Immunofluorescence in diagnosing Oral Lichen Planus

University of Groningen, Faculty of Medical Sciences, Antonius Deusinglaan 1 University Medical Centre Groningen, Department of Oral and Maxillofacial Surgery, Poortweg 2

09/02/2015-01/07/2015 Student: S.J. Geertman Studentnumber: s1826859 Supervisor: Dr. M.J.H. Witjes Dr. K.P. Schepman Drs. K. Delli Date: 01-07-2015

Abstract Background: Oral Lichen Planus (OLP) can be diagnosed based on several clinical criteria, which are highly variable. In the Dutch ‘Guideline Lichen Planus’ histopathology is the golden standard for additional research. The WHO criteria of 1978 have never been validated. Direct immunofluorescence (DIF) tests are used in the University Medical Centre Groningen (UMCG) to diagnose OLP. Objectives: To determine whether the Dutch ‘Guideline Lichen Planus’ (Dutch criteria) is followed by the department of Oral and Maxillofacial Surgery in the UMCG. To assess the additional value of direct immunofluorescence (DIF) in the process of diagnosing OLP. Methods: We included 358 patients from the medical database of the UMCG, who had a biopsy between June 2002 and January 2015 and where OLP was in the differential diagnosis. The patients, treated at the Department of Oral and Maxillofacial Surgery were scored on several clinical, histopathological and DIF features. The UMCG diagnostic work-up was compared with the ‘Dutch criteria’, ‘WHO criteria’ and the ‘Modified WHO criteria’. The additional diagnostic value of DIF was examined. Results: The ‘Dutch criteria’ are followed in 23.3% of the patients diagnosed with OLP by the clinicians in the UMCG. When DIF is used additionally to histopathology for differentiating OLP from OLL, 3.26 times more patients are diagnosed with OLP. The use of Levothyroxine was found in 11.5% of the patients with OLP or OLL. Conclusion: The Dutch ‘Guideline Lichen Planus’ is not always fully followed by the clinicians in the UMCG. The addition of DIF to the original ‘Dutch criteria’ would diagnose OLP in more patients. When the diagnostic work-up used in the UMCG is compared with abovementioned diagnostic criteria, there is a dramatic shift in the diagnosis of OLP to OLL. An additional finding was a high percentage of patients with OLP or OLL using suppletion of thyroid hormone.

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Samenvatting

Achtergrond: OLP kan worden gediagnosticeerd op basis van verscheidene klinische criteria, die zeer variabel zijn. In de Nederlandse ‘Richtijn Lichen Planus’ is histopathologie is de gouden standaard in aanvullend onderzoek. De WHO criteria uit 1978 zijn nooit gevalideerd. Direct immuunfluorescentie (DIF) onderzoek wordt in het Universitair Medisch Centrum Groningen (UMCG) gebruikt om OLP te diagnosticeren. Doel: Bepalen of de Nederlandse ‘Richtlijn Lichen Planus’(Nederlandse criteria) worden gevolgd door de afdeling Mond-Kaak- en Aangezichtschirugie in het UMCG. Vaststellen wat de toegevoegde waarde is van DIF in het diagnosticeren van OLP. Methode: We hebben 358 patiënten geïncludeerd uit de medische database van het UMCG, die zijn gebiopteerd tussen juni 2002 en januari 2015 en met OLP in de differentiaal diagnose. De patiënten, die behandeld zijn op de afdeling Mond-kaak- en Aangezichtschrirugie zijn gescoord op verschillende klinische, histopathologische en DIF kenmerken. Het diagnostische proces in het UMCG is vergeleken met de ‘Richtijn Lichen Planus’, de ‘WHO criteria’ en de ‘Modified WHO criteria’. De toegevoegde waarde van DIF is onderzocht. Resultaten: De Nederlandse ‘Richtlijn Lichen Planus’ wordt in 23.3% van de patiënten die zijn gediagnosticeerd met OLP door de clinici van het UMCG gevolgd. Wanneer DIF aanvullend wordt gebruikt naast de histopathologische criteria, om onderscheid te maken tussen OLP en OLL, worden er 3.26 meer patiënten met OLP gediagnosticeerd. Het gebruik van Levothyroxine is gevonden in 11.5% van de patienten met OLP of OLL. Conclusie: De Nederlandse ‘Richtlijn Lichen Planus’ wordt niet altijd volledig gevolgd door de clinici van het UMCG. Wanneer DIF aanvullend wordt gebruikt naast de ‘Nederlandse criteria’ om onderscheid te maken tussen OLP en OLL, worden er meer patiënten met OLP gediagnosticeerd. Wanneer het diagnostische proces in het UMCG wordt vergeleken met bovengenoemde diagnostische criteria, is er een dramatische verschuiving in de diagnose van OLP naar OLL. Een aanvullende bevinding was het hoge percentage patiënten met OLP of OLL die suppletie van schildklierhormoon gebruikten.

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Index Abstract ......................................................................................................................................................................... 2

Samenvatting ................................................................................................................................................................. 3

Index .............................................................................................................................................................................. 4

Introduction ................................................................................................................................................................... 5

Objectives ...................................................................................................................................................................... 9

Material and methods ................................................................................................................................................. 10

Results ......................................................................................................................................................................... 12

Discussion .................................................................................................................................................................... 16

Conclusion: .................................................................................................................................................................. 18

Tables:.......................................................................................................................................................................... 19

Figures: ........................................................................................................................................................................ 23

References: .................................................................................................................................................................. 25

Appendices .................................................................................................................................................................. 29

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Introduction Clinical aspects The typical clinical appearance of Oral Lichen Planus (OLP) is an intra oral bilateral lesion that is gray-white and has a slightly raised lace-like network (Striae of Wickham). This reticular type is often present at the buccal mucosa, lateral tongue or gingiva. Other clinical types of these lesions can be erythematous, atrophic/erosive, plaque like, ulcerative or bullous and multiple features can be present simultaneously. Less common locations in the oral cavity are the floor of mouth, the lips, pharynx and palate. In case of lesions without the typical OLP characteristics the Differential Diagnose (DD) can be quite extensive. The features are not pathognomic and can resemble several other oral diseases. This is due to the many different types of OLP that can coexist and blend with other diseases. The DD of OLP may consist of the following diseases depending on the clinical characteristics and the anamnesis (Table 1). The pain and number/size of oral lesions may exacerbate and go in remission from time to time. It is not unusual that patients are underdiagnosed (1,2). Etiology OLP is considered a chronic inflammatory condition of the oral mucosa and is assumed to be of autoimmune origin. There is substantial evidence that LP is immune mediated and that lymphocytes play an important role (3). Although the pathologic pathway is still unclear, it is probable that LP is a T-cel mediated auto-immune reaction in which (cytotoxic CD8+) T lymphocytes induce apoptosis of keratinocytes in the oral epithelium (4)(5)(6,7). Because Lichen Planus (LP) is found in the oral cavity, on the skin and at the genitalia it is likely that, at least, a part of the pathogenesis is systemic. Evidence that supports the theory of an auto-immune component is supported by the increased percentage of auto-antibodies against the thyroid gland and the high percentage of patients with concomitant OLP and thyroid diseases (8,9). Auto-antibodies against antigens in the basal membrane of the epithelium are found in patients with OLP and induce destruction of the epithelial attachment structures (10,11). Next to these auto-antibodies, there are several associations found with auto-immune diseases throughout the whole body in patients with OLP. Examples are Inflammatory Bowel disease, Coeliac disease, Vitiligo, Systemic Lupus Erythematosis, Rheumatoid arthritis and Thyroid diseases (3,8,9). It is believed that specific factors alter the function of the keratinocytes and the immune system, but which of the two is leading in the pathogenesis remains unclear. Although the etiology still remains unknown, there are indications for risk factors like stress, Hepatitis C virus infection and genetic factors which can trigger the path to OLP and Oral Lichen Like Lesions (OLL). Elevated stress levels and perception of stress are seen in patients with OLP. Whether the stress is the cause or the effect of the diseases is not clear (11,12). Several studies have found an association of OLP and Hepatitis C-virus (HCV) infections (13-16)(13-17). The relation is not yet completely understood and the incidence rates differ greatly from 0% to 32% of patients with OLP and a HCV infection. The effect of geographical differences seems to be great because of the differences found in several countries around the world (18,19). Especially in South-European, African and Asian countries the association is higher and thus screening for HCV infection in patients with

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OLP should be included in the diagnostic work-up. Screening in North-European countries, where the incidence of HCV infection is much lower, seems not indicated (20,21). A small role in the pathogenesis is probably played by a genetic component, although a significant relation with a single Human Leukocyte Antigen (HLA) has never been proven in families (22-24). Because of this lack of evidence for an immunopathogenetic pathway it is not likely that some people’s immune system is genetically destined to lead to OLP when certain stimuli are present. The major part of the pathogenesis is cell mediated by lymphocytes, cytokines which attract the lymphocytes to the peripheral blood vessels. The damage to the keratinocytes and the basement membrane is mostly driven by the cytotoxic T-lymphocytes. Exogenous agents induce OLP, but whether these agents influence the immune system or the keratinocyte function is not understood. Next to these exogenous agents associations with auto-immune diseases might be an indication that autoimmune factors initiate the cascade leading tot OLP (22,25). However the final parts of the whole cascade of developing OLP are for the greater part known, especially the ‘black box’ factors in the first stadium are still unclear. These different factors and probable combinations of factors might be the reason for the many different and often coexisting phenotypes of OLP and OLL (22). Epidemiology OLP is seen in all races and is present in 0.5-2.2% (mean 1.27%) of the adult population (26). However, there are demographic differences in the prevalence of the disease (27-30). The difference can be attributed to how the prevalence is measured (point, period or lifetime prevalence) and it is rather a raw estimate. In most populations and case control studies no difference was made between OLP and OLL, which are considered to have a different pathogenesis. OLP is mostly seen in patients from 30-70 years and can have a chronic presence or spontaneously disappear after several years (17). The male-female ratio for OLP is around 1:2, but there is little evidence about its exact ratio. Most papers report a tendency that OLP occurs more often in women (30). Long term follow-up studies were performed with the aim to investigate the possible premalignant character of OLP. There is evidence that the chance of developing a malignancy is higher in patients with OLP than in people without OLP. Malignant transformation might be seen in all types of OLP and the chance of malignant transformation is 0.4% per year higher than in patients without OLP. The location of the malignancy is not related to the location of the OLP lesions and that’s why the WHO proposed ‘possible premalignant condition’ as terminology in 2005. This is still controversial because there is evidence that OLL is premalignant, while OLP is not (31). Histopathological aspects Histopathological tests are used to exclude the possibility of malignancy. Next to this, there are several histopathological alterations coinciding in patients with OLP lesions (32). - Thickened ortho-or parakeratinized layer in sites with normally keratinized - Presence of Civatte bodies in basal layer, epithelium and superficial part of the connective tissue. - Presence of a well-defined bandlike zone of cellular infiltration that is confined to the superficial part of the connective tissue, consisting mainly of lymphocytes.

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- Signs of ‘liquefaction degeneration’ in the basal cell layer. - Acanthosis of the stratum spinosum. - Signs of epithelial atrophy - Presence of saw-toothed rete pegs. Not all of these aspects are required to be present in histological slides in order to ascertain the diagnosis OLP. Specific features should be present, but there is no consensus in the minimum of characteristics that defines OLP (33). The workgroup premalignant lesions of the WHO stated in 1978 that thickened parakeratinized layer, presence of Civatte bodies, bandlike lymphocytic infiltration and liquefaction of the the basal cell layer were the four essential criteria for ascertaining the diagnosis of OLP (34). The Dutch ‘Guideline Lichen Planus’ uses the same set histopathological criteria as proposed by the WHO in 1978. In 2003 a modified set of criteria was proposed in which bandlike lymphocytic infiltration, liquefaction of the basal cell layer and an absence of epithelial dysplasia were required to ascertain the diagnosis of OLP (35). Immunofluorescence Immunofluorescence (IF) has been suggested to have an additional value to the histopathological assessment and has been suggested to be decisive in unwinding the DD of OLP. IF has the ability to display the presence of various disease specific epitopes by using fluorescence labeled antibodies in tissue samples and in serum and may distinguish diseases like Pemfigus Vulgaris, Mucous Membrane Pemfigoid, Lupus Erythematosis and Linear IgA dermatose (2,36). Furthermore, when a specific pattern of anti-fibrinogen deposits is seen at the basement membrane zone (BMZ) displayed by immune fluorescence, it strongly supports the diagnosis of OLP or OLL. This characteristic can be seen in combination with cytoid bodies and immunoglobulins (37-39). The results of the Direct Immunofluoresence (DIF) testing are not enough to establish a diagnosis. Like the criteria in histopathological tests, these features are often characteristic, but not pathognomonic, and often IF is used to exclude other disease. The clinical, anamnestic, pathological and immunofluorescence information must be assessed and combined in order to establish a reliable diagnosis (40). Not all evidence supports the additional value of IF. There is a wide range in sensitivity results from 37-97% of DIF in patients with OLP. The sensitivity changes for different biopsy locations, but is not unequivocal (36-39,41-43). In 2012 the ‘Guideline Lichen Planus’ was developed in the Netherlands for caregivers. This guideline questions the value of IF in diagnosing OLP when Pemfigus Vulgaris, Mucous Membrane Pemfigoid or linear IgA dermatose are not in the DD (2). Diagnosis of OLP and OLL Because of the widespread clinical features in which oral lichenoid lesions can appear, the diagnosis of OLP is clinically often difficult to be established. Currently the diagnosis of OLP and OLL is based on clinical characteristics or on a combination of clinical characteristics and histopathological features as suggested by WHO (34). Although the criteria are used as a basis for the vast majority of the clinical research, the criteria have never been validated (35). The ‘Guideline Lichen Planus’ uses the set of histopathological criteria proposed by WHO. For the clinical criteria it acknowledges the bilateral presence of lesions and not just the presence of ‘Striae of Wickham’ as a minimal criterion for OLP. For scientific purposes,

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histopathological confirmation is considered a golden standard and is recommended for excluding possible malignancy (44). Unfortunately the histopathological assessment is not consistent among pathologists and different criteria for OLP are used all over the world (33,45,46). This is similarly for the clinical diagnosis of OLP and OLL (47). In some studies no distinction is made between OLP and OLL (30). Aim and outline of the present study The aims of this study is are 1) to determine if the diagnostic Dutch ‘Guideline Lichen Planus’ is followed by the department of Oral and Maxillofacial Surgery in the UMCG and 2) to identify the additional value of DIF in the diagnosis of OLP/OLL lesions. At the University Medical Center Groningen (UMCG), patients with OLP are usually diagnosed at the Department of Oral & Maxillofacial Surgery. Routinely, the medical history is noted and a clinical assessment is made of the oral lesions. In some cases the diagnosis is made solely at by the anamnesis and clinical features. Most cases undergo additional biopsies of the lesions. Routinely, when a biopsy is taken the sample is cut in half and one part is sent for histopathologal analysis and the other for IF. Therefore a unique database exists at the UMCG, that allows comparison of clinical, histopathological and IF data.

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Objectives I. The primary objective is to determine if the Dutch ‘Guideline Lichen Planus’ is followed by the department of Oral and Maxillofacial Surgery in the UMCG. II. Secondly we investigate the additional value of direct immunofluorescence (DIF) in the process of diagnosing OLP.

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Material and methods A retrospective database study was performed using medical records of patients referred to the Department of Oral & Maxillofacial Surgery of the UMCG. Patients with lesions of the oral mucosa where the DD included OLP/OLL and needed to be confirmed by histopathology were biopsied at the most representative area. Routinely, the sample tissue is cleaved in two parts. One part is submitted for routine H&E histopathological staining and examined by oral pathologists of the Department of Pathology, while (DIF) is performed on the other half by the Department of Dermatology. Tissue submitted for DIF is registered and given the so called STOrage IDentification number (STOID) thus allowing future retrieval of patients’ records. For this study medical records of patients were only included when Lichen Planus was in the DD and a sample, of the fresh frozen tissue was still available and was retrievable by STOID. It was checked whether the STOID number was linked correctly to the patient hospital IDnumber (UMCG number) and duplicates were excluded. In order to establish if the Dutch ‘Guideline Lichen Planus’ (2) for the diagnosis of OLP are followed in the normal clinical setting, we decided not to reevaluate the histological slides but to use the data as recorded in the patients file. Pathologists examined the histological slides and the pathology report contains a description of the microscopic findings as well as the most likely diagnosis or DD. In some cases the tissue was additionally stained with Periodic acid Schiff (PAS) for possible Candida infection. Patient characteristics were retrieved from the medical files (i.e. gender, age at the time of biopsy, use of medication) as well as characteristics of the oral lesions (clinical manifestations, experience of pain, location of lesions and location of biopsy, malignant transformation, final diagnosis) were retrieved from the medical files. The histopathological criteria for OLP in the pathology reports were noted. The WHO criteria (34) and the ‘Modified WHO criteria’ (35) were noted: - Thickened ortho or parakeratinized layer in sites with normally keratinized. - Presence of Civatte bodies in basal layer, epithelium and superficial part of the connective tissue. - Presence of a well-defined bandlike zone of cellular infiltration that is confined to the superficial part of the connective tissue, consisting mainly of lymphocytes. - Signs of ‘liquefaction degeneration’ in the basal cell layer. - Presence of epithelial dysplasia. Finally the diagnosis or the DD of the pathologist was noted. The second part of the material is analyzed in to the Department of Dermatology with DIF using the method described in Daniels et. al (36).The DIF results were scored on presence of a shaggy pattern of positive fluorescence of anti-fibrinogen at the epithelial basement membrane zone (EBMZ) and the amount of this fluorescence. The conclusion of the dermatologist who had performed the test was also scored. All scored criteria can be found in Appendix A.

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Statistical analysis: Statistical analyses were conducted with IBM SPSS Statistics 22 (SPSS, Chicago, Illinois, USA). In order to ascertain whether the different sets of criteria to distinguish between OLP and OLL are comparable, a Cochran’s Q test is conducted. To determine an association between clinical and histopathological variables, a Chi-square test was performed. P-values < 0.05 were noted as statistically significant.

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Results Clinical data After identifying 358 patients at the database with a STOID number, 115 patients were excluded because they were diagnosed with another disease than OLP or OLL. A total of 19 patients with unusable or incomplete histopathology reports and/or DIF results were excluded. 16 patients with a combination of OLP and other autoimmune diseases like Sjögren’s syndrome, Crohn’s disease, Systemic Lupus Erythematosus and Pemfigoid, were excluded. This led to a total of 208 patients included for this study (Figure 1). This number was assessed as sufficient for further analysis and therefore no samples were retrieved for repeating the histopathology or DIF. The analysis is performed on the 208 included patients. There are 61 male (29.3%) and 147 female (70.7%) patients (ratio 1:2.4) (Table 2). The mean age of these patients is 56.7 years old (median 56.0) with a standard deviation of 13.57 and a range of 9-88. The missing data is scored as unknown. 17.8% of the people used tobacco and 24.0% of these people used alcohol. 65.4% of the population used one or more types of medication. In 49.0 and 57.7% of the cases respectively, there were no records available about the use of tobacco and alcohol. Medical history was always noted. The type of medication used by the 136 patients was noted. The most common medication in this group was thyroid suppletion for hypothyroidism. Levothyroxine was used by 11.5% of the patients (n= 24). In the study of Vanderpump, the found prevalence of hypothyroidism was 0.2 – 7.0% (48). Symptoms 127 patients (61.1%) suffered from pain, which was chronic (i.e. more than 3 months) in 86 people (67.7%), acute (i.e. 3 months or less) in 31 people (24.4%) and. The duration of pain perception was constant in 21 patients (16.5%) and intermittent in 89 patients (70.1%). No information was found about the duration of the pain and the pain attacks in 10 (7.9%) and 17 (13.4%) patients, respectively. When patients complained of pain, most (n=94) had erosive lesions, combined with other type of lesions. Sixteen of these 94 patients had only erosive lesions. The reticular form alone was found in 53 patients and 25 (47.2%) of these patients experienced pain. Clinical subtyping and localization of the lesions The most frequent locations of the lesions were the mucosal part of the cheeks (n=166), the attached gingiva (n=92) and the lateral tongue (n=71). Lesions were predominantly present at multiple locations (n=130, 62.5%) in the mouth. The reticular type was reported the most often, followed by the erosive, ulcerative and hyperkeratotic. In 84 cases only one clinical type was seen, which was mainly the reticular form. The frequencies of multiple clinical types are shown in Table 3. When multiple types of OLP or OLL were present, the reticular and erosive types were most often observed (n=111; 89.5%). In four cases multiple types were present without the reticular form. In 182 patients (87.5%) the lesions were bilateral and in 26 (12.5%) unilateral (ratio 1:7). To differentiate between OLP and OLL according to the Dutch ‘Guideline Lichen Planus’

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(‘Dutch criteria), the ‘WHO criteria’ and the ‘Modified WHO criteria’, a bilateral, a reticular and a bilateral reticular pattern, respectively, must be clinically present in the oral cavity. A total of 182, 173 and 154 patients had lesions that met the clinical criteria for OLP according to the ‘Dutch criteria’, the ‘WHO criteria’ and the ‘Modified WHO criteria’, respectively. Additionally, 22, 30 and 46 patients, respectively, were diagnosed in the UMCG with OLP although they did not meet the clinical criteria. Malignant transformation Five out of 208 patients with OLP or OLL developed an oral squamous-cell carcinoma (SCC). One patient was already diagnosed with SCC at the first visit to the clinic together with OLP. All five patients were in the UMCG diagnosed with OLP. When the ‘Dutch criteria’, ‘WHO criteria’ or ‘Modified WHO criteria’ were used, all patients would have been diagnosed with OLL. SCC was found in four patients during recall appointments, after OLP or OLL was diagnosed (1.92%). On average, the patients with SCC were followed up for 58.7 months. Histopathological data In 58.2% of the patients a thickened parakeratinized layer was found. Civatte bodies were seen in 69.7%, bandlike lymphocytic infiltration in 67.8% and liquefaction of the basal cell layer in 57.2% of the patients. When patients without clinical characteristics for OLP, like unilateral lesions and without Striae of Wickham, were excluded, the aforementioned criteria were present in respectively 62.3%, 74.7%, 72.1% and 57.8% of the patients. 193 (92.8%) patients were diagnosed in the UMCG with OLP and 15 (7.2%) patients with OLL. All four histopathological WHO criteria were met in 48 patients with OLP as diagnosis. In the other 143 patients with OLP, three or less criteria were fulfilled. In 3 of these 143 patients, zero criteria were fulfilled. When the ‘Dutch criteria‘ were applied, 45 patients would be diagnosed with OLP and 163 with OLL (Table 4). This is 76.7% less patients with OLP compared with the diagnosis made by the clinicians in the UMCG. Next we assumed a patient positive for OLP if the lesions were clinically bilateral present in the oral cavity and if at least one of the following was present: 1. DIF, in case of presence of a shaggy pattern of positive fluorescence of anti-fibrinogen at the (EBMZ). 2. Four WHO histopathology criteria were met. Consequently 147 patients were diagnosed with OLP and 61 with OLL. Compared with the UMCG diagnosis the additional use of DIF leads to a decreased number of patients diagnosed with OLP by 23.9%. If the addition of DIF is compared with the original ‘Dutch criteria’, the number of patients diagnosed with OLP increases 3.26 times. In 38 (25.9%) of these patients with OLP, all three of the aforementioned criteria were met. DIF positivity was found independent to the number of histological criteria present. When the ‘WHO criteria‘ were applied, 45 patients would be diagnosed with OLP and 163 with OLL, which is 76.7% less patients with OLP compared with the diagnosis made by the clinicians in UMCG.

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Next we assumed a patient positive for OLP if the reticular type was clinically present in the oral cavity and if at least one of the following was present: 1. DIF, in case of presence of a shaggy pattern of positive fluorescence of anti-fibrinogen at the (EBMZ). 2. Four WHO histopathology criteria were met. Consequently 135 patients were diagnosed with OLP and 73 with OLL. Compared with the UMCG diagnosis the additional use of DIF leads to a decreased number of patients diagnosed with OLP by 30.1%. If the addition of DIF is compared with the original ‘WHO criteria’, the number of patients diagnosed with OLP increases 3 times. In 38 (28.1%) of these patients with OLP, all three of the aforementioned criteria were met. When the ‘Modified WHO criteria’ were used to distinguish between OLP and OLL, 70 patients would be diagnosed with OLP, leading to 63.73% less patients with OLP compared with the diagnosis established in the UMCG. Next we assumed a patient positive for OLP if the reticular type was clinically bilateral present in the oral cavity, epithelial dysplasia was absent on histopathological slides and if at least one of the following was present: 1. DIF was evaluated positive for the presence of a shaggy pattern of positive fluorescence of anti-fibrinogen at the (EBMZ). 2. Presence of bandlike lymphocytic infiltration and liquefaction of the basal cell layer in histopathological tests. Consequently 124 patients were now diagnosed with OLP and 104 with OLL. Compared with the UMCG diagnosis the additional use of DIF leads to a decreased number of patients diagnosed with OLP by 35.8%. If the addition of DIF is compared with the original ‘Modified WHO criteria’, the number of patients diagnosed with OLP increases 1.77 times. In 53 (42.7%) of these patients with OLP, all four of the aforementioned criteria were met. Again, DIF positivity was found independent to the number of histological criteria present. The proportions between OLP and OLL are statistically significant different (p < 0.05) when the UMCG diagnosis, ‘Dutch criteria’, ‘WHO criteria’ and ‘Modified WHO criteria’ are compared with each other (Figure 2). Only the ‘Dutch criteria’ and the ‘WHO criteria’ are comparable. The presence of certain histological characteristics in specific types of OLP is tested in the 84 patients who manifested only one type of OLP. The reticular (n=53) and erosive (n= 26) types of OLP were significantly associated with parakeratosis, (p = .003 and .031, respectively). For the absence of neutrophil granulocytes and granulocytes the p values were respectively .001 and .001 for the reticular type and for the erosive type .039 and .020. For the other types the sample size was too small to go get reliable results.

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Immunofluorescence data The presence of a shaggy pattern of positive fluorescence of anti-fibrinogen at the epithelial basement membrane zone (EBMZ) in OLP is highly variable because of the different criteria that can be used in differentiating OLP from OLL. Positive DIF was observed in 77-84% of the patients diagnosed with OLP when different sets of criteria were used (Table 4). Positive DIF was observed in 53-72% of the patients diagnosed with OLL when different sets of criteria were used. Patients diagnosed with OLP had the highest percentage of positive DIF, when the ‘Dutch criteria’ or the ‘WHO criteria’ were applied.

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Discussion

I. The Dutch ‘Guideline Lichen Planus’ is not always correctly followed by the clinicians in the UMCG. When these criteria for OLP were used, many patients would have been diagnosed with OLL instead of OLP. The diagnostic process of the clinicians is not retrievable in this retrospective research. It is unclear why the clinician diagnoses OLP or OLL, but it seems that it is mostly based on the conclusion of the pathologist, which is usually considered as ‘golden standard’. Officially the diagnosis of OLP can only be stated when all criteria are met. In this sample only 48 of the 193 patients with OLP, as confirmed by histology, met the four histological criteria that were scored. It seems that pathologists often arbitrary combines the different characteristics found in the biopsy with their own experience. This might explain why there exist differences between pathologists (45,49). To be unequivocal in the differentiating between OLP and OLL, the clinicians and the pathologists in the UMCG should follow the ‘Guideline Lichen Planus’ more strictly. II. It is not possible to determine the additional value of DIF. The sensitivity of DIF in diagnosing OLP is variable because of the different sets of criteria based on whom OLP is differentiated from OLL. The number of cases in which the DIF test is positive and OLP or OLL is diagnosed is variable. This changes when different criteria are used to differentiate between OLP and OLL. When DIF is positive and the clinical appearance is congruent with OLP, it is likely that the patient can be diagnosed with OLP. Still it is hard to say whether this is reliable. The percentage of positive DIF in cases with OLP is higher when the ‘Dutch criteria’, ‘WHO criteria’ and ‘Modified WHO criteria’ are used, compared with the UMCG diagnose. Still the same pattern is seen with the OLL cases and positive DIF. Apparently there are cases in which the DIF test is positive, but sufficient evidence to diagnose OLP is absent. Whether patients are diagnosed with OLP or OLL depends on the different criteria that are used in differentiating between OLP and OLL. The definition of OLP and OLL is not widely accepted and the terms are used interchangeably. The WHO criteria from 1978 have been tested on inter- and intraobserver variability and were found unreliable (35). Several other criteria have been developed to assist the diagnosis of OLP and OLL (50). Distinguishing OLP and OLL is difficult, because there are no specific clinical and histopathological characteristics in which OLP differs from OLL. When the suspected lesions appear in the area of restorations they are usually diagnosed as OLL (51). To be scientific reliable, the diagnosis of OLP should be verified by histopathological diagnostics. In 22 patients with OLP of our population, unilateral lesions were present, although it is known that OLP usually appears as bilateral and symmetrical lesions, in contrary to OLL which are found usually unilateral. This might imply that these patients were wrongly diagnosed with OLP instead of OLL (35). Following the ‘Dutch criteria’ and the ‘Modified WHO criteria’, a reticular pattern should be clinically present for the lesions to be called OLP. 39 patients (20.2%) had erosive or other clinical types of OLP without the reticular pattern. Following the criteria these patients should not have been diagnosed with OLP, but with OLL (34). The percentage of malignant transformation of patients with OLP or OLL is in this study is slightly higher than generally found. Whether these patients had OLP

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or OLL depends on the different criteria that are used in differentiate between OLP and OLL. Patients with OLL have an increased chance to evolve to malignancy (31). In this research the ‘Modified WHO criteria’ are used and it is unknown what the diagnosis would be if the ‘Dutch criteria’ or if the ‘WHO criteria’ were used. The used definitive diagnosis should not be the golden standard when seen from different points of view. With regard to the malignant transformation of OLP and OLL, the patients with a developed SCC should have been classified as OLL instead of OLP if the ‘Modified WHO criteria’ were used. It’s not fully illustrated why these specific histopathological features were used in composing the ‘Modified WHO criteria’ and why they are able to separate OLP lesions from an OLL. Whether the four histopathology criteria, proposed by Kramer et al. (34) in 1978, or the more recent criteria, proposed by van der Meij et al. (35) are followed, seems to be a matter of personal preference of clinicians. Among other things, in order to compare studies it is important that there are international accepted criteria for differentiating OLP from OLL and for the use of DIF. This needs to be discussed during a consensus meeting and accordingly processed in the guideline. Especially the way patients should be monitored for malignant transformation is of clinical relevance. From this study it becomes clear that the current diagnostic criteria are subjectively applied allowing substantial differences in diagnostic outcome. Whether OLP and OLL are truly different pathological entities cannot be reliably established by the existing diagnostic approaches. From this study it becomes clear that the current methods need to be supported by other diagnostic methods which reflect the pathological basis of the disease. Currently, the knowledge of the pathology of the disease is inadequate, limiting therefore progress in diagnostic possibilities (52). We observed a high use of Levothyroxine in patients with OLP or OLL. The reason of using Levothyroxine was always hypothyroidism, but the underlying mechanism as well as the daily dose could not be retrieved. At Lareb, the Dutch national centre where side effects of medication is recorded, one patient has been reported to manifest OLP while using Levothyroxine. In similar studies an increased number of patients with thyroid diseases and OLP were found (9,53-55). This finding supports the auto-immune background of OLP. Lo Muzio et al (9) advised to screen women over 40 years old with OLP for hypothyroidism. In our opinion, more research is required to elucidate whether Levothyroxine or hypothyroidism is associated with OLP and OLL patients, before concrete guidelines are applied. The age and gender distribution of the included patients is consistent to those found in similar studies. In many cases the use of tobacco and alcohol was not noted, so no reliable conclusions could be made. In contrary to other reports in the literature, patients with a reticular form of OLP or OLL often reported pain sensation (32,47). The reason for this discrepancy remains unclear, but since UMCG is reference center for oral diseases, it might be possible that more severe and complicated cases were referred. The presence of lesions in different locations in the oral cavity is rather comparable to the already existing literature (32,40). A relative higher amount of OLP and OLL was found on the attached gingiva. This is possible because lesions on the alveolar ridge of partially edentulous patients were counted as attached gingival.

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Conclusion: The Dutch ‘Guideline Lichen Planus’ is not always correctly followed by the clinicians in the UMCG. The addition of DIF to the original ‘Dutch criteria’ would diagnose OLP in more patients. There is a dramatic shift from diagnosis of OLP to OLL when other diagnostic criteria are applied than the diagnostic work-up used in the UMCG. An additional finding was a high percentage of patients using suppletion of thyroid hormone.

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Tables: Pemfigus Vulgaris (PV) Leukoedema Mucous Membrane Pemfigoid (MMP) Erythroplakia Erythema Exudativum Multiforme Graft Versus Host Disease Candidiasis Squamous cell carcinoma (SCC) Chronic Cheek biting/Morsicatio Candidiasis Discoid Lupus Erythematosis Lichenoid reaction (contactant or drug reaction) Systemic Lupus Erythematosis (SLE) White Sponge Nevus (Hairy) Leukoplakia Chronic Ulcerative Stomatitis.

Table 1 Differential Diagnose of OLP.

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Patients Data Total UMCG Dutch criteria WHO criteria Modified WHO OLP OLL OLP OLL OLP OLL OLP OLL

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total:

100%(208) 100(193) 100(15) 100(45) 163 45 163 70 138 Clinical: Male: %(n) 29(61) 30(57) 27(4) 31(14) 29(47) 29(13) 29(48) 33(23) 28(38) Female: %(n) 71(147) 70(136) 73(11) 69(31) 71(116) 71(32) 71(115) 67(47) 72(100) Lesions: Bilateral: %(n) 88(182) 89(171) 73(11) 100(45) 84(137) 89(40) 87(142) 100(70) 81(112)

Unilateral: %(n) 13(26) 11(22) 27(4) 0(0) 16(26) 11(5) 13(21) 0(0) 19(26) Location Cheeck: %(n) 75(156) 80(155) 73(11) 78(35) 80(131) 80(36) 80(130) 87(61) 76(105)

Attached gingiva: %(n) 44(92) 47(91) 7(1) 44(20) 44(72) 38(17) 46(75) 46(32) 43(60)

Lateral tongue: %(n) 34(71) 34(65) 40(6) 36(16) 34(55) 38(17) 33(54) 39(27) 32(44)

Floor of mouth: %(n) 8(16) 8(16) 0(0) 11(5) 7(11) 11(5) 7(11) 11(8) 6(8)

Lips: %(n) 8(16) 8(16) 0(0) 7(3) 8(13) 9(4) 7(12) 6(4) 9(12) Back tongue: %(n) 5(10) 5(9) 7(1) 9(4) 4(6) 9(4) 4(6) 9(6) 3(4) Tip tongue: %(n) 4(8) 4(7) 7(1) 2(1) 4(7) 2(1) 4(7) 1(1) 5(7) Soft palate: %(n) 4(8) 3(6) 13(2) 4(2) 4(6) 4(2) 4(6) 3(2) 4(6) Hard palate: %(n) 3(6) 3(5) 7(1) 2(1) 3(5) 2(1) 3(5) 1(1) 4(5) Table 2: Clinical data of OLP and OLL in different sets of criteria.

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Patients Data Total UMCG Dutch criteria WHO criteria Modified WHO OLP OLL OLP OLL OLP OLL OLP OLL

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Location Single: %(n) 38(78) 36(69) 60(9) 36(16) 38(62) 38(17) 38(61) 30(21) 41(57) Two: %(n) 32(67) 33(63) 27(4) 31(14) 33(53) 29(13) 33(54) 36(25) 31(42) Multiple: %(n) 30(63) 31(61) 13(2) 33(15) 29(48) 33(15) 29(48) 34(24) 28(39)

Pain <3months ago: %(n) 15(31) 15(29) 13(2) 2(1) 18(30) 2(1) 18(30) 9(6) 18(25)

>3 months ago: %(n) 41(86) 39(75) 74(11) 38(17) 42(69) 40(18) 42(68) 40(28) 42(58)

Clinical type Reticular: %(n) 83(173) 84(163) 67(10) 89(40) 82(133) 100(45) 79(128) 100(70) 75(103) Erosive: %(n) 67(140) 68(131) 60(9) 53(24) 71(116) 51(23) 72(117) 63(44) 70(96) Ulcerative: %(n) 6(13) 5(10) 20(3) 7(3) 6(10) 7(3) 6(10) 4(3) 7(10)

Hyperkeratotic: %(n) 6(13) 7(13) 0(0) 16(7) 4(6) 13(6) 4(7) 9(6) 5(7)

Number of Clinical types present One: %(n) 41(84) 39(75) 60(9) 47(21) 39(63) 40(18) 41(66) 33(23) 44(61) Two: %(n) 53(111) 55(106) 33(5) 42(19) 56(92) 49(22) 54(88) 57(40) 52(71) Three: %(n) 4(9) 4(8) 7(1) 7(3) 4(6) 7(3) 4(6) 7(5) 3(4) Four: %(n) 2(4) 2(4) 0(0) 4(2) 1(2) 4(2) 1(2) 3(2) 1(2) Table 3: Clinical data of OLP and OLL in different sets of criteria.

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Patients Data Total UMCG Dutch criteria WHO criteria Modified WHO OLP OLL OLP OLL OLP OLL OLP OLL

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Total in %(n)

Histopathological Lymphocytes: %(n) 96(200) 96(185) 100(15) 100(45) 94(153) 100(45) 94(153) 100(70) 93(128) characteristics Parakeratosis: %(n) 58(121) 60(115) 40(6) 100(45) 47(76) 100(45) 47(76) 63(44) 56(77) Civatte: %(n) 70(145) 72(138) 47(7) 100(45) 61(100) 100(45) 61(100) 89(62) 60(83) Liquefaction: %(n) 57(119) 60(115) 27(4) 100(45) 45(74) 100(45) 45(74) 100(70) 36(49) Bandlike inf: %(n) 68(141) 70(135) 40(6) 100(45) 59(96) 100(45) 59(96) 100(70) 51(71) dysplasia: %(n) 3(6) 3(6) 0(0) 2(1) 3(5) 2(1) 3(5) 0(0) 4(6)

Conclusion PA OLP: %(n) 65(136) 70(135) 7(1) 96(43) 57(93) 96(43) 57(93) 89(62) 54(74)

Conclusion PA possible OLP: %(n) 12(24) 10(20) 27(4) 4(2) 13(22) 4(2) 13(22) 6(4) 14(20)

Conclusion PA no LP: %(n) 23(48) 20(38) 67(10) 0(0) 29(48) 0(0) 29(48) 6(4) 32(44)

DIF α-fibrogen: %(n) 75(156) 77(148) 53(8) 84(38) 72(118) 84(38) 72(118) 81(57) 72(99) no fibrogen: %(n) 25(52) 23(45) 47(7) 16(7) 28(45) 16(7) 28(45) 19(13) 28(39) Table 4: Histopathological and direct immunofluorescene data of OLP and OLL in different sets of criteria.

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Figures:

Figure 1: Decision tree

23

Dermatology (n=852)

Patients with STOID (n=513)

Patients with UMCG number (n=506)

Patients without duplicates

(n=358)

Patients with OLP or OLL (n=243)

Patients with usable PA and IF (n=224)

Patients without concomitant

autoimmune diseases (n=208)

Excluded Concomitant

autoimmune diseases (n=16)

Excluded Unusable PA and/or IF

(n=19)

Excluded No OLP or OLL

(n=115) (n=115)

Excluded Duplicates (n=148)

Excluded Patients without UMCG

number (n=7)

Excluded Patients without STOID

(n=339)

Figure 2: Cochran’s Q test

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References: (1) Huber MA. Oral lichen planus. Quintessence Int 2004 Oct;35(9):731-752.

(2) Meijden van der WI, Burger MPM. Richtlijn Lichen Planus. 1st ed. Utrecht: Nederlandse Vereniging voor Dermatologie en Venereologie; 2012.

(3) Danielsson K, Ebrahimi M, Wahlin YB, Nylander K, Boldrup L. Increased levels of COX-2 in oral lichen planus supports an autoimmune cause of the disease. J Eur Acad Dermatol Venereol 2012 Nov;26(11):1415-1419.

(4) Sugerman PB, Satterwhite K, Bigby M. Autocytotoxic T-cell clones in lichen planus. Br J Dermatol 2000 Mar;142(3):449-456.

(5) Walton LJ, Thornhill MH, Farthing PM. VCAM-1 and ICAM-1 are expressed by Langerhans cells, macrophages and endothelial cells in oral lichen planus. J Oral Pathol Med 1994 Jul;23(6):262-268.

(6) Nickoloff BJ, Lewinsohn DM, Butcher EC. Enhanced binding of peripheral blood mononuclear leukocytes to gamma-interferon-treated cultured keratinocytes. Am J Dermatopathol 1987 Oct;9(5):413-418.

(7) Scully C, Beyli M, Ferreiro MC, Ficarra G, Gill Y, Griffiths M, et al. Update on oral lichen planus: etiopathogenesis and management. Crit Rev Oral Biol Med 1998;9(1):86-122.

(8) Cooper SM, Ali I, Baldo M, Wojnarowska F. The association of lichen sclerosus and erosive lichen planus of the vulva with autoimmune disease: a case-control study. Arch Dermatol 2008 Nov;144(11):1432-1435.

(9) Lo Muzio L, Santarelli A, Campisi G, Lacaita M, Favia G. Possible link between Hashimoto's thyroiditis and oral lichen planus: a novel association found. Clin Oral Investig 2013 Jan;17(1):333-336.

(10) Cooper SM, Dean D, Allen J, Kirtschig G, Wojnarowska F. Erosive lichen planus of the vulva: weak circulating basement membrane zone antibodies are present. Clin Exp Dermatol 2005 Sep;30(5):551-556.

(11) Shipman AR, Cooper S, Wojnarowska F. Autoreactivity to bullous pemphigoid 180: is this the link between subepidermal blistering diseases and oral lichen planus? Clin Exp Dermatol 2011 Apr;36(3):267-269.

(12) Allen CM, Beck FM, Rossie KM, Kaul TJ. Relation of stress and anxiety to oral lichen planus. Oral Surg Oral Med Oral Pathol 1986 Jan;61(1):44-46.

(13) Konidena A, Pavani BV. Hepatitis C virus infection in patients with oral lichen planus. Niger J Clin Pract 2011 Apr-Jun;14(2):228-231.

(14) Nagao Y, Sata M. A retrospective case-control study of hepatitis C virus infection and oral lichen planus in Japan: association study with mutations in the core and NS5A region of hepatitis C virus. BMC Gastroenterol 2012 Apr 10;12:31-230X-12-31.

25

(15) Halawani M. Hepatitis C virus genotypes among patients with lichen planus in the Kingdom of Saudi Arabia. Int J Dermatol 2014-02-01;53(2):171-177.

(16) Petti S, Rabiei M, De Luca M, Scully C. The magnitude of the association between hepatitis C virus infection and oral lichen planus: meta-analysis and case control study. Odontology 2011 Jul;99(2):168-178.

(17) Jaafari-Ashkavandi Z, Mardani M, Pardis S, Amanpour S. Oral mucocutaneous diseases: clinicopathologic analysis and malignant transformation. J Craniofac Surg 2011 May;22(3):949-951.

(18) Patil S, Khandelwal S, Rahman F, Kaswan S, Tipu S. Epidemiological relationship of oral lichen planus to hepatitis C virus in an Indian population. Oral Health Dent Manag 2012 Dec;11(4):199-205.

(19) Carrozzo M, Scally K. Oral manifestations of hepatitis C virus infection. World J Gastroenterol 2014 Jun 28;20(24):7534-7543.

(20) Lapane KL, Jakiche AF, Sugano D, Weng CS, Carey WD. Hepatitis C infection risk analysis: who should be screened? Comparison of multiple screening strategies based on the National Hepatitis Surveillance Program. Am J Gastroenterol 1998 Apr;93(4):591-596.

(21) Baccaglini L, Thongprasom K, Carrozzo M, Bigby M. Urban legends series: lichen planus. Oral Dis 2013-03-01;19(2):128-143.

(22) Lowe NJ, Cudworth AG, Woodrow JC. HL-A antigens in lichen planus. Br J Dermatol 1976 Aug;95(2):169-171.

(23) Porter SR, Kirby A, Olsen I, Barrett W. Immunologic aspects of dermal and oral lichen planus: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997 Mar;83(3):358-366.

(24) Bermejo-Fenoll A, Lopez-Jornet P. Familial oral lichen planus: presentation of six families. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006 Aug;102(2):e12-5.

(25) Sugerman PB, Rollason PA, Savage NW, Seymour GJ. Suppressor cell function in oral lichen planus. J Dent Res 1992 Dec;71(12):1916-1919.

(26) Carrozzo M. How common is oral lichen planus? Evid Based Dent 2008;9(4):112-113.

(27) Pindborg JJ, Mehta FS, Daftary DK, Gupta PC, Bhonsle RB. Prevalence of oral lichen planus among 7639 Indian villagers in Kerala, South India. Acta Derm Venereol 1972;52(3):216-220.

(28) Silverman S,Jr, Gorsky M, Lozada-Nur F. A prospective follow-up study of 570 patients with oral lichen planus: persistence, remission, and malignant association. Oral Surg Oral Med Oral Pathol 1985 Jul;60(1):30-34.

(29) Axell T, Rundquist L. Oral lichen planus--a demographic study. Community Dent Oral Epidemiol 1987 Feb;15(1):52-56.

(30) McCartan BE, Healy CM. The reported prevalence of oral lichen planus: a review and critique. J Oral Pathol Med 2008 Sep;37(8):447-453.

26

(31) van der Meij EH, Mast H, van der Waal I. The possible premalignant character of oral lichen planus and oral lichenoid lesions: a prospective five-year follow-up study of 192 patients. Oral Oncol 2007 Sep;43(8):742-748.

(32) Eisenberg E. Oral lichen planus: a benign lesion. J Oral Maxillofac Surg 2000 Nov;58(11):1278-1285.

(33) Au J, Patel D, Campbell JH. Oral Lichen Planus. Oral and Maxillofacial Surgery Clinics of North America 2013 2;25(1):93-100.

(34) Kramer IR, Lucas RB, Pindborg JJ, Sobin LH. Definition of leukoplakia and related lesions: an aid to studies on oral precancer. Oral Surg Oral Med Oral Pathol 1978 Oct;46(4):518-539.

(35) van der Meij EH, van der Waal I. Lack of clinicopathologic correlation in the diagnosis of oral lichen planus based on the presently available diagnostic criteria and suggestions for modifications. J Oral Pathol Med 2003 Oct;32(9):507-512.

(36) Daniels TE, Quadra-White C. Direct immunofluorescence in oral mucosal disease: a diagnostic analysis of 130 cases. Oral Surg Oral Med Oral Pathol 1981 Jan;51(1):38-47.

(37) Helander SD, Rogers RS,3rd. The sensitivity and specificity of direct immunofluorescence testing in disorders of mucous membranes. J Am Acad Dermatol 1994 Jan;30(1):65-75.

(38) Sano SM, Quarracino MC, Aguas SC, Gonzalez EJ, Harada L, Krupitzki H, et al. Sensitivity of direct immunofluorescence in oral diseases. Study of 125 cases. Med Oral Patol Oral Cir Bucal 2008 May 1;13(5):E287-91.

(39) Kulthanan K, Jiamton S, Varothai S, Pinkaew S, Sutthipinittharm P. Direct immunofluorescence study in patients with lichen planus. Int J Dermatol 2007 Dec;46(12):1237-1241.

(40) Eisen D. The clinical features, malignant potential, and systemic associations of oral lichen planus: a study of 723 patients. J Am Acad Dermatol 2002 Feb;46(2):207-214.

(41) Schiodt M, Holmstrup P, Dabelsteen E, Ullman S. Deposits of immunoglobulins, complement, and fibrinogen in oral lupus erythematosus, lichen planus, and leukoplakia. Oral Surg Oral Med Oral Pathol 1981 Jun;51(6):603-608.

(42) Laskaris G, Sklavounou A, Angelopoulos A. Direct immunofluorescence in oral lichen planus. Oral Surg Oral Med Oral Pathol 1982 May;53(5):483-487.

(43) Van Hale HM, Rogers RS,3rd. Immunopathology of oral mucosal inflammatory diseases. Dermatol Clin 1987 Oct;5(4):739-750.

(44) Eisenberg E, Krutchkoff DJ. Lichenoid lesions of oral mucosa. Diagnostic criteria and their importance in the alleged relationship to oral cancer. Oral Surg Oral Med Oral Pathol 1992 Jun;73(6):699-704.

(45) van der Meij EH, Reibel J, Slootweg PJ, van der Wal JE, de Jong WF, van der Waal I. Interobserver and intraobserver variability in the histologic assessment of oral lichen planus. J Oral Pathol Med 1999 Jul;28(6):274-277.

27

(46) Kamath VV, Setlur K, Yerlagudda K. Oral lichenoid lesions - a review and update. Indian J Dermatol 2015 Jan-Feb;60(1):102-5154.147830.

(47) van der Meij EH, Schepman KP, Plonait DR, Axell T, van der Waal I. Interobserver and intraobserver variability in the clinical assessment of oral lichen planus. J Oral Pathol Med 2002 Feb;31(2):95-98.

(48) Vanderpump MP. The epidemiology of thyroid disease. Br Med Bull 2011;99:39-51.

(49) Al-Hashimi I, Schifter M, Lockhart PB, Wray D, Brennan M, Migliorati CA, et al. Oral lichen planus and oral lichenoid lesions: diagnostic and therapeutic considerations. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007 Mar;103 Suppl:S25.e1-12.

(50) DeRossi SS, Ciarrocca KN. Lichen planus, lichenoid drug reactions, and lichenoid mucositis. Dent Clin North Am 2005 Jan;49(1):77-89, viii.

(51) Oliveira Alves MG, Almeida JD, Balducci I, Guimaraes Cabral LA. Oral lichen planus: A retrospective study of 110 Brazilian patients. BMC Res Notes 2010 Jun 3;3:157-0500-3-157.

(52) Farhi D, Dupin N. Pathophysiology, etiologic factors, and clinical management of oral lichen planus, part I: facts and controversies. Clin Dermatol 2010 Jan-Feb;28(1):100-108.

(53) Siponen M, Huuskonen L, Laara E, Salo T. Association of oral lichen planus with thyroid disease in a Finnish population: a retrospective case-control study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010 Sep;110(3):319-324.

(54) Robledo-Sierra J, Mattsson U, Jontell M. Use of systemic medication in patients with oral lichen planus - a possible association with hypothyroidism. Oral Dis 2013 Apr;19(3):313-319.

(55) Hirota SK, Moreno RA, dos Santos CH, Seo J, Migliari DA. Analysis of a possible association between oral lichen planus and drug intake. A controlled study. Med Oral Patol Oral Cir Bucal 2011 Sep 1;16(6):e750-6.

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Appendices Appendix A All scored criteria. UMCG Number Nagging pain Patient's gender Burning pain Patient's date of birth Stinging pain Date of bioption Irritating pain Patient's age at bioption Sensitive pain Use of tabacco For how long the patiënt feels pain Use of alcohol The duration of the pain Use of medication Localisation of the lesions Which type of medication Use of painmedication Use of Levothyroxine Does the patient describe pain as a complaint Lichen Planus is first in the DD of the bioption letter Thickened ortho or parakeratinized layer in sites with normally keratinized Presence of Civatte bodies in basal layer, epithelium and superficial part of the connective tissue Presence of a well-defined bandlike zone of cellular infiltration that is confined to the superficial part of the connective tissue, consisting mainly of lymphocytes Signs of ‘liquefaction degeneration’ in the basal cell layer Signs of dysplasia A shaggy patern of positive fluorescence with Presence of Lymfocytes antifibrinogen a the epithelial Presence of Plasmacells basement membrane zone (EMBZ) Presence of Neutrofile Granulocytes Degree of the shaggy patern Presence of Granulocytes The Diagnosis of immunofluorescence The Diagnosis of histopatology Appearance on lateral tongue Definitive diagnosis of LP Appearance on ventral tongue Reticular type of LP Appearance on tongue Erosive type of LP Appearance on the back of the tongue Plaque type of LP Appearance on the cheeks Bulleus type of LP Appearance on floor of the mouth Atrofic type of LP Appearance on the inside of the lips Hypertrofic type of LP Appearance on the hard palate Hyperkaratotic type of LP Appearance on the soft palate Ulcerative type of LP Appearance on attached gingiva Verruceus type of LP Appearance on pharynxbow

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