ⅠINTRODUCTION
Multiple oral ulcerations are sometimes serious complication to elderly peoples, deteriorating systemic condition due to infection, insufficiency of food intake, etc. The pathogenesis of oral ulceration is variable, including traumatism, superinfection, viral infection, autoimmune reaction, cancerous ulceration, drug-induced ulceration, etc.1). Therefore, intensive investigation is necessary to identify the ulcer etiology and to make a final differential diagnosis.
Oral aphtous ulcers associated with traumatism and superinfection most frequently occurred, but they are rapidly healed when the etiology factors are eliminated and patient’s general health becomes improved2). Multiple and recurrent oral ulcerations caused by viral infection are really serious, likely oral herpes simplex and herpes zoster which may undergo extensive vesicular ulcers3). For the diagnosis of viral infection the pathological examinations were performed through cytological observation, immunohistochemistry, and molecular genetic detection using PCR method.
Oral ulceration involved with autoimmune diseases such as pemphigus, pemphigoid, Behcet’s disease, etc., gradually progresses into severe painful ulcer depending on the immune hypersensitivity to specific antigens. These lesions are usually infiltrated with lots of lymphoid cells, which should be differentially diagnosed with immunological detection and could be treated with different anti-inflammatory agents4).
The cancerous ulceration which is gradually destroyed and aggravated by infiltrating tumor cells shows characteristic deep necrotic ulcer with marginal elevation and produces foul halitosis, while drug-induced ulceration is recently known to be occurred in elderly people who received multiple drugs for the treatment of hypertension, angina pectoris, myocardial infarction, etc.56). The pathogenetic mechanism of drug-induced ulceration is not clearly elucidated so far, but it is presumed that the oral mucosa keratinocytes which are active in their cellular turnover are vulnerable to the metabolic inhibitors such as calcium channel blockers7). However, as the present case showed acantholytic epithelial peeling off similar to Nikolsky sign of pemphigus/pemphigoid, the drug-induced pemphigoid was named and characterized by acantholytic erosive ulceration usually on the traumatic area of oral mucosa89). The drug-induced pemphigoid shows ambiguous clinical features of sudden and prolonged ulceration mimicking aphtous stomititis, herpes simplex, lichen planus, and even candidiasis due to the whitish epithelial peeling.
The present study demonstrated multiple oral mucosa ulcers clinically diagnosed as traumatic aphtous stomititis, candidiasis, lichen planus, and oral herpes simplex, but the lesions were not well responded with viral and fungal therapy and frequently recurred. With a series of negative confirmation for candidiasis, lichen planus, HSV infection, and autoimmune pemphigus/pemphigoid, the present case was finally diagnosed as drug-induced pemphigoid in the consideration of the patient’s medical history and literature review.
ⅡCASE REPORT
An 81-years-old woman presented multiple mucosa ulcers with a chief complaint of pain during wearing the lower denture. She had been wearing upper and lower complete dentures for five months. Pain had started three months ago. She received multiple drugs for the treatment of angina pectoris, constipation, neurosis, hypertension, and arthritis, but no history of immune-diseases and viral infection symptom. Those therapies might contain calcium channel blockers, furosemide, captopril, non-steroidal anti-inflammatory agents, and penicillamine.
On the clinical examination, there were multiple whitish lesions varying in size (2~5mm) on the right side of lower edentulous alveolar ridge, buccal mucosa and right ventral surface of tongue with small ulcer on right lingual side of lower posterior edentulous alveolar ridge (Fig. 1 A). Under the clinical diagnosis of denture traumatic ulcer the lower complete denture was relined with soft reliner, and the pain was much reduced but the ulcer lesions were not healed in two weeks. Therefore, the patient was referred to the Department of Oral Medicine. There, the lesions were treated with antifungal agent, but did not respond well, and followed by topical steroid therapy with 0.15% dexamethasone and lidocain gargle. And then the ulcer lesions increased in size (0.8 cm) with pseudo-necrotic floor and well defined margin in two months of follow-up check (Fig. 1 A2 and A3). Because the patient continuously complained of the unhealed ulcers which were gradually increased in size, an incisional biopsy was performed and the specimen was prepared for histological observation.
The biopsy specimen was fixed in 10% neutral buffered formalin, processed routinely, and embedded in paraffin. Histologic sections in 4 μm thickness were mounted on glass slides, routinely stained with hematoxylin and eosin and also stained with periodic acid Schiff (PAS) reaction for light microscopic examination. Sserial microsections were also prepared for IHC staining using antisera of IgG, E6, NFkB, p38, CD28, CD3, CD20 and IL28 (Santa Cruz Biotech. USA). IHC reaction protocols differed according to the target antigen and manufacturers’protocols. Briefly, after deparaffinization and rehydration of the tissue sections in xylene followed by ethanol, sections were incubated with 0.5% hydrogen peroxide in phosphatebuffered saline for 30 minutes. Primary anti-human (rabbit/mouse/goat) polyclonal antibodies were applied to each microsection using the triple sandwich indirect IHC methods10). The usage of biopsy specimens filed in the Department of Oral Pathology, GWNUDH was approved by our institutional review board (IRB2015-07).
The microsections showed a benign ulceration with pseudonerotic ulcer surface. The epithelium was partly keratinized, and gradually degenerated and ulcerated. In the high magnification the keratinocytes in suprabasal and spinous cell layers showed the beginning of acantholysis by separation of epithelial adhesion, and those in ulcer margin showed complete acantholysis, resulted in peeling off the epithelium (Fig. 1B). Thereby individual keratinocytes were scattered and destroyed, but epithelial regeneration for the ulcer wound healing was not found and much delayed.
The acantholysed keratinocytes still vivid with irregular plump nuclei, and did not easily undergo apoptosis. The nuclear matrices of the acantholysed keratinocytes were continuously hyperchromatic, but showed no feature of nuclear vacuolization and chromatin peripheralization as a sign of viral infection (Fig. 1 B5 and B6). Therefore, herpetic viral infection was not evident in the cytological observation.
In the histological observation the ulcer lesion showed no fungal hyphae in PAS stain, thereby oral candidiasis was easily ruled out. And also the characteristic features of lichen planus, i.e., epithelial degeneration accompanied with basement destruction by lymphocyte infiltration, were not found, so that oral lichen planus was also ruled out differentially.
In the IHC staining CD3 and CD28 were strongly positive in some lymphocytes in the subepithelial connective tissue (Fig. 1 C and D), but NFkB, and p38 were rarely/weakly positive in the ulcer lesion. These finding may indicate an immediate T cell mediated immune reaction rather than a chronic granulomatous inflammation. And the acantholytic epithelium was entirely negative for CD20, IgK, IL28 (Fig. 1 E), and E6 (human papilloma virus antigen), thus, this ulcer lesion was supposed to be far from autoimmune disease and viral infection.
In order to confirm HSV-1 infection, PCR was also performed using the biopsy specimen. Ten serial microsections were collected and deparaffinized, and followed by DNA extraction using DNA extraction kit (Bioneer Inc. Korea). The extracted DNA was explored with PCR method using primer sets (HSV-1 outer primers, forward; 5'-TGCTGGAGGATCACGAGTTT-3', reverse; 5'-CATCGTCTTTGTTGGGAACT-3', HSV-1 inner primers, forward; 5'-TGCAGAGCAACCCCATGAAG-3', reverse; 5'-ATGACCATGTCGGTGACCTT-3')1112,). However, the PCR showed no amplication of HSV-1 DNA. Therefore, it became clear that the present case was not relevant to HSV-1 infection.
Although the present lesion was primarily diagnosed as traumatic ulcer, candidiasis and lichen planus in the clinical observation, and then conservatively treated with denture relining, antifungal agent, and steroidal agent, the ulcer lesion was not healed for two months and rather increased in size. The histological, IHC, and PCR examinations provided no evidences for the diagnosis of candidiasis, lichen planus, autoimmune pemphigus/pemphigoid, and HSV infection differentially. With the patient’s medical history received multiple drug intake for the treatment of angina pectoris, constipation, neurosis, hypertension and arthritis the ulcer lesion was finally diagnosed as drug-induced pemphigoid which was not originated from infection, granulomatous and autoimmune inflammation but relevant to the acantholytic degeneration of epithelial cells caused by multiple drug abuse13). However, the present ulcer lesion was prolonged for three months, and gradually healed with careful conservative treatment of adhesive ointment.
ⅢDISCUSSION
It had been reported that the mucous membrane pemphigoid ulceration usually occurred in adult patients receiving different medical drugs, including anticonvulsant and mood-stabilizing agent (carbamazepine, tegretol14)), anti-inflammatory agent (sulphasalazine15)), antihypertensive agents (calcium channel blocker; nifedipine, atenolol7)), and anti-glaucoma agent (topical administration including 0.5% timolol maleate, 0.1% dipivefrine hydrocholoride, 0.1% fluorometholone, and 0.3% ofloxacin16)). The demonstration of biochemical acantholysis to nifedipine from in vitro cultured normal human skin explants study found a biochemical reaction that leads to desmosomal function loss, but this skin susceptibility to nifedipine may be genetically determined17).
In the present study the patient received different medical therapies for angina pectoris, constipation, neurosis, hypertension, and arthritis for more than ten years, using multiple drugs of calcium channel blockers, furosemide, captopril, nonsteroidal anti-inflammatory agents, and penicillamine. Although the causative drug for the oral pemphigoid ulceration could not be identified in this study, it was highly suspected that the multiple drug abuse might aggravate the mucosal ulceration via acantholytic desquamation of oral epithelium where the traumatic denture injury occurred. For the differential diagnosis of the drug-induced pemphigoid ulceration the present study performed a series of pathological examinations to rule out the diagnosis of candidiasis, lichen planus, and herpes simplex infection.
The candidiasis and lichen planus were easily ruled out in the histological observation due to the absence of fungal hyphae in PAS stain and no features of basement destruction accompanied by lymphocyte infiltration, respectively. In the high magnification the ulcerated mucosa showed severe acantholytic degeneration of keratinocytes exhibiting plump nuclei containing homogeneous dense chromatin. But the features of nuclear vacuolization and chromatin peripheralization, which are usually seen in viral infection, were not found in the acantholytic keratinocytes. These findings may reduce the presumption of oral herpes simplex infection. However, in order to rule out the HSV infection, PCR detection was performed using template DNAs extracted from the microsections and HSV-1 primer sets1118,), but resulted negative reaction. Therefore, it was confirmed that present oral ulceration was not related to the HSV-1 infection.
Although it was known that the anti-inflammatory agents, such as topical and systemic steroids (dexamethasone), combination of nicotinamide and tetracycline19), are effective in the treatment of bullous pemphigoid, the autoimmune pemphigus/pemphigoid eruption is still serious in patient management. And the drug-induced pemphigoid ulceration should be clearly distinguished from the autoimmune pemphigus/pemphigoid. Especially the present patient was once treated with dexamethasone ointment, because the ulcer lesion was prolonged and not healed in ordinary conservative treatment. The ulcer lesion was not responded to the steroid therapy, and rather it became more painful and its size was enlarged. But the ulcer margin was relatively clear and showed mild inflammatory reaction only with no deep granulomatous necrosis, where showed relatively weak/rare immunoreaction of CD20, IgK, NFkB, p38, and IL28. Therefore, the present case could be ruled out the diagnosis of traumatic aphtous ulcer and autoimmune disease.
Taken together, the present lesion was primarily diagnosed as traumatic ulcer, candidiasis, and lichen planus in the clinical observation, and conservatively treated with denture relining, antifungal agent, and steroidal agent. The ulcer lesion was not healed for two months of treatment and rather increased in size. With the possibility of viral infection, the IHC staining of IL28 and E6 were done, but showed almost negative reaction. More PCR detection also showed negative reaction. Therefore, the IHC findings exhibiting strong positive reaction of CD3 and CD2820), but rare/weak reaction of NFkB, CD20, IgK, and p38 might be supportive for the finally diagnosis of drug-induced pemphigoid ulceration together with the patient’s medical history of multiple drug intake for the treatment of angina pectoris, constipation, neurosis, hypertension, and arthritis.
Because the present study made a final diagnosis of drug-induced pemphigoid through negative confirmation of other differential diagnosis, i.e., traumatic aphtous ulcer, candidiasis, lichen planus, autoimmune pemphigus/ pemphigoid, and HSV infection, there might still remain a possible other pathogenesis evoking these multiple oral ulcers. Therefore, further investigation should be followed to elucidate the true pathogenesis of oral mucosa ulceration.
