NORMATIVA DE COMERCIO ELECTRÓNICO Y CONTRATACIÓN

When looking at brushings of the tumour site from p16 positive cases, 100% (3/3) from patients under anaesthetic were positive for HPV 16 DNA. Of the tumour site brushings from conscious patients, 63% (17/27) were positive for HPV 16 DNA. Further analysis was not possible due to the low numbers of brushings taken from patients under anaesthetic. Of the remaining ten HPV 16 DNA negative samples; four were base of tongue samples, two had recently had the affected tonsil removed and two had abnormal cytology.

There were 24 cases where brushings were taken from a tonsillar tumour site. Of these, HPV 16 DNA was detectable in 75% (18/24). There were nine brushings from a base of tongue tumour site in which HPV 16 DNA was detectable in 44% (4/9).

134 Figure 5.3: Images of abnormal cytology. Magnification 40x, ThinPrep® Papanicolaou Stain. A & B) Low grade changes. Low grade cells show nuclear enlargement and hyperchromasia relative to surrounding normal cells. C - F) High grade changes. High grade cells are small parabasal sized cells with nuclear hyperchromasia and irregular nuclear membranes. G & H) Invasive carcinoma. Cells are pleomorphic with marked nuclear atypia with tumour diathesis in the background.

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5.5 Discussion

The detection of HPV 16 DNA in tonsillar brushings from known oropharyngeal cancer patients seen in this study is consistent with other published studies [125,167]. HPV DNA has previously been detected in the tonsils from cancer cases and cancer free individuals by either brushings taken under anaesthetic [123,125] or the use of ex vivo brushings [126,127]. Approaches using patients under anaesthetic limit the interpretation of how brushings could be applied as a screening test as the procedure imparts significant financial and time costs, and risks for the patient. Our results show in brushings from conscious oropharyngeal cancer patients HPV 16 detection appears to be superior for tumours of the tonsillar sub-site over base of tongue carcinomas with 18/24 and 4/9 respectively having detectable HPV 16 DNA. The base of tongue is defined as “the posterior attached portion of the tongue extending from the line of the circumvallate papillae to the junction of the base of the epiglottis”[169]. However, it is possible that samples that were taken from the base of tongue from conscious patients may have been posterior oral tongue specimens as access to the base of tongue is often prevented by the pharyngeal reflex [126]. Interestingly, half of the base of tongue samples were positive for HPV 16 DNA, and had abnormal cytology compared to all four HPV 16 negative base of tongue samples having normal cytology. This suggests the periphery of the tumour may have been able to be sampled in half the cases even though on visual examination there was no abnormality seen.

This study detected HPV 16 DNA in 83% (10/12) of samples taken from p16 positive cases with visible tumours. Of the remaining two samples one case had high grade cytology and therefore may be attributed to a non-HPV 16 type. The final brushing was recorded as taken from the right tonsil and the appearance recorded as visible tumour.

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However, the pathology reports state the tumour was on the left tonsil. It is therefore uncertain if the tumour was actually sampled. Regardless of this sample, these results from visible tumours and the overall 21/30 (70%) of HPV 16 positive brushings from p16 positive samples are comparable to the 28/32 (88%) of p16 positive oropharyngeal cancer samples with high risk HPV in brushings taken under anaesthetic during parendoscopy seen by Kofler et al [125]. Although the number of cases in our study was small, the results show HPV 16 DNA can frequently be detected in superficial brushings from the tonsils of conscious p16 positive OPC patients.

This study had one p16 negative case that had detectable HPV 16 in the brushing sample. This case also had low grade changes on cytology, however adequate RNA was not able to be extracted. This patient was an 87 year old woman with a metastatic poorly differentiated carcinoma. When reviewing the pathology report, it was noted that the p16 immunohistochemistry was performed on a cell block from a fine needle aspiration sample. There are no set guidelines for what constitutes a positive p16 result in a cell block specimen, and if the cut off of 70% of tumours cells stained in the histology specimens is applied, there is likely to be a high rate of false negatives [170]. It is also possible the HPV detected was simply a transient infection and unrelated to the tumour [47,136].

HPV 16 mRNA was detectable in 55% of HPV 16 DNA positive samples after excluding 25% of samples due to a lack of amplifiable mRNA. To the best of our knowledge we are the first to apply RNA based tests to brushings for oropharyngeal cancer. The advantage of an RNA over a DNA based test is a higher specificity for disease due to the detection of transcriptionally active HPV [93]. However, results from this study

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showed the detection of HPV 16 DNA correlated well with both p16 results and the brushing site. HPV 16 DNA detection was rare in samples taken from a non-tumour site. Overall HPV 16 DNA detection was superior to cytology. This scenario is also seen in cervical screening which is likely to result in a move to primary HPV testing in the near future [121,171].

In this study, 1mL of sample for was used for RNA extractions. However, the high number of beta-globin negative samples suggests using more sample volume may be required. It should also be noted that the gross appearance of brushings varied considerably from transparent to very turbid or heavily blood-stained. This likely occurred from a combination of whether the tumour or a normal tonsil was sampled and the patient’s ability to tolerate the procedure. It is also possible that factors such as patient age, time since last meal or drink, and oral health may have played a role. A limitation of our study was we had multiple people collecting the samples. Although all were trained on how to do the procedure, it may have had an impact on the quality of the specimens collected.

This study describes cytological changes comparable to cervical pre-cancerous lesions. A continuum of cytological changes, ranging from low to high grade dysplasia, high grade dysplasia to invasive carcinoma, or low grade dysplasia to invasive carcinoma was seen in eight of the 18 samples with abnormal cytology. The continuum of changes was seen only in p16 positive cases, and may suggest progression similar to that seen in cervical cancer. None of the low grade changes observed in this study included koilocytes. This is consistent with finding by Francheschi et al who found only one koilocyte in a study of 200 tonsillectomy samples removed due to benign diseases

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[127]. Koilocytes are a common finding in low grade cervical neoplasia, and are a result of productive HPV infection and the production of virions in a mature squamous cell [5]. Koilocytes are not generally associated with malignancy as they are found in productive infection. By comparison, in cervical screening it is common to see non- koilocytic low grade changes in a smear with a high grade lesion. To date, studies of tonsillar specimens from cancer free individuals have not found any evidence of pre- cancerous lesions for OPC [123,127,166]. However, areas of dysplasia and carcinoma in situ associated with oropharyngeal cancer have been described [81]. This may support the idea that although HPV infection in the tonsil is rare, the proportion of lesions that progress may be higher than in the cervix [166]. Further research into the histological occurrence of and appearance of dysplastic changes in HPV positive OPC is required, and should be conducted on those with OPC. The natural history of HPV in the tonsil remains uncertain and describing precancerous lesions adjacent to an existing tumour is more feasible than detecting pre-cancers in studies of the general population. Until the lesions seen in this study are well described histologically, their significance cannot be ascertained.

The absence of well-defined pre-cancerous lesions is a major issue when looking at the potential for screening for oropharyngeal cancer. Fakhry et al found associations between HPV 16 detection and abnormal cytology in those with visible oropharyngeal lesions. They then attempted to see if similar associations were present using a nested case-control study of 401 human immunodeficiency virus (HIV) infected individuals [123], a group known to be at higher risk HPV positive oropharyngeal cancer [155]. This case-control study found no association between the presence of HPV 16 DNA and the presence of cytological abnormality. They attribute this to not being able to

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adequately sample the site of infection and therefore cytological abnormality in the tonsils [123]. Similar criticisms of any brush based pap-test equivalent have been described by several authors [124,127,165]. Although HIV infected individuals have a higher risk for oropharyngeal cancer, other factors need consideration. For example, the study population used by Fakhry et al consisted of 60% males and almost 90% African Americans, and had a median age of 46 years [123]. By comparison, a study of 271 oropharyngeal cancer cases from 1984-2004 in the United States with an overall HPV prevalence of 44%, found 80% of patients were male, 63% were European and 80% of cases were aged over 50 [46]. Therefore, the Fakhry et al study population may not have been adequately designed for the early detection of OPC.

It is currently unknown if the early detection of HPV positive OPC would decrease the mortality from the disease [108]. A focus on the early detection of OPC to minimise the significant morbidities from current treatment regimens may prove more useful than the quest for precancerous lesions. Treatment for OPC often involves high dose radiation and side effects of treatment have significant impact on patient quality of life [53]. The detection of micro-lesions confined to the tonsil by narrow band imaging has been previously described and although the follow-up time is limited the treatment was a surgical approach with no requirement for radiation [172,173].

Overall, this study is the first to use brushings from conscious patients to detect HPV 16 DNA, RNA, and cytological abnormalities which has shown to be a simple procedure that is acceptable to patients. We have described cytological abnormalities comparable to cervical precancerous lesions and shown a continuum of dysplasia in a

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number of p16 positive cases. Further research and histological confirmation of these apparent precancerous lesions is required before their significance can be determined.

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