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Methods: 30 consecutive pathological specimens, ranging from conjunctival intraepithelial neoplasia, carcinoma in situ, to invasive squamous cell carcinoma were retrieved from tissue archives. Two masked pathologists studied haematoxylin and eosin stains on paraffin embedded conjunctival tissues. Elastic stain for solar elastosis was blindly interpreted in comparison with negative and positive controls.
HPV infection was studied by polymerase chain reaction and dot hybridisation. The male to female ratio was Solar elastosis was seen in Conjunctival squamous cell neoplasia CSCN is the most common malignant tumour of the ocular surface.
The tumours then grow slowly, invading the nearby tissues including the eyeball, eyelids, and orbital tissues leading to severe visual loss, loss of the eye, and severe facial deformities. Similar to the squamous cell carcinoma of the uterine cervix, the stagings of conjunctival squamous cell carcinoma are classified by the thickness of epithelial dysplastic changes and the tumour invasion into the substantia propria. The disease severity varies from conjunctival intraepithelial neoplasia CIN , carcinoma in situ CIS , to invasive squamous cell carcinoma.
So far the causes of the disease are not adequately understood. There have been a number of investigations on the relation between the tumour to several factors, including solar exposure 5— 7 and human papillomavirus HPV. Newton et al 5 related the prevalence of this malignancy to tropical location of the patient dwellings. Sun et al 6 found an association between ultraviolet exposure and the prevalence of squamous cell neoplasia of the conjunctiva and the eyelids. However, these two studies were conducted as non-comparative studies.
Lee et al 7 found a relation of sun exposure to squamous cell dysplasia in a case-control study, but did not include the pathological study on the actinic damage of the tissue. This study was designed to identify the association between sun exposure and human papillomavirus infection to CSCN. The earliest case was from May and the latest one was from November Controls were disease-free conjunctiva from age and sex matched patients who underwent planned extracapsular cataract extraction.
Age matching was done by stratifying age groups of cases into 5 year strata—50—54, 55—59, 60—64, etc. The controls were recruited from consecutive patients whose age at the time of operation fell into the similar stratum of the carcinoma cases. The recruitment process took place from July to January All control patients agreed to participate in the study and provided informed consent.
The study protocol was reviewed and approved by the ethics committee of the Faculty of Medicine, Chulalongkorn University. Staining with haematoxylin and eosin was performed. Specimens of both the cases and the controls were coded and mixed in order to blind the interpreting pathologists. Two masked pathologists interpreted haematoxylin and eosin stained slides.
Carcinoma cases were classified into different stages. If the readings disagreed, sections were reviewed to make the diagnosis and staging under consensus. Solar elastosis Conjunctival sun exposure has been demonstrated by solar elastosis in the substantia propria. External positive controls were three pterygium tissues.
Internal controls could be demonstrated only in some cases where the arterial walls were identified. Internal elastic lamina, shown as concentric rings of dark wavy lines, was seen if the cross sections of the arteries were found. Negative controls were from normal appearing conjunctival tissues of other irrelevant diagnosis. All slides were coded to mask the interpreters. The codes were revealed after the interpretation was complete, before statistical analysis.
The tissue was carefully handled to avoid viral DNA contamination to other specimens. Each microtome blade was singly used for a specimen. Quantitative analysis of DNA was performed by ultraviolet spectrophotometer at and nm. The statistical analyses were performed under stata 6.
Mild dysplasia was found in one case and severe dysplasia in six others. Positive staining of elastic tissue in the substantia propria, representing pathologically proved solar damage of the conjunctiva, was seen in There were only 3. The odds ratio, calculated from the discordant pairs, was DNA was not quantifiable in 14 specimens three cases, 11 controls.
However, in this study, all samples were measured. DISCUSSION Conjunctival squamous cell neoplasia is a serious problem with a high impact on public health owing to its relatively high prevalence and the potential to cause severe disability. We conducted a study to find the association of sun exposure and HPV infection and the neoplasm.
The study was designed in such a way to minimise biases. Sampling bias may occur if only selected cases were included. In this study we searched the archival tissue for the most recent cases. We found a variety of stages of diagnosis. However, these cases were from a hospital based population, which is an unrepresentative sample of the general population. In order to obtain a comparable group of samples with equal opportunity to reach medical attention, the controls were recruited from the same hospital.
With a further attempt to minimise any unknown confounders, control specimens were age and sex matched to the cases. Matching increased the power of the comparison between groups by balancing the number of cases and controls at each level of the constitutional factors.
Elastic staining and HPV DNA detection were compared with the positive and negative controls to enhance the validity of measurement. Two pathologists interpreted the results to increase the reliability of the study. We blinded the outcome assessors of the case-control status in order to prevent ascertainment bias.
This finding was used as a pathognomonic sign in the pathological diagnosis of degenerative diseases of the conjunctiva such as pinguecula and pterygia. This material changes its colour to dark brown after elastic staining. We found different proportions of positive solar elastosis between neoplastic cases and non-diseased conjunctival controls.
The odds ratio showed that solar elastosis is related to malignant changes of the conjunctiva. Owing to the small sample size, the confidence interval of the odds ratio was rather large.
The dose-response relation is one of the criteria to establish a causal effect of exposure to the disease. We, however, did not measure the solar elastosis quantitatively because the retrieved specimens were previously sectioned to generate pathological reports, and later to detect HPV infection.
We extracted DNA from paraffin embedded specimens with the most appropriate technique as previously described by Chan et al. This might be because of low prevalence of the viral infection. Multiple factors may contribute to the development of the disease. Further studies to explore all factors in a single situation would contribute to more useful information. Since HIV infection is a possible confounding factor, 26— 30 the investigation on HPV and HIV infections together with special characteristics on pathological figures will predict the diseases more definitely.
In conclusion, sun exposure, as demonstrated by pathologically proved solar elastosis of the subconjunctival tissue, is one of the risk factors to develop conjunctival squamous cell neoplasia. More studies need to be performed to explore the relation of this tumour to multiple factors. Tumors of the conjunctiva. In: Tumors of the eye and ocular adnexa. Third series. Fascicle Conjunctival lesions in adults. A clinical and histopathologic review. Cornea ;— Taweepanich S.
Eye, ear nose and throat malignant tumours in Siriraj hospital in — Siriraj Hospital Gazette ;— Malignant tumours of the eye and ocular adnexa at King Chulalongkorn Memorial Hospital: a twelve year review — Chulalongkorn Med J ;— Effect of ambient solar ultraviolet radiation on incidence of squamous-cell carcinoma of the eye.
Lancet ;—1. Epidemiology of squamous cell conjunctival cancer. Cancer Epidemiol Biomarkers Prev ;—7. Risk factors in the development of ocular surface epithelial dysplasia. Ophthalmology ;—4. Demonstration of papilloma capsid antigen in human conjunctival neoplasia. Arch Ophthalmol ;—5. DNA of humanpapilloma type 16 in dysplastic and malignant lesions of the conjunctiva and cornea.
N Engl J Med ;—6. Published erratum in N Engl J Med ; Human papillomavirus type 18 in conjunctival intraepithelial neoplasia. Am J Ophthalmol ;—7. Detection of human papillomavirus infection in squamous tumours of the conjunctiva and lacrimal sac by immunohistochemistry, in situ hybridisation, and polymerase chain reaction.
Lab Methods in Histotechnology
Laboratory methods in histotechnology