Virchows Arch (2007) 451:815–821 DOI 10.1007/s00428-007-0481-9
ORIGINAL ARTICLE
Ligneous conjunctivitis: a clinicopathological, immunohistochemical, and genetic study including the treatment of two sisters with multiorgan involvement M. Teresa Rodríguez-Ares & Ihab Abdulkader & Ana Blanco & Rosario Touriño-Peralba & Clara Ruiz-Ponte & Ana Vega & José Cameselle-Teijeiro
Received: 21 May 2007 / Revised: 7 July 2007 / Accepted: 18 July 2007 / Published online: 15 August 2007 # Springer-Verlag 2007
Abstract Ligneous conjunctivitis (LC) is a rare disease characterized by wood-like pseudomembranes developing on the ocular and extraocular mucosae secondary to plasminogen (PLG) deficiency. In this paper, we report two cases of LC in two sisters of 57 and 62 years of age that presented with recurrent, bilateral pseudomembranes on conjunctiva and a history of consanguinity and deafness. Pseudomembranes showed superficial and/or subepithelial deposits of eosinophilic amorphous hyaline, amyloid-like material with a variable proportion of granulation tissue, and inflammatory cells. The eosinophilic deposits were negative for Congo red stain, immunoreactive for fibrinogen, and consistently negative for amyloid A component, transthyretin, β2-microglobulin, albumin, fibronectin, collagen type IV, vimentin, and cytokeratins. Among inflammatory cells, a percentage of positivity of roughly 60% for lymphocytes T (CD3+) and 40% for lymphocytes B (CD8+), with a relation of cytotoxic/helper (CD8/4) T cells of 3:2, was found. In one case, nasal polyps and M. T. Rodríguez-Ares : R. Touriño-Peralba Anterior Segment Unit, Department of Ophthalmology, Hospital de Conxo-Complexo Hospitalario Universitario de Santiago, Galicia, Spain I. Abdulkader : J. Cameselle-Teijeiro (*) Department of Anatomic Pathology, Clinical University Hospital, SERGAS, University of Santiago de Compostela, Choupana s/n, 15706 Santiago de Compostela, Galicia, Spain e-mail:
[email protected] A. Blanco : C. Ruiz-Ponte : A. Vega Unidade de Medicina Molecular, Fundación Pública Galega de Medicina Xenómica, SERGAS, Grupo de Medicina Xenómica-University of Santiago de Compostela, Galicia, Spain
recurrent gastric peptic ulcer were also characterized by the same subepithelial hyaline deposits. A novel homozygous point mutation c.1856 C>T was found in exon 15 of the PLG gene in both patients. Amniotic membrane transplantation was done in one case with promising results. Keywords Ligneous conjunctivitis . Plasminogen gene . Nasal polyps . Gastric ulcer . Mutation analysis Introduction The term ligneous conjunctivitis (LC) was coined in 1933 by Borel [4] to describe a rare form of chronic, bilateral, recurrent conjunctivitis characterized by the development of firm fibrin-rich, woody-like pseudomembranous lesions mainly on the upper tarsal conjunctivae, and less frequently in the lower eyelid and the bulbar conjunctive [38]. LC was first described by Bouisson [5] as early as 1847, and the microscopic appearance of pseudomembranes presented as inflammatory fungus-shaped masses first reported in 1924 [19]. Because similar lesions can occur on other mucous surfaces indicating a systemic disease, Mingers et al. [23] proposed the term pseudomembranous disease for this entity; the same group [22] also demonstrated severe inherited type I plasminogen (PLG) deficiency as the cause of this disease in 1994. Familial occurrence was also reported [4, 38], and distinct homozygous and compound-heterozygous mutations in the PLG gene have been demonstrated by Schuster et al. [35, 39] and other groups since 1997. Extraocular lesions were reported occurring in the oral cavity (mainly gingiva) [14, 40], the upper gastrointestinal tract [38], the ear (including temporal bone) [7, 15], the nasopharynx, larynx, tracheobronchial tree, and lung [10,
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27], the female genital tract (sometimes with infertility) [8, 17, 28], and the renal collecting system [37]. A new syndrome or a coincidental association of non-Hodgkin’s lymphoma, auricular hypoplasia, and juvenile colloid milium with LC were recently noted [16]. In very rare cases, congenital occlusive hydrocephalus has been associated with the more severe forms of the disease and may even precede LC [1, 10, 36]. More recently, the report of a single patient with extensive woody deposits in her eyes, hydrocephalus, gingival hyperplasia, otitis media, sinusitis, and genital tract involvement exemplified the various manifestations of the pseudomembranous disease characteristic of severe PLG deficiency [21]. In 1998, it was shown that repeated infusions of PLG could resolve ligneous pseudomembranes [34], and effective treatment with topical PLG was reported in 2002 [47]. More recently, amniotic membrane transplantation has shown promising results in conjuntival reconstruction in difficult-to-manage cases of LC [2, 3]. In this paper, we report the clinicopathological, immunohistochemical, and genetic data of two sisters that presented with LC in adulthood, one of whom was treated with amniotic membrane transplantation.
Clinical history Two sisters of 57 (Case 1) and 62 (Case 2) years of age were referred to the Clinical University Hospital with a Fig. 1 LC. a–b A thick membrane covers the upper palpebral conjunctiva in both eyes (Case 1). c–d Postoperatively, amniotic membranes have disappeared, and there are no pseudomembranous lesions
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diagnosis of bilateral epidemic conjunctivitis and unsatisfactory responses to different treatments. Consanguinity and moderate deafness were found in both patients. In Case 2, there had been a previous history of deafness at age 32, recurrent gastric peptic ulcer at age 57, and recurrent nasal polyps at age 60. No other additional relevant data were found in the clinical records of the two patients. The patients showed abundant mucopurulent secretions with wood-like pseudomembranes on the upper and lower tarsal conjunctiva, with Staphilococcus epidermidis in culture. A putative diagnosis of LC was made based on recurrences during the previous 6 months to 1 year period (in both cases) and on the clinical appearance of the lesions (Fig. 1a and b). Surgical excision of pseudomembranes with a thorough cleaning of the surgical field using 5,000 IU/ml heparin eye drops was carried out. Pathological examination of the removed tissue supported a diagnosis of LC, and a PLG deficiency was confirmed with a level of 0.061 U/ml in Case 1 and of 0.045 U/ml in Case 2 (normal range, 0.80–1.20). After surgery, both patients underwent topic treatment with dexametasone at 0.1% every 4 h, cyclosporine A at 2% every 6 h, heparin 5% eye drops every 2 h, and ofloxacine every 6 h until complete epithelization was achieved. Unfortunately, topical PLG drops were unavailable. Six weeks later, both patients presented with a new recurrence of pseudomembranes, and similar surgical and medical treatment was undertaken. Twelve weeks later, the Case 1 patient presented again with serious lesions in tarsal and nasal conjunctiva of both eyes with no corneal involvement. Excision of all inflamed tissue
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together with amniotic membrane transplantation for conjunctival reconstruction under local anesthesia was decided upon. Previous to surgery, the ethics committee of our institution approved treatment, and the patient gave her written informed consent. The human amniotic membrane was prepared according to the method of Tseng et al. [46]. The corneal surface was covered by a therapeutic contact lens to prevent abrasion, and the amniotic membrane epithelium was secured up to the conjunctival edge with interrupted sutures. Perioperative topical treatment with intensive (every 30 to 60 min) heparin (5,000 U/ml) was carried out. The patient received dexamethasone and tobramicine eye drops four times daily for 6 weeks, heparin (5,000 U/ml) eye drops gradually tapered down from six times daily over 3 months, and topical cyclosporine A (2%) every 6 h daily for 6 weeks. Two months postoperatively, the amniotic membranes had dissolved, and the conjunctiva appeared free of pseudomembranous lesions (Fig. 1c and d). The patient has been examined monthly by the same ophthalmologist, and no recurrence of membranes has occurred in 39 months. Case 2 patient was lost on followup due to death caused by an ovarian poorly differentiated adenocarcinoma.
Materials and methods Histological, histochemical, and immunohistochemical analyses The surgical specimens were fixed in buffered formaldehyde and routinely embedded in paraffin. The 4-μm-thick sections were stained with hematoxylin and eosin and Congo red stain. Immunohistochemical studies were also performed on paraffin sections using a universal second antibody kit that utilized a peroxidase-conjugated labeleddextran polymer (Dako EnVision Peroxidase/DAB; Dako, Glostrup, Denmark), to avoid misinterpreting endogenous biotin or biotin-like activity in tissues [9] as positive staining. The following antibodies were used: amyloid A component (clone mc1, dilution 1:50, Novocastra, Newcastle upon Tyne, UK), transthyretin (polyclonal, 1:1,000, antigen retrieval water bath, Dako, Glostrup, Denmark), β2microglobulin (polyclonal, 1:2,000, Dako), fibrinogen (polyclonal, 1:200, microwave, Dako), albumin (polyclonal, 1:2,000, BioGenex, San Ramon, CA, USA), fibronectin (polyclonal, 1:10,000, water bath, Dako), collagen type IV (CIV, 1:10, microwave oven and EDTA), vimentin (V9, 1:5,000, microwave oven, BioGenex), cytokeratins (AE1– AE3, 1:20, water bath and proteinase K, Dako), immunoglobulin A (IgA; polyclonal, 1:10,000, microwave oven, Dako), IgD (polyclonal, 1:1,000, water bath, Dako), IgG
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(polyclonal, 1:10,000, microwave oven, Dako), IgM (R1/ 69, 1:500, microwave oven, Dako), light chains kappa (polyclonal, 1:50,000, water bath, Dako), light chains lambda (polyclonal, 1:50,000, water bath, Dako), CD3 (SP7, 1:50, water bath and EDTA, Master Diagnóstico, Granada, Spain), CD4 (4B12, 1:4, water bath and EDTA, Master Diagnóstico), CD8 (4B11, 1:20, water bath and EDTA, Novocastra), and CD20 (L26, 1:50, water bath, Dako). Negative control samples (the primary antibody was replaced by nonimmune mouse serum) and positive tissue samples were included in each slide run. Mutation analysis Informed consent was obtained for this study, which was conducted according to the Spanish law including adherence to the Helsinki Principles of 1975, as revised in 1983. For Case 1, genomic DNA was extracted from peripheral white blood cells by standard methods. For both cases (Case 1 and Case 2), to confirm our results, genomic DNA was extracted from paraffin-embedded tissue using QIAamp DNA Kit and QIAamp DNA Blood Mini Kit (QIAGEN: www.qiagen.com), following the manufacturer’s instructions. Thereafter, all coding regions and exon/intron boundaries of the PLG gene were amplified by polymerase chain reaction (PCR). To prevent the coamplification of homologous genes, we used primers and strategy described in Siboni et al. [41]. After the amplification, PCR products were purified. Both strands were subjected to cycle sequencing with the same PCR primers that covered the complete gene, both exons and introns, using the BigDye terminator kit and run in the 3730 xl DNA Analyser (Applied Biosystems, Foster City, CA, USA).
Results Pathological and immunohistochemical findings Histological examination revealed similar findings in both patients. Pseudomembranes showed superficial and/or subepithelial deposits of eosinophilic amorphous hyaline, amyloid-like material with a variable proportion of granulation tissue, and inflammatory cells, mainly plasma cells and lymphocytes (Fig. 2a). Ulceration and hyperplastic changes in the residual epithelium were observed. The eosinophilic deposits were negative for Congo red stain, immunoreactive for fibrinogen (Fig. 2b), and consistently negative for amyloid A component, transthyretin, β2microglobulin, albumin, fibronectin, collagen type IV, vimentin, and cytokeratins. A similar proportion of immu-
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Fig. 2 Ligneous disease (a–d). An inflammatory cell component with amyloid-like material is noted in the histological section of the conjunctiva (a). Immunohistochemical analysis of the deposits demonstrates positive localization of fibrinogen (b). Subepithelial deposits of amorphous eosinophilic material in nasal polyp (c) and in gastric mucosa (d)
noreactivity for light chains was found in plasma cells, but no stain was found in the hyaline material. Among inflammatory cells, a percentage of positivity of roughly 60% for lymphocytes T (CD3+) and 40% for lymphocytes B (CD8+), with a relation of cytotoxic/helper (CD8/4) T cells of 3:2, was found. Reevaluation of biopsies in Case 2 showed both exuberant granulation tissue and subepithelial fibrin deposits in gastric biopsies (Fig. 2d) and in metastatic vaginal lesions from the ovarian adenocarcinoma. The nasal polyps were also composed of subepithelial deposits of amorphous eosinophilic material instead of the loose mucoid stroma characteristic of nasal inflammatory polyps (Fig. 2c).
cleavage site that is necessary for the conversion of PLG to plasmin [29]. The same mutation was also present in homozygous state on the paraffin samples of the patient and her deceased sister.
Genetic analysis Direct sequencing of both single strands of PLG exon 15 from patient 1 showed a homozygous point mutation c.1856 C>T (using GenBank X05199 as reference sequence and starting with +1 at the A of the ATG translation initiation codon), which leads to an amino acid change ACT (Thr) to ATT (Ile) at position 600: p.Thr600Ile (the amino acid residues are numbered starting with the aminoterminal glutamic acid residue of the mature PLG as number +1) (Fig. 3). This mutation lies in the beta chain of PLG, 39 amino acids downstream of the Arg561-Val562
Fig. 3 Electropherogram of the direct sequence of exon 5 PLG gene. Homozygous novel germline mutation (c.1856 C>T) in DNA from patient’s blood sample (top). Wild-type sequence corresponding to a healthy control DNA (bottom)
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Discussion According to the excellent revision of Shuster and Seregard [38], the diagnosis of LC is based on the clinical picture (pseudomembranous lesions), the typical histological findings, and eventually on a positive family history. All of these criteria were present in the two patients here presented, with some unusual features that deserve consideration. The prevalence of LC may be roughly estimated in the range of 1.6 per 1 million people [38], at least in Europe, but in countries where consanguinity is more frequent, the prevalence of LC may be higher [10]. Although most cases of LC are sporadic, some familial cases have been reported with an autosomal-recessive pattern [4, 38]. In most cases, infants and children are affected, but late onset manifestation of LC may occur, as in our patients, ranging from birth to 85 years [18, 38]. Both eyes are affected in up to 51% of cases, and corneal involvement, a complication that may lead to blindness, occurs in 20–30% of cases [38]. Systemic signs and symptoms may precede or concomitantly occur with the ocular disease. Histopathological findings of the ocular and extraocular lesions in LC are identical. The excessive depositions of fibrinogen in LC should be differentiated from very rare cases of amyloidosis of the eye [20]. While amyloid deposits are positive with the Congo red stain, fibrinogen is negative. It must be noted that, in some extraocular biopsies, the nature of the disease may be overlooked by the pathologist, particularly if there is no clinical suspicion of LC, as occurred in our Case 2. Immunohistochemical studies for fibrinogen can definitively confirm the nature of the eosinophilic material in LC as occurred in our biopsies. PLG plays an important role in fibrinolysis and wound healing. In patients with LC, wound healing in mucous surfaces is impaired and seems arrested at the stage of granulation tissue [30, 38]. Histopathological and immunohistochemical findings indicate that fibrinogen is the major constituent of the membranes in LC. As was referred to in the literature, and as we confirmed in our patients, severe type I PLG deficiency (hypoplasminogenemia) is a major cause of LC [23, 24, 38]. The observation of cases of LC developed during treatment with tranexamic acid, an antifibrinolytic drug, also supports this idea [12]. Several mutations have been detected in the PLG gene of patients affected with LC [10, 33, 38, 43, 45]. PLG is the proenzyme of the serine protease plasmin and is a 791amino-acid residue single-chain glycoprotein with a molecular weight of 93 kDa [26]. Its gene spans approximately 52.5 kb on chromosome 6q26–27 and consists of 19 exons [11]. The proteolytic activity of plasmin contributes to the digestion of the insoluble fibrin clot and thrombi by which normal recanalization and tissue repair can be accomplished
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[31]. The mutation p.Thr600Ile of the PLG gene is very close to His-603, one of the three amino acid residues that constitute the serine protease triad. Moreover, p.Thr600Ile is next to p.Ala601Thr, a mutation present in 2–4% of Japanese subjects that predisposes them to thrombophilia and is known as Plasminogen Tochigi [25]. But, as stated for ligneous conjunctivitis [35], the mutation found in our patients does not predispose them to thromboembolic episodes [6]. In these patients, however, the risk of thrombotic occlusions of implanted catheters may be markedly increased [21, 38]. To the best of our knowledge, this is the first report of this mutation described in patients with LC. LC has also been reported in different animal species [38]; mice in which both PLG genes have been knocked out (PLG-deficient mice, Plg−/− mice) will develop conjunctival lesions indistinguishable from human lesions [13]. The clinical phenotype in Plg−/− was reported to differ from that in humans with LC, in that, in humans, gastric, colon, and rectal ulcers, rectal polyps, and liver involvement have not been reported so far. We believe, however, that our Case 2 patient is, as far as we know, the first description of recurrent gastric ulcer and LC. Shuster and Seregard [38] and Tefs et al. [44] concluded that a potentially useful initial approach for LC is the topical application of a PLG concentrate, possibly in combination with a PLG activator (uPA or tPA), which may soften the pseudomembranes and facilitate removal. To prevent recurrence, manipulations of the conjunctiva should be kept to a minimum, and treatment with heparin and corticosteroids should be undertaken, sometimes combined with topical cyclosporine A [3, 38, 44]. Topical and subconjunctival fresh frozen plasma may also help in the prevention of membranes in susceptible PLG-deficient patients [42]. Therapy with intravenous infusion of lysplasminogen was favorable in one case [34], but impossible as a long-term solution due to the short half-life of the preparation, the risk of intravascular fibrinolysis activation, and the high costs [3]. In rare cases, LC may resolve itself spontaneously [38]. Amniotic membrane transplantation is effective in some corneal and conjunctival lesions because of production of antiangiogenic and antiinflammatory proteins; Barabino and Rolando [2, 3] has proposed this as a promising therapy for LC. Although other experimented researchers [44] have not found this procedure adequate, we have had no recurrences of pseudomembranes in our patient for 39 months after amniotic membrane transplantation nor did any appear in the patient reported by Barabino and Rolando [3] for at least 3 years. Oral contraceptive therapy deserves attention as an alternative therapy in selected cases of LC [32]. In summary, the present paper, describing two cases of LC shows that LC is a rare disease characterized by wood-
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like pseudomembranes developing on the ocular and extraocular mucosae secondary to PLG deficiency. Pseudomembranes are subepithelial deposits of amorphous eosinophilic material (fibrinogen) with a variable proportion of granulation tissue and inflammatory cells. The same subepithelial deposits occurred as nasal polyps and recurrent ulcers in gastric mucosa. A novel homozygous point mutation c.1856 C>T in exon 15 of the PLG gene was found in both patients, and amniotic membrane transplantation was done in one case with promising results.
Acknowledgment We thank Ms. Elena Couso Folgueiro for her technical assistance.
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