- Email: [email protected]
Usefulness of a direct immunofluorescence in the diagnosis of plaque type oral lichen planus
Accepted Manuscript Usefulness of a direct immunofluorescence in the diagnosis of plaque type oral lichen planus
Patricia Alejandra Masquijo-Bisio, Mariana Silvia Gandolfo, Alicia Keszler, Maria Elina Itoiz, María Luisa Paparella PII: DOI: Reference:
S1092-9134(17)30138-7 doi: 10.1016/j.anndiagpath.2017.05.008 YADPA 51177
To appear in: Revised date: Accepted date:
###REVISEDDATE### ###ACCEPTEDDATE###
Please cite this article as: Patricia Alejandra Masquijo-Bisio, Mariana Silvia Gandolfo, Alicia Keszler, Maria Elina Itoiz, María Luisa Paparella , Usefulness of a direct immunofluorescence in the diagnosis of plaque type oral lichen planus, (2017), doi: 10.1016/j.anndiagpath.2017.05.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title Usefulness of a direct immunofluorescence in the diagnosis of plaque type oral lichen planus. Authors names and affiliations Patricia Alejandra Masquijo-Bisio1-2 [email protected]
T
Mariana Silvia Gandolfo1 [email protected]
IP
Alicia Keszler2 [email protected] María Luisa Paparella2 [email protected]
CR
Maria Elina Itoiz2 [email protected]
US
1. Cátedra de Clínica Estomatológica, Facultad de Odontología, Universidad de Buenos Aires, Argentina.
AN
2. Cátedra de Anatomía Patológica, Facultad de Odontología, Universidad de Buenos Aires, Argentina.
M
Corresponding author Patricia Masquijo Bisio
ED
Cátedras de Anatomía Patológica. Facultad de Odontología. Universidad de Buenos
M. T. de Alvear 2142 C1122AAH
PT
Aires.
Argentina.
CE
Ciudad Autónoma de Buenos Aires.
AC
[email protected]
1. Introduction
Oral lichen planus (OLP) is a chronic inflammatory condition of unknown etiology, affecting 0.5 to 2% of the world population (1). It most frequently affects women aged 40 to 60 years (2-7), though it can also present in young adults and children (8-11). Despite advances in the knowledge about the etiopathogenesis of OLP, there are no conclusive
ACCEPTED MANUSCRIPT studies on the etiological factors and triggering mechanisms involved in the disease. There is evidence that OLP is a T-lymphocyte (CD4+ and CD8+) mediated immunological process triggered by an unidentified antigen, that alters the basal keratinocytes of the oral mucosa, causing lesion and cell death by apoptosis (5, 6, 8, 9). Clinically, OLP can present in six patterns: reticular, papular, plaque-like, atrophic, erosive, or bullous (2, 5, 6, 8-14). The reticular pattern is the most frequent, followed by
T
the erosive and atrophic forms (5, 10-12, 14, 15). Plaque-like OLP is the most difficult
IP
form to differentiate from leukoplakia (2, 7, 9, 12, 13, 16).
There is consensus that diagnosis of OLP must be made based on the clinical and
CR
histopathological features of the lesion (2, 3, 7, 9, 22, 23). Accepted clinical criteria for diagnosis of OLP include the presence of mucosal lesions, usually multiple, and often,
US
though not always, symmetrically distributed. They commonly take the form of minute white papules which gradually enlarge and coalesce to form either a reticular, annular, or plaque pattern. A characteristic feature is the presence of slender white lines
AN
(Wickham’s striae) radiating from the papules. In the reticular form, there is a lacelike network of slightly raised gray-white lines, often interspersed with papules or rings (13).
M
Erosive, atrophic, bullous and plaque-type lesions are accepted only as an OLP subtype in the presence of reticular lesions elsewhere in the oral mucosa (22). Nevertheless, the
ED
reticular areas can be small and difficult to interpret upon clinical examination, and can go undetected when erosive, bullous or plaque-type lesions are present.
PT
The histopathologic criteria for OLP are: hyperkeratosis and parakeratosis of epithelium surface layers (13), sings of liquefactive degeneration in the basal cell layer, absence of
CE
epithelial dysplasia, and presence of a well-defined bandlike zone of cellular infiltration that is confined to the superficial part of the connective tissue, consisting mainly of
AC
lymphocytes (13, 22). Diagnosis can be difficult in some cases on account of the lack of clinicopathologic correlation in the diagnosis of OLP (2, 3, 7, 22-24). Leukoplakia is predominantly a white lesion on clinical examination, which cannot be clearly diagnosed as any other disease or disorder of the oral mucosa. Although there are no pathognomonic microscopic features, a biopsy is mandatory. Definitive diagnosis is made when no etiological cause other than tobacco/areca nut use has been identified, and histopathology does not confirm any other specific disorder (16). Oral lichen planus and leukoplakia are potentially malignant disorders of the oral mucosa (16). It is well accepted that the annual malignant transformation rate of leukoplakia ranges from 2 to 3% (17), whereas the potentially malignant character of OLP is still
ACCEPTED MANUSCRIPT under discussion, with most authors estimating the annual malignant transformation rate of OLP to be less than 0.7% (18-21). Discrepancy regarding OLP premalignancy may stem from the lack of clear clinical and histopathologic diagnostic criteria for OLP. Direct immunofluorescence (DIF) is widely used in immunological mucocutaneous disorders as a diagnostic tool (9, 25, 26). This technique allows demonstrating the presence of fibrinogen at the basement membrane zone (BMZ) in OLP biopsies.
T
Different case-series studies have reported 60 to 100% fibrinogen positive cases within
IP
their series (4, 25-31). There are no studies on the variations in fibrinogen deposition among the different patterns of OLP, and this could account for the variation among
CR
reports.
The aim of the present study was to analyze fibrinogen expression in plaque-like OLP
US
and homogenous leukoplakia, which are lesions that may pose problems for differential
AN
diagnosis.
2. Materials and Methods
M
The study included 85 biopsies of patients who had definitive diagnosis of OLP (reticular and plaque-like) and homogenous leukoplakia based on clinical findings and histological
ED
confirmation, according to World Health Organization (WHO) (13) and modified WHO diagnostic criteria (16, 22). Cases of oral lesions suspected of being associated with the
PT
use of drugs or with oral restorations (oral lichenoid lesions) and cases with doubtful diagnosis were excluded. Medical and dental clinical records and medication charts
CE
were reviewed. The study was approved by the Ethics Committee of the School of Dentistry of the University of Buenos Aires, Argentina (Res CD 325/02).
AC
Three groups were studied: reticular OLP (n=37), plaque-like OLP (n=30) and homogenous leukoplakia (n=18). The clinical data of the studied cases are shown in Table 1.
The biopsies were obtained with a 0.6 cm punch. In all cases of reticular OLP, they were taken from a white patch. As regards cases of plaque-like OLP, the biopsies were taken from a white patch region in 14 cases, from a white patch-plaque in 7 cases, and from a plaque in 9. In the group of homogenous leukoplakia, the biopsies were taken from a white patch in 7 cases and from a plaque in 11 cases. The specimens were divided in half. One half was routinely embedded in paraffin and stained with hematoxylin-eosin for histopathological diagnosis. The other half was embedded unfixed in CRYO-OCT
ACCEPTED MANUSCRIPT (Fisher Scientific) and sectioned in a cryostat for DIF analysis. Serial sections were incubated in a dark humid chamber, with fluorescein isocyanate-conjugated rabbit antihuman IgG, IgA, IgM, C3, and fibrinogen in a 1:50 dilution (DAKO). After incubation, the sections were rinsed in PBS buffer, counterstained with Evans blue, and mounted in glycerine-PBS. All batches included a recent positive biopsy as controlof the technique. The slides were observed under a fluorescence microscope (Axioskop 2MOT; Zeiss,
T
Germany) with a UV light source and 490 nm excitation and 525 nm emission filters.
IP
3. Results
CR
Fibrinogen deposition was found at the BMZ in 27 cases of reticular OLP (72.97%) and 17 cases of plaque-like OLP (56.66%). Deposition of fibrinogen showed thread-like
US
(66.66%), thread-like and linear (22.22%), and thread-like and band-like (11.11%) patterns in the reticular OLP group. Deposition of fibrinogen at the BMZ was observed in
AN
56.66% of cases in the plaque-like OLP group, and showed thread-like (58.82%), bandlike (11.76%), linear (11.76%), thread-like and band-like (11.76%), and thread-like and linear (5.88%) patterns. Deposition of C3 and IgM was also observed at the BMZ in
M
some cases of lichen planus. Immunostainings were negative in all cases of homogenous leukoplakia (Figure 1, Table 2). A statistically significant difference in the
ED
frequency of fibrinogen deposition was observed between plaque-like oral lichen planus
4. Discussion
PT
and homogenous leukoplakia. Pearson's Chi-Square 15.79; p (Pearson's) 0.0001.
CE
The importance of differential diagnosis between OLP and leukoplakia lies mainly in their different potential for malignant transformation, which in turn implies using different
AC
treatments and involves different follow up regimens accordingly. Although the WHO has established the necessary clinical and histological criteria for diagnosing OLP, clinicopathologic correlation is not always observed. Clinical manifestations of plaquelike OLP can be difficult to distinguish from leukoplakia when there is no conclusive histopathologic diagnosis. In such cases, DIF can be a valuable diagnostic adjunct. DIF is a useful, widely used procedure for diagnosing mucocutaneous diseases (9, 31), and shows fibrinogen deposition at the BMZ in OLP. According to reports in the literature, the positive yield of DIF in OLP is in the range of 60 to 100% of cases (4, 2531). It is of note, however, that the cited studies did not evaluate the possible differences among the clinical patterns of OLP. The results of the present study are in agreement
ACCEPTED MANUSCRIPT with reported findings on reticular OLP (73%), which is the most frequent clinical pattern. Fibrinogen deposition decreased to 57% in plaque-like OLP, and was negative in all cases of leukoplakia. This finding implies that fibrinogen detection in plaque-like OLP could yield false negative results but not false positive results. Daniels et al (26) found a threadlike pattern of fibrinogen deposition in 97% of OLP cases, and suggest that this pattern is sufficiently unique to be used as a diagnostic
T
criterion for OLP. Our results showed similar patterns of fibrinogen deposition in both
IP
reticular and plaque-like OLP, with the thread-like pattern being the most frequent in both types. It would seem that the diagnostic value of DIF lies in showing the presence
CR
of fibrinogen deposition, regardless of the fluorescence reaction pattern. In keeping with results reported in the literature (4, 26-28, 30), our results showed
US
deposition of IgM, and C3 to be less frequent. Nevertheless, the presence of these deposits could be useful to establish differential diagnosis from other autoimmune
AN
lesions.
We consider that DIF is a simple diagnostic adjunct, routinely available at histopathology
M
laboratories, that contributes greatly to the still difficult task of correctly diagnosing OLP.
ED
Aknowledgement
This work was supported in part by the grant from the UBACyT program, University of
CE
Conflict of interest
PT
Buenos Aires.
AC
The authors declare no conflicts of interest.
References
1. Di Stasio D, Guida A, Salerno C, et al. Oral lichen planus: a narrative review. Front Biosci (Elite Ed) 2014;6:370-6. 2. Al-Hashimi I, Schifter M, Lockhart PB, et al. Oral lichen planus and oral lichenoid lesions: diagnostic and therapeutic considerations. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103 (suppl.1):S25.e1-S25.e12.
ACCEPTED MANUSCRIPT 3. Eisen D, Carrozzo M, Bagan Sebastian JV, Thongprasom K. Number V Oral lichen planus: clinical features and management. Oral Dis 2005;11:338-49. 4. Kulthanan K, Jiamton S, Varothai S, Pinkaew S, Sutthipinittharm P. Direct immunofluorescence study in patients with lichen planus. Int J Dermatol 2007;46:1237-41. 5. Rodrigues Payeras M, Cherubini K, Figueiredo MA, GonçalvesSalum F. Oral
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lichen planus: focus on etiopathogenesis. Arch Oral Biol 2013;58:1057-69.
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6. Siar CH, Lim JSM, Tang SP, Chia HS, Loh YM, Ng KH. Identifying factors associated with diagnostic concordance/discordance in oral lichen planus. J Oral
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Maxillofac Surg 2013;71:1688-93.
7. Scully C, Carrozzo M. Oral mucosal disease: Lichen planus. Br J Oral Maxillofac
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Surg 2008;46:15-21.
8. Roopashree MR, Gondhalekar RV, Shashikanth MC, George J, Thippeswamy
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SH, Shukla A. Pathogenesis of oral lichen planus – a review. J Oral Pathol Med 2010;39:729-34.
9. Ismail SB, Kumar SKS, Zain RB. Oral lichen planus and lichenoid reactions:
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etiopathogenesis, diagnosis, management and malignant transformation. J Oral Sci 2007;49:89-106.
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10. Scully C, El-Kom M. Lichen planus: review and update on pathogenesis. J Oral Pathol 1985;14:431-58.
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11. Scully C, Beyli M, Ferreiro MC, et al. Update on oral lichen planus: etiopathogenesis and management. Crit Rev Oral Biol Med 1998;9:86-122.
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12. Andreasen JO. Oral lichen planus. 1 A clinical evaluation of 115 cases. Oral Surg Oral Med Oral Pathol 1968;25:31-42.
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13. WHO Collaborating Centre for Oral Precancerous Lesions. Definition of leukoplakia and related lesions: an aid to studies on oral precancer. Oral Surg Oral Med Oral Pathol 1978;46:518-39. 14. Ingafou M, Leao JC, Porter SR, Scully C. Oral lichen planus: a retrospective study of 690 British patients. Oral Dis 2006;12:463-8. 15. Bajaj DR, Khoso NA, Devrajani BR, Matlani BL, Lohana P. Oral lichen planus: a clinical study. J Coll Physicians Surg Pak 2010;20:154-7. 16. Warnakulasuriya S, Johnson NW, van der Waal I. Nomenclature and classification of potentially malignant disorders of the oral mucosa. J Oral Pathol Med 2007;36:575-80.
ACCEPTED MANUSCRIPT 17. Brouns EREA, Baart JA, Karagozoglu KH, Aartman IHA, Bloemena E, van der Waal I. Malignant transformation of oral leukoplakia in a well-defined cohort of 144 patients. Oral Dis 2014;20:e19-e24. 18. van der Waal I. Oral potentially malignant disorders: Is malignant transformation predictable and preventable?. Med Oral Patol Oral Cir Bucal 2014;19:e386-90. 19. Carbone M, Arduino PG, Carrozzo M et al. Course of oral lichen planus: a
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retrospective study of 808 northern Italian patients. Oral Dis 2009;15:235-43.
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20. Bombeccari GP, Guzzi G, Tettamanti M et al. Oral lichen planus and malignant transformation: a longitudinal cohort study. Oral Surg Oral Med Oral Pathol Oral
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Radiol Endod 2011;112:328-34.
21. Fitzpatrick SG, Hirsch SA, Gordon SC. The malignant transformation of oral
US
lichen planus and oral lichenoid lesions: a systematic review. J Am Dent Assoc 2014;145:45-56.
AN
22. 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;32:507-12.
M
23. Patel KJ, De Silva HL, Tong DC, Love RM. Concordance between clinical and
2011;69:125-33.
ED
histopathologic diagnoses of oral mucosal lesions. J Oral Maxillofac Surg
24. Rad M, Hashemipoor MA, Mojtahedi A, et al. Correlation between clinical and
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histopathologic diagnoses of oral lichen planus based on modified WHO diagnostic criteria. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
CE
2009;107:796-800.
25. Raghu AR, Nirmala NR, Sreekumaran N. Direct immunofluorescence in oral
AC
lichen planus and oral lichenoid reactions. Quintessence Int 2002;33:234-9. 26. Daniels TE, Quadra-White C. Direct immunofluorescence in oral mucosal disease: a diagnostic analysis of 130 cases. Oral Surg Oral Med Oral Pathol 1981;51:38-47. 27. Schiødt 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;51:603-8. 28. Rinaggio J, Crossland DM, Zeid MY. A determination of the range of oral conditions submitted for microscopic and direct immunofluorescence analysis. J Periodontol 2007;78:1904-10.
ACCEPTED MANUSCRIPT 29. Sano
SM,
Quarracino
MC,
Aguas
SC,
et
al.
Sensitivity
of
direct
immunofluorescence in oral diseases. Study of 125 cases. Med Oral Patol Oral Cir Bucal 2008;13:E287-91. 30. Laskaris G, Sklavounou A, Angelopoulos A. Direct immunofluorescence in oral lichen planus. Oral Surg Oral Med Oral Pathol 1982;53:483-7. 31. Firth NA, Rich AM, Radden BG, Reade PC. Assessment of the value of
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immunofluorescence microscopy in the diagnosis of oral mucosal lichen planus.
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CE
PT
ED
M
AN
US
CR
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J Oral Pathol Med 1990;19:295-7.
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US
CR
IP
T
ACCEPTED MANUSCRIPT
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Table 1: Clinical data of the studied cases
Leukoplakia
(n=37)
(n=30)
(n=18)
55.4 (27-83)
60.36 (19-81)
48.72 (30-76)
4.25:1
1.72:1
0.8:1
92 %
90 %
72 %
PT
F:M ratio
Plaque-like OLP
ED
Average age (range)
Reticular OLP
Localization in cheek
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mucosa (%)
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Table 2: Frequency of antibody deposition at the basement membrane zone IgG
IgA
IgM
Fibrinogeno
C3
Reticular OLP (n=37)
0
0
3 (8.1%)
27 (72.97%)
4 (10.81%)
Plaque-like OLP (n=30)
0
0
0
17(56.66%)*
4 (13.33%)
Leukoplakia (n=18)
0
0
0
0*
0
ACCEPTED MANUSCRIPT
Legend to figure 1 Fig.1: Plaque-type oral lichen planus (A, B, C) and homogenous leukoplakia (D, E, F). A and D. Cheek mucosa exhibiting white patch and plaque-type lesions. B and E. Microscopy. H-E. Mag Orig X400. C. Direct immunofluorescence; note the thread-like
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pattern of fibrinogen deposition at the basement membrane zone. F. No deposition in
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CE
PT
ED
M
AN
US
CR
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leukoplakia specimens. Counterstaining with Evans blue.
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AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
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PT
ED
Fig. 1
ACCEPTED MANUSCRIPT
Highlights
Plaque-like oral lichen planus and leukoplakia may pose problems for differential diagnosis
Oral lichen planus, but not leukoplakia, present subepithelial fibrinogen
IP
Fibrinogen demonstration is an routinely available technique at histopathology
CE
PT
ED
M
AN
US
CR
laboratories
AC
T
deposition
Patricia Alejandra Masquijo-Bisio, Mariana Silvia Gandolfo, Alicia Keszler, Maria Elina Itoiz, María Luisa Paparella PII: DOI: Reference:
S1092-9134(17)30138-7 doi: 10.1016/j.anndiagpath.2017.05.008 YADPA 51177
To appear in: Revised date: Accepted date:
###REVISEDDATE### ###ACCEPTEDDATE###
Please cite this article as: Patricia Alejandra Masquijo-Bisio, Mariana Silvia Gandolfo, Alicia Keszler, Maria Elina Itoiz, María Luisa Paparella , Usefulness of a direct immunofluorescence in the diagnosis of plaque type oral lichen planus, (2017), doi: 10.1016/j.anndiagpath.2017.05.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title Usefulness of a direct immunofluorescence in the diagnosis of plaque type oral lichen planus. Authors names and affiliations Patricia Alejandra Masquijo-Bisio1-2 [email protected]
T
Mariana Silvia Gandolfo1 [email protected]
IP
Alicia Keszler2 [email protected] María Luisa Paparella2 [email protected]
CR
Maria Elina Itoiz2 [email protected]
US
1. Cátedra de Clínica Estomatológica, Facultad de Odontología, Universidad de Buenos Aires, Argentina.
AN
2. Cátedra de Anatomía Patológica, Facultad de Odontología, Universidad de Buenos Aires, Argentina.
M
Corresponding author Patricia Masquijo Bisio
ED
Cátedras de Anatomía Patológica. Facultad de Odontología. Universidad de Buenos
M. T. de Alvear 2142 C1122AAH
PT
Aires.
Argentina.
CE
Ciudad Autónoma de Buenos Aires.
AC
[email protected]
1. Introduction
Oral lichen planus (OLP) is a chronic inflammatory condition of unknown etiology, affecting 0.5 to 2% of the world population (1). It most frequently affects women aged 40 to 60 years (2-7), though it can also present in young adults and children (8-11). Despite advances in the knowledge about the etiopathogenesis of OLP, there are no conclusive
ACCEPTED MANUSCRIPT studies on the etiological factors and triggering mechanisms involved in the disease. There is evidence that OLP is a T-lymphocyte (CD4+ and CD8+) mediated immunological process triggered by an unidentified antigen, that alters the basal keratinocytes of the oral mucosa, causing lesion and cell death by apoptosis (5, 6, 8, 9). Clinically, OLP can present in six patterns: reticular, papular, plaque-like, atrophic, erosive, or bullous (2, 5, 6, 8-14). The reticular pattern is the most frequent, followed by
T
the erosive and atrophic forms (5, 10-12, 14, 15). Plaque-like OLP is the most difficult
IP
form to differentiate from leukoplakia (2, 7, 9, 12, 13, 16).
There is consensus that diagnosis of OLP must be made based on the clinical and
CR
histopathological features of the lesion (2, 3, 7, 9, 22, 23). Accepted clinical criteria for diagnosis of OLP include the presence of mucosal lesions, usually multiple, and often,
US
though not always, symmetrically distributed. They commonly take the form of minute white papules which gradually enlarge and coalesce to form either a reticular, annular, or plaque pattern. A characteristic feature is the presence of slender white lines
AN
(Wickham’s striae) radiating from the papules. In the reticular form, there is a lacelike network of slightly raised gray-white lines, often interspersed with papules or rings (13).
M
Erosive, atrophic, bullous and plaque-type lesions are accepted only as an OLP subtype in the presence of reticular lesions elsewhere in the oral mucosa (22). Nevertheless, the
ED
reticular areas can be small and difficult to interpret upon clinical examination, and can go undetected when erosive, bullous or plaque-type lesions are present.
PT
The histopathologic criteria for OLP are: hyperkeratosis and parakeratosis of epithelium surface layers (13), sings of liquefactive degeneration in the basal cell layer, absence of
CE
epithelial dysplasia, and presence of a well-defined bandlike zone of cellular infiltration that is confined to the superficial part of the connective tissue, consisting mainly of
AC
lymphocytes (13, 22). Diagnosis can be difficult in some cases on account of the lack of clinicopathologic correlation in the diagnosis of OLP (2, 3, 7, 22-24). Leukoplakia is predominantly a white lesion on clinical examination, which cannot be clearly diagnosed as any other disease or disorder of the oral mucosa. Although there are no pathognomonic microscopic features, a biopsy is mandatory. Definitive diagnosis is made when no etiological cause other than tobacco/areca nut use has been identified, and histopathology does not confirm any other specific disorder (16). Oral lichen planus and leukoplakia are potentially malignant disorders of the oral mucosa (16). It is well accepted that the annual malignant transformation rate of leukoplakia ranges from 2 to 3% (17), whereas the potentially malignant character of OLP is still
ACCEPTED MANUSCRIPT under discussion, with most authors estimating the annual malignant transformation rate of OLP to be less than 0.7% (18-21). Discrepancy regarding OLP premalignancy may stem from the lack of clear clinical and histopathologic diagnostic criteria for OLP. Direct immunofluorescence (DIF) is widely used in immunological mucocutaneous disorders as a diagnostic tool (9, 25, 26). This technique allows demonstrating the presence of fibrinogen at the basement membrane zone (BMZ) in OLP biopsies.
T
Different case-series studies have reported 60 to 100% fibrinogen positive cases within
IP
their series (4, 25-31). There are no studies on the variations in fibrinogen deposition among the different patterns of OLP, and this could account for the variation among
CR
reports.
The aim of the present study was to analyze fibrinogen expression in plaque-like OLP
US
and homogenous leukoplakia, which are lesions that may pose problems for differential
AN
diagnosis.
2. Materials and Methods
M
The study included 85 biopsies of patients who had definitive diagnosis of OLP (reticular and plaque-like) and homogenous leukoplakia based on clinical findings and histological
ED
confirmation, according to World Health Organization (WHO) (13) and modified WHO diagnostic criteria (16, 22). Cases of oral lesions suspected of being associated with the
PT
use of drugs or with oral restorations (oral lichenoid lesions) and cases with doubtful diagnosis were excluded. Medical and dental clinical records and medication charts
CE
were reviewed. The study was approved by the Ethics Committee of the School of Dentistry of the University of Buenos Aires, Argentina (Res CD 325/02).
AC
Three groups were studied: reticular OLP (n=37), plaque-like OLP (n=30) and homogenous leukoplakia (n=18). The clinical data of the studied cases are shown in Table 1.
The biopsies were obtained with a 0.6 cm punch. In all cases of reticular OLP, they were taken from a white patch. As regards cases of plaque-like OLP, the biopsies were taken from a white patch region in 14 cases, from a white patch-plaque in 7 cases, and from a plaque in 9. In the group of homogenous leukoplakia, the biopsies were taken from a white patch in 7 cases and from a plaque in 11 cases. The specimens were divided in half. One half was routinely embedded in paraffin and stained with hematoxylin-eosin for histopathological diagnosis. The other half was embedded unfixed in CRYO-OCT
ACCEPTED MANUSCRIPT (Fisher Scientific) and sectioned in a cryostat for DIF analysis. Serial sections were incubated in a dark humid chamber, with fluorescein isocyanate-conjugated rabbit antihuman IgG, IgA, IgM, C3, and fibrinogen in a 1:50 dilution (DAKO). After incubation, the sections were rinsed in PBS buffer, counterstained with Evans blue, and mounted in glycerine-PBS. All batches included a recent positive biopsy as controlof the technique. The slides were observed under a fluorescence microscope (Axioskop 2MOT; Zeiss,
T
Germany) with a UV light source and 490 nm excitation and 525 nm emission filters.
IP
3. Results
CR
Fibrinogen deposition was found at the BMZ in 27 cases of reticular OLP (72.97%) and 17 cases of plaque-like OLP (56.66%). Deposition of fibrinogen showed thread-like
US
(66.66%), thread-like and linear (22.22%), and thread-like and band-like (11.11%) patterns in the reticular OLP group. Deposition of fibrinogen at the BMZ was observed in
AN
56.66% of cases in the plaque-like OLP group, and showed thread-like (58.82%), bandlike (11.76%), linear (11.76%), thread-like and band-like (11.76%), and thread-like and linear (5.88%) patterns. Deposition of C3 and IgM was also observed at the BMZ in
M
some cases of lichen planus. Immunostainings were negative in all cases of homogenous leukoplakia (Figure 1, Table 2). A statistically significant difference in the
ED
frequency of fibrinogen deposition was observed between plaque-like oral lichen planus
4. Discussion
PT
and homogenous leukoplakia. Pearson's Chi-Square 15.79; p (Pearson's) 0.0001.
CE
The importance of differential diagnosis between OLP and leukoplakia lies mainly in their different potential for malignant transformation, which in turn implies using different
AC
treatments and involves different follow up regimens accordingly. Although the WHO has established the necessary clinical and histological criteria for diagnosing OLP, clinicopathologic correlation is not always observed. Clinical manifestations of plaquelike OLP can be difficult to distinguish from leukoplakia when there is no conclusive histopathologic diagnosis. In such cases, DIF can be a valuable diagnostic adjunct. DIF is a useful, widely used procedure for diagnosing mucocutaneous diseases (9, 31), and shows fibrinogen deposition at the BMZ in OLP. According to reports in the literature, the positive yield of DIF in OLP is in the range of 60 to 100% of cases (4, 2531). It is of note, however, that the cited studies did not evaluate the possible differences among the clinical patterns of OLP. The results of the present study are in agreement
ACCEPTED MANUSCRIPT with reported findings on reticular OLP (73%), which is the most frequent clinical pattern. Fibrinogen deposition decreased to 57% in plaque-like OLP, and was negative in all cases of leukoplakia. This finding implies that fibrinogen detection in plaque-like OLP could yield false negative results but not false positive results. Daniels et al (26) found a threadlike pattern of fibrinogen deposition in 97% of OLP cases, and suggest that this pattern is sufficiently unique to be used as a diagnostic
T
criterion for OLP. Our results showed similar patterns of fibrinogen deposition in both
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reticular and plaque-like OLP, with the thread-like pattern being the most frequent in both types. It would seem that the diagnostic value of DIF lies in showing the presence
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of fibrinogen deposition, regardless of the fluorescence reaction pattern. In keeping with results reported in the literature (4, 26-28, 30), our results showed
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deposition of IgM, and C3 to be less frequent. Nevertheless, the presence of these deposits could be useful to establish differential diagnosis from other autoimmune
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lesions.
We consider that DIF is a simple diagnostic adjunct, routinely available at histopathology
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laboratories, that contributes greatly to the still difficult task of correctly diagnosing OLP.
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Aknowledgement
This work was supported in part by the grant from the UBACyT program, University of
CE
Conflict of interest
PT
Buenos Aires.
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The authors declare no conflicts of interest.
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Table 1: Clinical data of the studied cases
Leukoplakia
(n=37)
(n=30)
(n=18)
55.4 (27-83)
60.36 (19-81)
48.72 (30-76)
4.25:1
1.72:1
0.8:1
92 %
90 %
72 %
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F:M ratio
Plaque-like OLP
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Average age (range)
Reticular OLP
Localization in cheek
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mucosa (%)
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Table 2: Frequency of antibody deposition at the basement membrane zone IgG
IgA
IgM
Fibrinogeno
C3
Reticular OLP (n=37)
0
0
3 (8.1%)
27 (72.97%)
4 (10.81%)
Plaque-like OLP (n=30)
0
0
0
17(56.66%)*
4 (13.33%)
Leukoplakia (n=18)
0
0
0
0*
0
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Legend to figure 1 Fig.1: Plaque-type oral lichen planus (A, B, C) and homogenous leukoplakia (D, E, F). A and D. Cheek mucosa exhibiting white patch and plaque-type lesions. B and E. Microscopy. H-E. Mag Orig X400. C. Direct immunofluorescence; note the thread-like
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pattern of fibrinogen deposition at the basement membrane zone. F. No deposition in
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leukoplakia specimens. Counterstaining with Evans blue.
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Fig. 1
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Highlights
Plaque-like oral lichen planus and leukoplakia may pose problems for differential diagnosis
Oral lichen planus, but not leukoplakia, present subepithelial fibrinogen
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Fibrinogen demonstration is an routinely available technique at histopathology
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laboratories
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deposition