tobacco in various forms have carcinogenic impact in oral cavity including smoking, chewing and in betel quid etc.
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Figure 2 Etiological factors for oral cancer
Smoking and tobacco chewing are the most common form of use of tobacco. The various forms in which tobacco is used as smoke are cigarettes, cigars, pipe and bidi etc.
In some countries of Asia including India, hookah or chillum (a clay pipe used to keep the burning tobacco) are other most common forms of smoking. Tobacco smoke is dissolved in water (‘‘smoke on the water’’) which is another form of tobacco use in some part of India like Mizoram.
Alcohol
According to numerous studies alcohol is one of the major risk factor for OC.
There is some degree of controversy if alcohol alone may have carcinogenic impact. This is because of simultaneous alcohol and tobacco intake of study subjects in various
10
epidemiological studies. Studies have shown that individuals taking more than 170 g of alcohol daily have ten times higher risk of OC than the light drinkers15. Alcohol may have additive effect because it alters the metabolism of oral mucosal cells by facilitating the entry of carcinogens into the exposed cells. However, the current evidences do not implicate that pure alcohol intake alone is carcinogen for the development16.
Betel
Betel products, which are derivative of the nut of the areca palm, prove to be impending carcinogens. They are used frequently in Indian subcontinent and Southeast Asia. Preparations generally consist of a combination of betel leaf, tobacco, betel nut and slaked lime (calcium hydroxide). Adding of lime increases the carcinogenicity besides giving an euphoric effect. Prolonged intake may lead to the development of submucous fibrosis.
Gutkha and pan masala are in more demand among younger and old age groups.
Betel quid chewing with or without tobacco is proved to be carcinogenic in humans.
Gutkha is the combination of areca nut and tobacco with addition of cardamom, lime, spices, catechu, and flavouring agents. Gutkha is established to be accountable for a number of oral diseases and has addictive effects that lead to the addiction due to the presence of tobacco and areca nut 17, 18.
Biological Factors Viruses
Role of oncogenic viruses in human cancer is a promising area of research.
Viruses are capable of hijacking host cellular machinery and modifying DNA and the
11
structure of the chromosome. And also it induces the proliferative changes in the cells.
HPV and Herpes simplex virus (HSV) have been established as causative agents of OC in recent years. HPV have been found in about 23.5% of OC cases. The most common type of HPV virus identified in head and neck squamous cell carcinoma (HNSCC) is HPV-16, which has been confirmed in 90–95% of all HPV positive HNSCC cases, followed by HPV-18, 31 and 33. The prognostic importance of HPV in pre-cancerous oral lesion is not clear. However, few studies have shown improved disease specific survival and better prognosis for HPV positive OC.
HSV-1 or ‘‘oral herpes’’ is usually associated with sores around the mouth and lip and has been recommended to be a causative agent of OC. Epidemiological studies found that higher level of IgG and IgM antibodies to OC patients compared to control subjects . Kassim et al also found oncogenic association between HSV-1 and oral squamous cell carcinoma (OSCC). A population based study showed HSV-1 to enhance development of OSCC in HPV affected patients and individuals having adverse habit of smoking.
Risk of pharyngeal cancer and oral cancer is two-fold higher in human immunodeficiency virus (HIV) affected patients signifying a link between HIV and OSCC. Epstein Barr Virus (EBV), cytomegalovirus and human herpes virus-8 (HHV-8) have also been reported as risk factors of OSCC in different studies.
Syphilis
The data on underlying association between syphilis and OC is weak. There are reports of 19 and 6% serological positivity for syphilis among oral cancer patients.
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Candida
Candida has been recommended to play a role in initiation of OC. various studies have found that nodular leukoplakia infected with Candida has a propensity for higher rate of dysplasia and malignant conversion. It has also been revealed that epithelium of the chick embryo, when infected with Candida albicans demonstrate squamous metaplasia and higher proliferative phenotype. The causal relationship of Candida infection and OC is still divisive and needs more proof.
Dental Hygiene and Related Factors
There is inverse connection between oral hygiene and incidence of OC. Poor oral hygiene and sharp teeth with prolonged irritation have been viewed for their potential role in the development of OC. Poor oral hygiene and dental sepsis is considered to promote carcinogenic action of tobacco. There are several spotted reports on the role of oro-dental factors in the causation of OC, but it lacks major evidence.
Nutritional Factors
Dietary deficiencies are also found to play a role in the development of OC. This however, needs more experimental and clinical evidence for establishment of causal connection with the development of OC. Some workers have reported lower incidence of OC with more intake of fruits and vegetables 15, 16.
Immunosuppression:
In immunocompromised individuals the risk of malignancy is very high in throughout the body, as also in oral cavity. HIV-infected patients are very much prone to develop Kaposi sarcoma, Non-Hodgkin lymphoma and oral Squamous cell carcinoma.
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Likewise transplant patients are associated with increased risk for oral malignancies.
Dyskeratosis congenita, an inherited disorder related with progressive bone marrow failure causing aplastic anemia and presents with dystrophic nail changes, skin hyper pigmentation, and leukoplakia which has more risk of malignant transformation. Yet another uncommon bone marrow failure syndrome Fanconi anemia is also associated with increased risk for oral Squamous cell carcinoma 19, 20.
Molecular Genetics
The common genetic alterations seen in oral squamous cell carcinoma are mutations involving p53, p16, cyclin D1, p63, PTEN, Rb, and epidermal growth factor receptor (EGFR). P16 mutations are present in 80% of oral cancers. The critical pathways involve primarily p53 inactivation by inhibition and mutation of HPV-16 E6 protein, EGFR activation and over expression of signal transducer and activator of transcription 3 (STAT3) and vascular endothelial growth factor receptor (VEGFR). Carcinogens in tobacco lead to increased TP53 mutations. HPV is the major causative agent in oropharyngeal cancer with more than 50% showing HPV DNA. HPV associated E6 protein inactivates p53, whereas E7 protein by inactivation of Rb gene causes over expression of p16. Gene expression report identifies transcriptional signatures which assist in predicting the overall survival and possibility of nodal metastasis.
Carcinogenic agents in tobacco products and cigarette smoke are mainly benzopyrene and nitrosamines, and same as are aresoline in areca nut. These products cause modification in genes mainly in 3p, 17 and 9p 21-26.
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HIGH-RISK SITES Buccal mucosa
Among tobacco users most common site for occurrence of oral carcinomas is buccal mucosa. This is because of tobacco quid or betel products which have been kept for quite long time in the mandibular vestibule. This further causes absorption of the carcinogenic agents and predisposes to cancer.
Tongue
The tongue is the next common site for oral cancer with more than half of lesions occurring on the oral tongue, and the rest presenting in the tongue base. In the oral cavity proper, lesions are most frequently seen on the ventral and lateral surfaces, and these areas stay to be the high-risk sites. Tongue base tumours appear to be more highly developed at the time of diagnosis, frequently presenting with metastasis to regional lymph nodes.
Lip
The vermillion border of the lip is another main site for occurrence of oral cancer, but the incidence is low. Majority of the labial carcinomas take place on the lower lip. It presents more common in men than women. The main risk factor is Ultraviolet radiation exposure. It may also present in pipe smokers where the pipe stem repeatedly contacts the lip for relatively an extended period of time. Generally lip cancers are diagnosed early because of its easy visibility 21, 23.
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INVESTIGATIONS
The investigatory modalities generally used in diagnosis of oral cancers are 1. Tissue biopsy and histopathology, assisted with or without immunohistochemistry.
2. Exfoliative cytological analysis
Most of the malignancies develop in the setting of premalignant lesions.
Premalignant and malignant oral lesions can mimic a number of benign oral lesions which appear as a white patchy or red lesion (leukoplakia and erythroplakia). In histopathology the degree of dysplasia is assessed. The lesions are graded as mild, moderate, and severe dysplasia. So far, tissue biopsy and following histological examination remains the gold standard for the diagnosis of premalignant and malignant oral disorders.
Surgical biopsy from the lesion is an invasive procedure and creates both psychological implications for the patient and technical difficulty for the person doing the procedure. Widespread lesions may lead to sampling error. In addition, there will be inter-observer variation in interpreting dysplasia and reproducibility in morphological features of mild dysplasia is poor.
ORAL EXFOLIATIVE CYTOLOGY History of evolution of oral cytology
The Papanicolaou and Traut studied the cells exfoliated from cervical mucosa for precancerous and cancerous lesion created a way for oral cytology. This work proved a valuable tool for screening malignant lesions of the cervix. In the early stages,
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comparative studies of cervical and oral cytology was done .They noted cellular changes based on the phase of menstrual cycle.
In late nineteenth century, exfoliated malignant cells in the sputum were indicative of oropharyngeal carcinoma. The diagnosis of nasopharyngeal carcinoma was done by using pap staining in oral smears. Cytology of the oral cavity was measured as a useful tool for detecting oral lesions by many researchers. Later, Sandler, by his major work named Veterans Administration studies of oral cytology, opined that this technique helps in early detection of oral malignancies.
Important events in oral exfoliative cytology
1860 - Beale - In a case of pharyngeal carcinoma showed malignant cells in sputum.
1940 - Weinmann – Done study on Cytological examination of oral cellular keratinisation 1941 - Ziskin et al. - oral cellular morphology associated with menstrual cycle
1942 - Papanicolaou - Introduction of a pap staining procedure for cytological smears 1943 - Papanicolaou and Traut – Exfoliative cytological diagnosis of uterine cancer 1949 - Morrison - Cytological interpretation of nasopharyngeal malignancies
Cytology techniques and their modifications
Over a period of time, the field of oral cytology begun to grow, many investigators including Montgomery and Von haam found the limitations of oral cytology so felt the need for improvements. They devised numerous modifications to obtain larger amount of cells, to sample a large cellular area and also to progress the quality of cell staining.
However these modifications were not set to widespread use. Special stains have been advocated, either to identify the best area for cell collection in a diffuse lesion or to
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improve the definition of malignant cells for the examining pathologist. In order to attain the cells of the basal and parabasal layers, the atypical keratotic cell layers need to be removed. It is for this very purpose, the use of a sharp spoon or a metal spatula was suggested by some authors.
In addition to these techniques, several supportive analytical methods for light microscopy were used. Possible use of fluorescence microscopy and phase contrast microscopy was investigated. Fluorescent dyes specific for DNA like acridine orange were utilized to measure the cellular DNA content. Analysis of nucleolar diameter and size, as added parameters for malignancy, was carried out with the aid of image cytometry. Moreover the classical applications of the oral exfoliative cytological studies, recognition of Epstein–Barr virus in oral lesions of hairy leukoplakia has also been done, thereby widening its possibilities.
Oral cytology in cancer detection
Oral cytology is a hopeful diagnostic tool for early identification of malignant lesions. The topic of whether oral cytology could be appropriate for mass population screening is rather unsettled. Thus, investigators came up with several modifications in the techniques, some of which have been described above.
Conventional exfoliative oral brush cytology has sensitivity value ranging between 79% and 97% and specificity between 95.1% and 99.5%. Even if there is an increase in the accuracy but this have not extensively increased compared with conventional exfoliative cytology. Thus, they require improving analytics, which make use of
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technology like image analysis and automated machines, remains a challenge for the investigators.
Oral cytology techniques
The instrument using for a nice cytological smear have to show the following features
1. Ease to use at difficult sites in the mouth 2. Minimal traumatic damage should be there 3. Helps in obtaining adequate number of cells Collecting instruments in oral exfoliative cytology Wooden/metallic spatula
Metal spatula, wooden tongue depressor, and cotton-tipped applicator were the most commonly used instruments for oral exfoliative cytology. But these instruments were related with some disadvantages. When they are scraped or rolled over sensitive oral mucosa may cause pain and tenderness. So they are applicable only in selected intraoral locations such as the lingual, mandibular and gingival regions and they should be flexible and long handles. In addition, the quantity and quality of epithelial cells collected with these instruments can vary strikingly. Epithelial cells obtained with the metal spatula and wooden tongue depressor often displays significant cytoplasmic and nuclear distortion.
The wooden spatula in particular lacks enough flexibility in collecting diagnostic cells from areas of the oral cavity which are not easy to reach, such as the ventral surface of the tongue. The transfer of the amount of obtained cells from either a glass slide or
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wooden spatula is not satisfactory and producing non representative and paucicellular smears with clumping of the cells. Furthermore a fair amount of mechanical damage to the cells can occur. It is resulting from the rubbing of the rigid spatula surfaces with the glass slide during the transport and causing troubles in interpretation.
Brush biopsy
Brush biopsy is a safe, simple and highly sensitive, comparatively cheaper method of screening for cancer. A full transepithelial cellular sample is obtained with ease with the brush. The combination of smears and computer based image analysis system go a long way in recognizing oral epithelial abnormalities. If full thickness sample has been obtained there should be evidence of pinpoint bleeding.
The introduction of the oral brush is significant in the history of oral cytology. Use of a brush for obtaining cervical cytology showed better cell spreading on objective slides. It also proved that improvement in validity and quality of smears compared with smears collected by using a wooden spatula. This instrument is more convenient, than the wooden tongue depressor in oral cavity lesions. This technique is an easy and painless investigation that can be used to evaluate oral premalignant lesion.
Many others opined that brush cytology could discover lesions that were not clinically suspicious of carcinoma or pre-invasive disease. A study clubbed conventional oral brush cytology and application of toluidine blue to identify the site for brushing in suspicious mucosal regions. Mehrotra et al., in his study recently, impressed on the utility of automated analysis in minimally suspicious lesions.
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Indication of brush biopsy
1. Evaluation of lesions of unknown significance
2. Larger surface mucosal lesions, which have been duly noted and have remained under observation only and should be analyzed on a periodic basis
Advantages of oral brush biopsy
• Relatively simple, inexpensive, highly sensitive, and risk-free method of screening for cancer
• Improved accuracy due to ease in obtaining full transepithelial cellular samples
• Exposes lesions not clinically suspicious of carcinoma or pre-invasive disease
• Cytobrush is more convenient to clinician than the wooden tongue depressor during oral exfoliative cytology
•Provides a more even distribution of epithelial cells on a glass slide than the wooden/tongue depressor
• Cellular samples obtained by cytobrush can be used for cytomorphometry, DNA cytometry, and immunocytochemical analysis 27.
NORMAL ORAL CYTOLOGY Oral epithelial cells
The squamous epithelium of oral cavity resembles the squamous epithelium of cervix and vagina. They also have superficial and intermediate cells but the nuclear pyknosis is not seen. The nuclear chromatin is longitudinally condensed in the form of a nuclear bar with lateral extensions, similar to the Anitschkow cells of myocardium in rheumatic heart disease. Such cells are commonly seen in smears of the mucosal surface
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of the lower lip and the adjacent floor of the mouth in perfectly healthy people. The change is probably related to nuclear creases but its significance is unknown. Similar cells may be observed in mesothelial cells in the pericardium surface of the conjunctiva and in other organs.
Other cells
Mucus producing columnar cells originating in the nasopharynx or the salivary gland ducts may occasionally be observed. A vigorous scrape of the tonsillar area or the base of the tongue may result in shedding of lymphocytes, singly or in clusters.
Oral flora
Oral flora, especially in patients with poor oral hygiene, is rich in a variety of saprophytic fungi and bacteria. A protozoon, Entamoeba gingivalis, is fairly common. It is a multinucleated organism larger than Amoeba histolytica, from which it differs because it does not phagocytize red blood cells .The presence of these organisms, does not necessarily indicate an inflammatory process in the oral cavity. An unusual organism, Simonsiella species, was described in smears of oropharynx, sputum, and gastric aspirates. The large bacteria form caterpillar-like chains, each composed of 10 to 12 individual bacteria. The bacterial chains are readily observed overlying squamous cells.
The organism is non-pathogenic, most likely to be observed in mouths of people with rich dietary intake, particularly fat and proteins
Buccal squamous cells in genetic counseling and as a source of DNA
Buccal smears are the cheapest and easiest to use laboratory test to determine genetic sex, by observing and counting sex chromatin (Barr bodies) in oral squamous
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epithelial cells. The Barr bodies can be recognized as a half-moon shaped chromatin condensation at the nuclear membrane. Although, theoretically, in genetic females all squamous cells with non pyknotic, open vesicular nuclei should contain a Barr body, in practice, it can be identified in fewer than half of these cells by light microscopy of oral smears stained with Papanicolaou's stain.
Peripherally placed chromocenters and focal thickening of the nuclear membrane may mimic Barr bodies. Occasionally, malignant cells may contain two or more Barr bodies, reflecting aneuploidy. The presence of Barr bodies in a phenotypic male strongly suggests Klinefelter's syndrome (47 chromosomes, YXX). The absence of Barr bodies in a phenotypic female suggests Turner's syndrome.
Buccal cells collected by mouthwash or by other techniques acts as a source of DNA for various tests, including person identification.
INFLAMMATORY DISORDERS
The diffuse inflammatory processes and poor oral hygiene produces ulceration or erosions. The large Parabasal squamous cells come into picture than the superficial and intermediate cells which are normally seen. They exhibit multiple, round or oval vesicular nuclei. Small nucleoli and chromo centers are noted. The cytoplasm is often poorly preserved.
Multinucleated macrophages may occur in chronic inflammatory process. These conditions express purulent exudates and leukocytes. Plasma cells are also seen, more commonly with smears obtained from posterior pharynx
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Specific inflammatory disorders Oral Herpes
This common disorder, characterized by blisters and painful ulcerations, is caused by Herpes virus type 1. Kobayashi et al (1998) observed the pathognomic cell changes in smears of only 4 out of 11 patients in whom the diagnosis could be confirmed by culture.
Moniliasis (Thrush)
Clinically, Moniliasis forms a characteristic white coating of the oral cavity. This organism may be identified by finding the characteristic fungal spores and pseudohyphae.
It occurs in diabetics, HIV patients. In HIV patients, it may be the first clinical symptom.
CHANGES IN ORAL SQUAMOUS CELLS IN DEFICIENCY DISEASES
In deficiencies of vitamin B12 and folic acid, both the nucleus and the cytoplasm of the squamous cell show increase in size. Megaloblastic anemia, Tropical sprue may show identical changes. Vitamin B12 and folic acid are vital ingredients for DNA synthesis. If either one is inadequate, the DNA synthesis is altered, exhibiting cell enlargement.
Other benign disorders Benign leukoplakia
Heavy keratin formation on the surface of epithelium is a process localized to areas like the palate, parts of gingiva. The benign leukoplakia is milky white appears histologically as a benign squamous epithelium, with layers of keratin. The precancerous leukoplakia may have a similar clinical appearance. Cytology of benign leukoplakia is
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fully keratinized, yellow stained cells without nuclei being characteristic of this disorder.
It may also be seen in normal oral smears; therefore, the cytological diagnosis should always be correlated with clinical findings.
MALIGNANT LESIONS
Invasive Squamous Cell Carcinoma and its Precursors Cytology
The biopsy of clinically suspicious lesions yields a diagnosis of invasive squamous cell carcinoma. The ulcerated invasive lesions can be diagnosed cytologically.
It is necessary to remove necrotic material before cytological sampling. The background always shows blood, numerous leukocytes and necrotic material. The degree of keratinization can be assessed by cytology. The cancer cells are large with orange and yellow staining cytoplasm. They also have pyknotic, dark staining irregular nuclei.
“Ghost”cells have no nuclear material. The mean AgNOR value of a squamous cell carcinoma is the highest in comparision with benign and precursor lesions.
The cytological diagnosis of non ulcerated, invasive, keratinizing carcinomas, like verrucous carcinoma, is not clear as abundant “ghost” cells hinder the observation of malignant cells. Reddy and Kameswari (1974) made a study with 165 patients with keratinizing carcinoma of the hard palate in reverse smokers in India and diagnosis was made in only 60% of the patients.70 similar results were reported by Banoczy and Rigo (1976). A detailed observation of nuclear abnormalities was made, which occurs in only a few cells. Irregularity of outline, nuclear hyperchromasia, nuclear enlargement, is of diagnostic significance. A biopsy is advised if diagnosis is suspicious.
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In poorly differentiated squamous cell carcinomas, keratinization is not prominent, but coarse chromatin and nucleoli which is large is seen. In oral cancer, nucleocytoplasmic ratio is usually modified in favor of the nucleus
PRECURSOR LESIONS IN ORAL SQUAMOUS CELL CARCINOMA
Identifying precursor lesions may be lifesaving in case of oral cancers.
Precancerous lesions of the squamous epithelium of origin invariably precede invasive cancer.
Two types of precancerous lesions in the oral cavity
The common white lesions with irregular, jagged borders, usually referred to as precancerous leukoplakia, similar to the benign leukoplakia, and correspond to precancerous lesions with a heavily keratinized surface and nuclear abnormalities in well differentiated squamous cells . The white color of the lesion is due to the opaque surface layer of keratinized epithelium. Mild or Moderate dysplasia is often attached to such lesions.
The less common red lesions (erythroplakia), corresponding to the non- keratinizing precursor epithelial lesions, are usually composed of smaller cancer cells with minimal or absent keratinization of surface (carcinomas in situ or severe dysplasia).
The red colour is because of vascularized stroma underlying the often thin epithelium.
The lesion is a precursor of invasive squamous cancer. It is recognized in the studies by Sandler (1962, 1963), Shafer et al (1975), and Mashberg et al (1977). Niebel and Chomet (1964) suggested in vivo staining of the oral mucosa with toluidine blue to demarcate the territories of these lesions. Incidentally discovered, there are no visible oral lesions.
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Acetowhite areas, after application of 3% acetic acid solutions, may be observed in such patients.
It is difficult to diagnose precancerous leukoplakia and keratinising carcinoma in situ. The abnormal cells in smears are obscured by anucleated squames or keratinized benign cells. Few visualized cells suggest either a borderline squamous lesion or a well- differentiated squamous cell cancer with keratinized cytoplasm and nuclear enlargement.
In these conditions, it is of prime importance to note the clinical finding .The information from cytology may be minimal, is an indication to proceed for a biopsy. Hong et al opined that a beneficial effect on the size and degree of cellular abnormalities in oral precancerous leukoplakias of some patients is seen with administration of 13-cis retinoic acid.
Non keratinizing Lesions
Oral carcinoma in situ or severe dysplasia, are not similar to precancerous leukoplakia. They are seen without significant keratin formation on their surfaces and have malignant epithelial cells. All these lesions present clinically as areas of redness (erythroplakia).
Scrape smears from such lesions are characterized by the presence of both malignant parabasal and intermediate cells with marked nuclear enlargement, hyperchromasia with a translucent cytoplasm. A few squamous cancer cells show marked nuclear abnormality and keratinized cytoplasm. The smear pattern in oral carcinoma in situ is remarkably similar to that of a high-grade squamous precursor lesion of carcinoma of the uterine cervix of well-differentiated type.
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Stahl et al (1964), observed the implications of dyskaryosis in oral mucosal lesions, pointed out the necessity of long-term follow up of patients showing such cells in their smears.
Results of cytological screening for occult carcinoma and precursor lesions
The difficulty in clinical identification of precancerous leukoplakia and carcinoma in situ, both easily curable precursor stages of oral cancer, was appreciated in an extensive cytological study of mouth lesions which was conducted by the Veterans administration, guided by Dr. H. Sandler. There were 2,758 patients with visible mouth lesions identified by cytology, and there were 287 histologically documented cases of invasive carcinoma.
Many of these lesions were very small, many were not ulcerated, not indurated, and not fixed to the underlying tissue. There were 28 patients with carcinoma in situ.
Thirteen lesions were reddish in colour, 6 were white, and the rest were of various colours; only 6 were ulcerated and only 5 were indurated. Redness of circumscribed areas of oral epithelium, erythroplakia is frequently characteristic of carcinoma in situ.
Shafer studied the clinical and histologic data on 82 oral carcinomas in situ diagnosed by biopsy only. The comparison of clinical findings of both the studies showed roughly 50% of Sandler's lesions were red, whereas there were only 16% of such lesions in Shafer's survey, suggesting that even competent observers consider red oral lesions as benign and do not biopsy them. Such lesions should be the prime target for cytological screening.
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A survey by Stahl et al (1967) confirmed that cytological screening for oral cancer is feasible. It does not appear feasible or reasonable to cytologically screen all dental patients. A scrape smear of an oral lesion may well permit more conservative surgery for earlier lesions and may be lifesaving.
AgNOR count in precursor lesion is on the higher side in comparison with benign lesions. Shiboski et al (2000) recently made an observation of deficiency in the education of professional and public education for early diagnosis of oral cancer.
Sandler's, Shafer's, and Mashberg and Meyer's studies pointed out that the floor of the mouth was the most frequently affected site of oral squamous cancer, followed by lateral surface of tongue and soft palate. These areas deserve a careful inspection during routine dental examination.
Within recent years, there has been a revival of interest in cytological detection of oral cancers based on evaluation of oral smears by a semi-automated cell analysis system oralcdx (Sciubba, 1999). A specially designed brush was used to secure cell samples from the visible lesions of the oral cavity. Of the 945 lesions sampled by cytology, 131 were “dysplastic” lesions or carcinomas confirmed by biopsies. In these cases, the smears were judged to be either “positive” or “atypical”.
Extensive surveys are necessary, in which increased risk of oral cancer exists. One such study by Wahi, impressed upon the value of cytological techniques among betel-nut chewers. The data strongly suggest that high-risk candidates for oral cancer are primary target for screening by cytological techniques for oral malignancies.
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Cytological diagnosis of recurrent oral cancer After treatment
A close follow up of all patients is essential when local recurrences after treatment are possible. They have an increased chance of treated patients to have a second malignancy or a recurrence. The addition of cytological techniques with the follow-up examination may result in the diagnosis of a recurrent or new cancer, before it is suspected clinically.
Hutter and Gerold (1966) used cytological techniques in the follow-up of patients previously treated by surgery. The application of cytology to the patients without visible lesions, uncovered clinically unsuspected recurrent cancer in 10 of 177 patients investigated. They used material scraped from the general area of prior surgery by an endometrial curette.
This work, as well as the results of cancer detection surveys described above, strongly suggest that the silent stage of carcinoma in situ, whether primary or recurrent, is not readily identifiable clinically and precedes invasive squamous cell carcinoma of the oral cavity. This stage of cancer may last for several months, and possibly much longer, before producing a visible lesion. Carcinoma in situ may be accurately identifiable by cytology 28.
HISTOPATHOLOGY
The biopsy is the gold standard for diagnosis of oral cancers. An excision biopsy is definitely sufficient for pathologic analysis of small 0.5–1.0 cm lesions. However, multiple incision biopsies are more appropriate for a large lesion, which may also be
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stained with toluidine blue to define highly suspected locations. It is common to find cases with no definite lesion location but rather a picture of pan orally premalignant
cancerization in high-risk patients with a history of BQ use and smoking for 20–30 years 29, 30.
WHO HISTOLOGICAL CLASSIFICATION OF TUMOURS OF THE ORAL CAVITY AND MOBILE TONGUE (4th edition, 2017) 2
Epithelial tumours and lesions
Squamous cell carcinoma 8070/3 Oral epithelial dysplasia
Low grade 8077/0 High grade 8077/2 Proliferative Verrucous leukoplakia
Papilloma
Squamous cell papilloma 8052/0 Condyloma acuminatum
Verruca vulgaris
Multifocal epithelial hyperplasia
Tumours of uncertain histogenesis Congenital granular cell epulis
Ectomesenchymal chondromyxoid tumour 8982/0
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Soft tissue and neural tumours
Granular cell tumour 9580/0 Rhabdomyoma 8900/0 Lymphangioma 9170/0 Haemangioma 9120/0 Schwannoma 9560/0 Neurofibroma 9540/0 Kaposi sarcoma 9140/3 Myofibroblastic sarcoma 8825/3 Oral mucosal melanoma 8720/3 Salivary type tumours
Mucoepidermoid carcinoma 8430/3 Pleomorphic adenoma 8940/3
Haematolymphoid tumours
CD30-positive T- cell lymphoproliferative disorder 9718/3 Plasmablastic lymphoma 9735/3 Langerhans cell histiocytosis 9751/3 Extramedullary myeloid sarcoma 9930/3
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Oral potentially malignant disorders (OPMDs)
Oral potentially malignant disorders are clinical presentations that carry a risk of cancer development in the oral cavity, whether in a clinically definable precursor lesion or in clinically normal oral mucosa.
List of Oral potentially malignant disorders
Erythroplakia Erythroleukoplakia Leukoplakia
Oral submucous fibrosis Dyskeratosis congenita Smokeless tobacco keratosis
Palatal lesions associated with reverse smoking Chronic candidiasis
Lichen planus
Discoid lupus erythematosus Syphilitic glossitis
Actinic keratosis (lip only)
The global prevalence of leucoplakia is 2% to 3%. In western countries it ranges from 1% to 4%. Higher prevalence rates are reported in south-eastern Asia. In contrast oral erythroplakia is a rare lesion, with prevalence between 0.02% and 0.83%. Men are affected more commonly than women. Other OPMDs can be common, but have lower transformation rates.
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The transformation risk in many OPMDs is low, and many regress. For leucoplakia a mean global transformation rate of 1-2% has been estimated. Presence of oral epithelial dysplasia is the most important prognostic factor for malignant transformation.
ORAL EPITHELIAL DYSPLASIA
OED is defined as a spectrum of architectural and cytological changes caused by accumulation of genetic changes, associated with an increased risk for progression to squamous cell carcinoma.
Dysplasia is graded into three histological types and is designated as mild, moderate, and severe dysplasia. The microscopic features of dysplasia are as follows Architectural disturbance
(1) Irregular stratification
(2) Basal cell crowding with loss of polarity (3) Drop-shaped epithelial ridges
(4) Premature keratinization in single cells (5) Reduced intercellular adhesion
(6) Keratin pearls within rete ridges (7) Increased mitotic figures
(8) Abnormal superficial mitosis Cytological atypia
1. Nuclear pleomorphism and hyperchromatism 2. Anisonucleosis
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3. Increased nuclear size
4. Anisocytosis and cellular pleomorphism 5. Increased nucleus to cytoplasmic ratio 6. Abnormal mitotic figures
7. Increased number and size of nucleoli 2 Mild dysplasia
Mild dysplasia is defined as the architectural disturbance limited to the lower third of the epithelium with associated cytological atypia. It is characterized by basal zone hyperplasia with mild increase in thickness. Cellular crowding is seen only in the lower one third of the epithelium and mitosis is absent. Nuclei show mild degree of pleomorphism.
Moderate dysplasia
Moderate dysplasia is related with loss of polarity in the lower two third of the lining epithelium. Anyhow, it is upgraded in view of increased cytologic atypia. There is moderate increase in thickness with basal zone hyperplasia. Cellular crowding involves the lower two thirds of the epithelium with no loss of polarity. Nuclear grooves and lobulations may be seen with moderate degree of cellular and nuclear pleomorphism.
Mitosis is appreciated in the lower one third.
Severe dysplasia
Severe dysplasia involves architectural irregularity and increased cytologic atypia in more than two thirds of the epithelium. Markedly increased thickness with basal zone expansion is noted. There is loss of polarity with mitosis in the lower two thirds of the
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epithelium. Cells demonstrate high nuclear cytoplasmic ratio, nuclear folding, coarse chromatin and prominent nucleoli with increased pleomorphism.
CARCINOMA IN SITU
Carcinoma in situ is defined as architectural irregularity and increased cytological atypia involving full thickness of the epithelium, but invasion is absent. There is epithelial disarray with cellular crowding, loss of polarity and full thickness atypia.
Marked degree of cellular and nuclear pleomorphism is noted and mitotic figures are seen throughout the epithelium. No flattening of surface layer is seen. The basement membrane is intact, but may have thinning when seen with immunostains4, 36.
SQUAMOUS CELL CARCINOMA
Almost 90% of the oral tumours come under this category of squamous cell carcinoma. In India, tumours tend to arise in the vicinity of leukoplakia, whereas in western countries, red lesion or normal epithelium is much more common. SCC is characterized by lobules of squamous cells with cytological pleomorphism and abundant keratinization. The malignant epithelial cells proliferate and invade the stroma singly or as islands or cords. There is increase in nuclear cytolopasmic ratio with increased apoptotic bodies. Mitosis is increased with many abnormal mitotic figures. Variable amounts of desmoplasia and inflammatory infiltration with lymphocytes and eosinophils are seen. Perineural and vascular invasion is noted. The adjacent epithelium shows dysplastic changes or features of carcinoma in situ.
Squamous cell carcinoma is graded based on the degree of differentiation of the epithelium, nuclear pleomorphism and mitotic activity. SCC is usually graded into three
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categories viz., well differentiated, moderately differentiated and poorly differentiated grade.
Well differentiated SCC
In this type, the tumour resembles normal squamous epithelium and consists of large differentiated keratinocyte like squamous cells with the periphery of the tumour having small basal type cells. Keratinisation is present throughout. It is characterized by nests, cords and islands of large cells with pink cytoplasm, prominent intercellular bridges and round nuclei. Dyskeratotic cells and squamous pearls are prominent. Only few mitoses are noted.
Moderately differentiated SCC
In this type of SCC, there is increase in nuclear pleomorphism and keratinization is reduced. Mitosis is increased with many abnormal mitotic figures.
Poorly differentiated SCC
Here the basal type cells predominate. Mitoses are numerous with increased abnormal mitotic figures. The intercellular bridges are barely recognized and keratinization is usually minimal. So it requires immunohistochemical conformation – AE1/AE3, CK5/6, p63 and p40 are useful markers 2, 30-34.
Variants of squamous cell carcinoma
There are many variants of squamous cell carcinoma. It is important to distinguish them as they differ in their prognosis. Only few tumours present entirely of their classical features. Most of them occur in combination with conventional SCC presenting as mixed tumours. However, it is better to mention the histological variation, which would be
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possible for the clinician to delineate the tumours with aggressive behavior or poor prognosis.
Verrucous carcinoma
Verrucous carcinoma (Ackerman tumor) is a variant of well differentiated squamous cell carcinoma. Verrucous carcinoma has predilection for buccal mucosa, alveolar ridge and mandibular sulcus with occurrence in other sites like gingiva, tongue, soft palate and tonsillar fossa. The clinical manifestation is that of an exophytic growth with warty or papillary surface.
The histologic features are the epithelium showing marked acanthosis, marked parakeratosis and hyperkeratosis. There is papillary exophytic and endophytic proliferation of well differentiated squamous epithelium. The bulbous frond like rete ridges push downward into the submucosa with parakeratin plugging. Only minimal cytologic atypia is noted. The prognosis is comparatively better 5, 34-39.
Basaloid squamous cell carcinoma
Basaloid SCC in oral cavity seems to be associated with HPV 16. It is a highly aggressive tumour, mostly presenting in stage iii or iv with regional metastasis.
Microscopically, it is classically arranged in nests, lobules and cribriform pattern with basaloid cells having squamous differentiation in the nests. The cells are small, with scant cytoplasm, indistinct cell borders and dark nuclei exhibiting pleomorphism. Mitotic activity is increased. Admixed with them are larger cells having abundant cytoplasm, vesicular nuclei with small nucleoli. Nuclear palisading may be seen in the periphery of the nests. The characteristic is the presence of necrosis with both single cell and comedo
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necrosis. Some of them may have pseudo glandular pattern filled with hyaline or mucoid material. The stroma may be hyalinized or myxoid. It has to be differentiated from basaloid lesions like salivary duct carcinoma, solid variant of adenoid cystic carcinoma and peripheral ameloblastoma. It commonly metastasize to the lungs. The three year survival rate is only 35% 5, 36-38,40.
Papillary squamous cell carcinoma
It is uncommon in the oral cavity and presents as an exophytic, cauliflower like mass with broad base or as finger like papillary projections with slender fibrovascular core. Microscopically it consists of non keratinizing epithelial proliferation in papillary exophytic or endophytic pattern with considerable cytological atypia, para and ortho keratosis and often micro abscess at the tips of bulbous rete ridges. Hyperkeratosis is minimal and stromal invasion is not well defined.
Depending on the maturation of the overlying epithelium, two patterns of proliferations are identified. One type is undifferentiated variant with close resemblance to small cell carcinoma in situ, and consists of non keratinizing basal and parabasal like cells with dysplastic features. The proliferation is seen in the entire thickness of the epithelium. This variant is frequently seen in tonsils and oropharynx. The next variant comprises of varying degrees of keratinization with dysplastic changes in the epithelium.
It should be differentiated from verrucous carcinoma, exophytic conventional SCC and squamous papilloma. The prognosis is only 44% and most of them die within 2 years 5, 37, 38, 41.
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Spindle cell (sarcomatoid) carcinoma
Spindle cell squamous carcinoma is commonly seen in the head and neck region, but is rare in the intraoral site. The tumour presents as red lesions, non healing ulcer associated with pain or as exophytic bosselated mass. It is a spindle cell tumour and consists predominantly of sheets of spindled pleomorphic cells. This tumour may resemble malignant fibrous histiocytoma and hence needs to be differentiated from other soft tissue sarcomas. However, the spindle cell component is admixed with conventional squamous cell carcinomatous areas. The metastatic deposits of the sarcomatoid carcinoma show pure carcinomatous or mixed components in lymph node and distant sites 5, 37, 38, 42.
Adenoid / Acantholytic squamous cell carcinoma
In this variant, the tumour shows pseudo glandular or alveolar architecture. This tumour is more common in men. The usual site involved is the lip, but also seen in tongue and gingiva. The main etiological factor is irradiation. The tumour is arranged in biphasic pattern with proliferation of malignant epithelial cells associated with acantholysis and formation of pseudo glandular structures. Here, there is loss of intercellular adhesion within the tumor cell nests creating a glandular pattern. The tumour is more aggressive with poor prognosis 5,37,38,43.
Adenosquamous carcinoma
This is a rare tumour which exhibits both squamous and glandular differentiation.
The glandular differentiation is thought to have arisen from minor salivary glands. The involvement of the salivary gland ducts in these tumors support the hypothesis, but is still
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controversial. The currently favoured explanation is the derivation from surface epithelium. This neoplasm has to be differentiated from mucoepidermoid carcinoma and conventional SCC invading the normal salivary gland. These tumors have an aggressive course with poor prognosis. There is increased tendency for local recurrence (45%) and
early nodal metastasis (65%). The five year survival rate is 13% and at 10 years it is only 4.5%.
Microscopically adenosquamous carcinoma is characterized by three distinct components a) squamous cell carcinoma b) adenocarcinoma c) admixture of glandular mucous cells with squamoid differentiation which resemble mucoepidermoid carcinoma.
Many densely keratinized glassy cells are seen. Adjacent areas show foci of dysplasia or carcinoma in situ. There is widespread and extensive permeation into adjacent soft tissues. Perineural invasion is also noted 5, 37, 38, 44
. Small cell carcinoma
Small cell carcinoma has histologic features akin to lung carcinoma. This is an aggressive tumour. The tumour consists purely of small cells or has an admixture of squamous component. Few of them have Merkel cell carcinoma features 5, 36-38, 45.
Lymphoepithelioma like carcinoma
This is a rare tumour of oral cavity. The appearance is similar to that of the tumour found elsewhere in head and neck like nasopharynx and tonsil 2, 5, 46.
Clear cell carcinoma
This is a rare variant of squamous cell carcinoma 5, 47.
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Nut (midline) carcinoma
This is a newly recognized type involving midline structures, mainly in the head and neck region. This is frequently seen in children and young adults, but affects all age groups. It is characterized by rearrangement of the NUT gene on chromosome 15q14.
The tumour consists of dual population of cells composed of islands of undifferentiated cells and islands of keratinization. There is a sharp distinction between these two regions.
The diagnosis is confirmed immunohistochemically by nuclear expression of NUT in undifferentiated cells. NUT carcinoma has an aggressive course, but a very good response to chemotherapy 2, 5.
Cuniculate carcinoma
This is a rare variant seen commonly on plantar aspect of foot and skin lesions. In oral cavity, this lesion is seen in gingiva and alveolar ridge and most of them have intra osseous extension. It has an indolent course. Histologically, the tumour is characterized by proliferation of stratified squamous epithelial cells in trabecular or ribbon like pattern with complex arborizing architecture and variable cytological atypia. Many convoluted irregular cysts or crypts filled with keratin which may burrow into bone are noted.
Obvious cytological malignant features are not seen. This tumour has to be differentiated from verrucous carcinoma. They usually do not metastasize 2, 48 .
IMMUNOHISTOCHEMISTRY Premalignant lesions
Keratosis without dysplasia: show keratin 19, epidermal growth factor And Ki-67 expression limited to basal layer.
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Keratosis with dysplasia: show keratin 19, epidermal growth factor, p16ink4a and Ki-67
expression extending to suprabasal cells. Ki-67 is the most important marker helpful in identification and Grading of dysplasia in premalignant lesions. Few cases of dysplasia show p53 over expression.
Squamous cell carcinoma
Cytokeratin: CK5/6, CK8 and CK19 positive but CK20 negative. Over expression of TP53 oncogene is seen in 30% to 50% cases. Basaloid squamous cell carcinoma show immunoreactivity for high Molecular weight keratin (detected with the 34ße12 antibody) Adenosquamous carcinoma shows positivity for CEA, CAM 5.2 and CK7.80 2, 5, 37, 38. CANCER STAGING
Squamous cell carcinoma is graded according to TNM Staging of tumours by AJCC. The tumour staging is of important Prognostic significance and aids in treating the patient.
PROGNOSIS
The most important prognostic indicators of oral cancers are as follows
1. Location: The overall five year survival rates vary depending upon the Site of tumour.
Lip has a good prognosis with 90% survival rate for Tumors of lower lip. Next is the anterior tongue with 60% survival rate and floor of the mouth, posterior tongue, tonsil, hard palate and gingival having 40%. The poor prognostic tumors are those in the soft palate with only 20- 30% survival rate.
