Upregulated Expression of Activation-Induced Cytidine Deaminase in Ocular Adnexal Marginal Zone Lymphoma with IgG4-Positive Cells.

Immunoglobulin G4-related disease (IgG4-RD) is a systemic disorder characterized by tissue fibrosis and intense lymphoplasmacytic infiltration, causing progressive organ dysfunction. Activation-induced cytidine deaminase (AID), a deaminase normally expressed in activated B-cells in germinal centers, edits ribonucleotides to induce somatic hypermutation and class switching of immunoglobulin. While AID expression is strictly controlled under physiological conditions, chronic inflammation has been noted to induce its upregulation to propel oncogenesis. We examined AID expression in IgG4-related ophthalmic disease (IgG4-ROD; n = 16), marginal zone lymphoma with IgG4-positive cells (IgG4+ MZL; n = 11), and marginal zone lymphoma without IgG4-positive cells (IgG4- MZL; n = 12) of ocular adnexa using immunohistochemical staining. Immunohistochemistry revealed significantly higher AID-intensity index in IgG4-ROD and IgG4+ MZL than IgG4- MZL (p < 0.001 and = 0.001, respectively). The present results suggest that IgG4-RD has several specific causes of AID up-regulation in addition to inflammation, and AID may be a driver of oncogenesis in IgG4-ROD to IgG4+ MZL.

Sustained activation of ERK1/2 MAPK in Schwann cells causes corneal neurofibroma.

Recent studies have shown that constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in Schwann cells (SCs) increases myelin thickness in transgenic mice. In this secondary analysis, we report that these transgenic mice develop a postnatal corneal neurofibroma with the loss of corneal transparency by age six months. We show that expansion of non-myelinating SCs, under the control of activated ERK1/2, also drive myofibroblast differentiation that derives from both SC precursors and resident corneal keratocytes. Further, these mice also harbor activated mast cells in the central cornea, which contributes to pathological corneal neovascularization and fibrosis. This breach of corneal avascularity and immune status is associated with the growth of the tumor pannus, resulting in a corneal stroma that is nearly four times its normal size. In corneas with advanced disease, some axons became ectopically myelinated, and the disruption of Remak bundles is evident. To determine whether myofibroblast differentiation was linked to vimentin, we examined the levels and phosphorylation status of this fibrotic biomarker. Concomitant with the early upregulation of vimentin, a serine 38-phosphorylated isoform of vimentin (pSer38vim) increased in SCs, which was attributed primarily to the soluble fraction of protein-not the cytoskeletal portion. However, the overexpressed pSer38vim became predominantly cytoskeletal with the growth of the corneal tumor. Our findings demonstrate an unrecognized function of ERK1/2 in the maintenance of corneal homeostasis, wherein its over-activation in SCs promotes corneal neurofibromas. This study is also the first report of a genetically engineered mouse that spontaneously develops a corneal tumor.

Abrogating TNF-α expression prevents bystander destruction of normal tissues during iNOS-mediated elimination of intraocular tumors.

Although intraocular tumors reside in an immune privileged site, some tumors are rejected nonetheless. For example, intraocular adenovirus-induced (Ad5E1; adenovirus type 5 early region 1) tumors are rejected in syngeneic C57BL/6 mice by one of two pathways. One pathway leads to extensive necrosis of innocent bystander cells and culminates in destruction of the eye, a condition called phthisis. The second pathway is characterized by piecemeal tumor cell death that rids the eye of the tumor while preserving the architecture and function of the eye. To study the mechanisms of phthisical tumor rejection, we isolated a cell clone-designated clone 2.1 that consistently undergoes rejection in a phthisical manner. CD4(+) T cells and macrophages were required for phthisical rejection of intraocular clone 2.1 tumors and M1 macrophages were involved in mediating tumor rejection. In vitro and in vivo inhibition of iNOS (inducible nitric oxide synthase) abolished macrophage-mediated killing of tumor cells and rejection of intraocular tumors. A role for M1 macrophages was further supported by investigations showing that intraocular tumors grew progressively in IFN-γ KO (knockout) mice. Studies in mice deficient in TNF-α, TNF receptor-1, or TNF receptor-2 revealed that although TNF-α was not needed for tumor rejection, it was required for the development of necrotizing inflammation and phthisis of tumor-bearing eyes. Together, our findings suggest new strategies to successfully eliminate ocular tumors while preserving the integrity of the eye.

Cyclooxygenase-2 expression in the normal human eye and its expression pattern in selected eye tumours.

PURPOSE: Cyclooxygenase-2 (COX-2) is an enzyme involved in neoplastic processes. The purpose of the present study is to investigate COX-2 expression in the normal human eye and the expression pattern in selected eye tumours involving COX-2 expressing cells. METHODS: Immunohistochemical staining using antibodies against COX-2 was performed on paraffin sections of normal human eyes and selected eye tumours arising from cells expressing COX-2. RESULTS: Cyclooxygenase-2 expression was found in various structures of the normal eye. Abundant expression was seen in the cornea, iris, ciliary body and retina. The COX-2 expression was less in tumours deriving from the ciliary epithelium and also in retinoblastoma. CONCLUSION: Cyclooxygenase-2 is constitutively expressed in normal human eyes. The expression of COX-2 is much lower in selected eye tumours involving COX-2 expressing cells.

Effect of human cell malignancy on activity of DNA polymerase iota.

An increased level of mutagenesis, partially caused by imbalanced activities of error prone DNA polymerases, is a key symptom of cell malignancy. To clarify the possible role of incorrect DNA polymerase iota (Pol iota) function in increased frequency of mutations in mammalian cells, the activity of this enzyme in extracts of cells of different mouse organs and human eye (melanoma) and eyelid (basal-cell skin carcinoma) tumor cells was studied. Both Mg2+, considered as the main activator of the enzyme reaction of in vivo DNA replication, and Mn2+, that activates homogeneous Pol iota preparations in experiments in vitro more efficiently compared to all other bivalent cations, were used as cofactors of the DNA polymerase reaction in these experiments. In the presence of Mg2+, the enzyme was active only in cell extracts of mouse testicles and brain, whereas in the presence of Mn2+ the activity of Pol iota was found in all studied normal mouse organs. It was found that in cell extracts of both types of malignant tumors (basal-cell carcinoma and melanoma) Pol iota activity was observed in the presence of either Mn2+ or Mg2+. Manganese ions activated Pol iota in both cases, though to a different extent. In the presence of Mn2+ the Pol iota activity in the basal-cell carcinoma exceeded 2.5-fold that in control cells (benign tumors from the same eyelid region). In extracts of melanoma cells in the presence of either cation, the level of the enzyme activity was approximately equal to that in extracts of cells of surrounding tumor-free tissues as well as in eyes removed after traumas. The distinctive feature of tissue malignancy (in basal-cell carcinoma and in melanoma) was the change in DNA synthesis revealed as Mn2+-activated continuation of DNA synthesis after incorrect incorporation of dG opposite dT in the template by Pol iota. Among cell extracts of different normal mouse organs, only those of testicles exhibited a similar feature. This similarity can be explained by cell division blocking that occurs in all normal cells except in testicles and in malignant cells.

COX-1 and COX-2 expression in canine cutaneous, oral and ocular melanocytic tumours.

In order to evaluate the potential value of non-steroidal anti-inflammatory drugs (NSAIDs) in the treatment of canine malignant melanoma, expression of cyclooxygenase (COX)-1 and COX-2 was determined in 20 cutaneous, nine oral and two ocular malignant melanomas, and in nine cutaneous melanocytomas. Almost all tumours expressed COX-1, but COX-2 expression was restricted to the malignant tumours being found in 11 of the 20 cutaneous malignant melanomas, all oral malignant melanomas and in one of two ocular malignant melanomas. COX-1 expression did not differ significantly between benign and malignant skin lesions, but COX-2 expression was significantly greater in cutaneous malignant melanoma compared with melanocytoma (P=0.047). COX-2 labelling was particularly intense in the more highly malignant oral tumours. The results of the study suggest that NSAIDs, particularly COX-2 inhibitors, may be useful in the treatment of canine malignant melanoma.

Expression of cyclooxygenase-2 by equine ocular and adnexal squamous cell carcinomas.

OBJECTIVE: To investigate whether cyclooxygenase-2 (COX-2) is expressed by equine ocular and adnexal squamous cell carcinomas (SCC). METHODS: Forty-three samples of histologically confirmed cases of ocular SCC or carcinoma in situ (CIS) from 34 horses presented to the Animal Health Trust between 1992 and 2004 were subjected to a standard, two-layered, indirect immunohistochemical method using a rabbit polyclonal antihuman COX-2 antibody. Ten formalin-fixed, paraffin-embedded tissue samples taken from recognized predilection sites for SCC, from the grossly normal eyes of 10 horses euthanized for reasons unrelated to this study, were used as negative controls. Samples of equine fetal kidney were used as positive controls. Following immunolabeling, the number of normal and neoplastic epithelial cells exhibiting positive COX-2 expression was recorded along with staining intensity and distribution. RESULTS: Of 43 tumors, 34 were defined as first presentation tumors. When compared with control tissue, in which 0% (0/10) of samples expressed COX-2, significantly more of these samples with SCC (58.6%, 17/29: P = 0.002), CIS (60%, 3/5: P = 0.022) or either tumor type (58.8%, 20/34: P = 0.001) exhibited positive cytoplasmic and perinuclear immunohistochemical staining for COX-2. Of the samples exhibiting positive immunohistochemical staining, only 10% (2/20) showed staining in 2%-10% of neoplastic cells, while 90% (18/20) showed staining in 1% of neoplastic cells. About 70% (14/20) of those positively immunolabeled samples exhibited an intensity of staining greater than or equal to the staining exhibited by the equine fetal kidney positive control. CONCLUSION: Neoplastic tissue from both equine ocular SCC and CIS exhibit COX-2 expression at significantly higher levels than normal control ocular tissue. However, the percentage of cells expressing positive immunohistochemical staining is consistently low. On the basis of this study, it is unlikely that anti-COX-2 therapy would be of benefit in the treatment of equine ocular and adnexal SCC.

TNFAIP3 is the target gene of chromosome band 6q23.3-q24.1 loss in ocular adnexal marginal zone B cell lymphoma.

The genomic aberrations in extra nodal marginal zone B cell lymphoma vary according to their anatomical origin. This polarization is a reflection of the participation of different genes in the lymphomagenesis of marginal zone B cell lymphoma. We previously demonstrated by means of genome-wide array comparative genomic hybridization (CGH) that the genomic profile of ocular adnexal marginal zone B cell lymphoma is distinct from that of pulmonary or nodal marginal zone B cell lymphoma. The novel finding was a recurrent deletion of a 2.9-Mb region at chromosome band 6q23.3-q24.1, including homozygous loss, in ocular adnexal marginal zone B cell lymphoma. For a more detailed examination of the deletions of 6q23.3-24.1, we used contig bacterial artificial chromosome (BAC) array CGH, containing 24 BAC clones covering the 2.9-Mb region, to analyze nine cases with 6q23.3-q24.1 loss. We narrowed the minimal common region down to a length of 586 kb with two genes and four expressed sequence tags (ESTs). All of these genes and ESTs were subjected to RT-PCR and real-time quantitative RT-PCR. Correlation between genomic loss and expression level was found only for TNFAIP3, demonstrating that TNFAIP3 is a target gene of 6q deletion in ocular adnexal marginal zone B cell lymphoma. TNFAIP3 is an inhibitor of NF-kB signaling so that loss of this gene may play an important role in lymphomagenesis and suggests that TNFAIP3 may act as a tumor suppressor gene in ocular adnexal marginal zone B cell lymphoma.

Anti-tumor activity of arginine deiminase via arginine deprivation in retinoblastoma.

In spite of recent advances in the treatment of retinoblastoma, chemotherapy is still challenging in high-stage intraocular retinoblastoma or metastatic retinoblastoma. Here, we investigated whether arginine deprivation via arginine deiminase (ADI) could be a new anti-tumor therapy in retinoblastoma cells. Expression of argininosuccinate synthetase (ASS) was detected in human retinoblastoma tissues. Even with a high expression of ASS, ADI effectively inhibited the proliferation of retinoblastoma cells and induced retinoblastoma cell death in a dose-dependent manner. These results indicate that arginine deprivation via ADI could be another treatment option for retinoblastoma due to low ASS activity in retinoblastoma cells.

Cyclooxygenase-2 immunoreactivity in equine ocular squamous-cell carcinoma.

Squamous-cell carcinoma (SCC) is the second most common tumor in horses, and 40%-50% may occur in ocular and adnexal structures. Cyclooxygenase (COX)-2 is an inducible enzyme responsible for the production of prostaglandins that control cell growth and the development and progression of cancer. Mechanisms responsible for the initial upregulation of COX-2 in neoplasia are unclear; prolonged sunlight exposure and mutations in the p53 gene may be possibilities. Because the etiopathogenesis of ocular SCC in horses may involve ultraviolet sunlight and p53 mutations, the purpose of this study was to characterize the immunoreactivity of COX-2 in these tumors. Cyclooxygenase-2 expression was found in 6 of 22 (27%) paraffin-embedded equine SCCs. Cyclooxygenase-2 immunoreactivity was associated with the mitotic index (P < 0.001). Strategies to inhibit COX-2 by the use of topical or systemic COX-2 inhibitors might prove to be a safe and economical treatment in some horses with SCC.

Expression of matrix metalloproteinases and their inhibitors in retinoblastoma.

Tumor invasion is the critical step that could lead to metastasis in retinoblastoma (RB), a common childhood cancer. Matrix metalloproteinases (MMPs) degrade extracellular matrix, which is a crucial step involved in various stages of tumor progression, including tumor angiogenesis, tumor growth, and also local invasion and subsequent distant metastasis. We investigated the role of extracellular MMP inducer (EMMPRIN), MMP-2, MMP-9 and tissue inhibitor of metalloproteinases (TIMPs): TIMP-1, TIMP-2 in RB and correlated clinicopathologically. Among 60 tumors, EMMPRIN was expressed in 40 (64%), MMP-2 in 41 (66%), MMP-9 in 38 (61%), TIMP-1 in 35 (56%), and TIMP-2 in 33 (53%) tumors. EMMPRIN was positive (3+) in 13 (39%) out of 33 tumors with invasion and was positive (3+) in only 1 (3%) out of 29 tumors without invasion. MMP-2 (P<0.0001) and MMP-9 (P<0.0001) were significantly positive (3+) in 7 (21%) and 12 (36%) out of 33 tumors with invasion, whereas positive (3+) in 3 (10%) and faint (1+) in 10 (34%) tumors, respectively, out of 29 tumors without invasion. TIMP-1 (P<0.0001) and TIMP-2 (P=0.04) were significantly positive (3+) in 7 (21%) and 10 (30%), respectively out of 33 tumors with invasion, whereas positive (3+) in only 1 (3%) tumor each out of 29 tumors without invasion. Immunoblotting of tumors confirmed the presence of EMMPRIN, MMPs, and TIMPs. In conclusion, both MMPs and TIMPs may be involved RB invasion and EMMPRIN could play a role in up-regulation of MMP-2 in invasive RB.

Inhibition of urethane-induced carcinogenicity in cyp2e1-/- in comparison to cyp2e1+/+ mice.

Urethane is an established animal carcinogen and has been classified as "reasonably anticipated to be a human carcinogen." Until recently, urethane metabolism via esterase was considered the main metabolic pathway of this chemical. However, recent studies in this laboratory showed that CYP2E1, and not esterase, is the primary enzyme responsible for urethane oxidation. Subsequent studies demonstrated significant inhibition of urethane-induced genotoxicity and cell proliferation in Cyp2e1-/- compared to Cyp2e1+/+ mice. Using Cyp2e1-/- mice, current studies were undertaken to assess the relationships between urethane metabolism and carcinogenicity. Urethane was administered via gavage at 1, 10, or 100 mg/kg/day, 5 days/week, for 6 weeks. Animals were kept without chemical administration for 7 months after which they were euthanized, and urethane carcinogenicity was assessed. Microscopic examination showed a significant reduction in the incidences of liver hemangiomas and hemangiosarcomas in Cyp2e1-/- compared to Cyp2e+/+ mice. Lung nodules increased in a dose-dependent manner and were less prevalent in Cyp2e1-/- compared to Cyp2e+/+ mice. Microscopic alterations included bronchoalveolar adenomas, and in one Cyp2e1+/+ mouse treated with 100 mg/kg urethane, a bronchoalveolar carcinoma was diagnosed. Significant reduction in the incidence of adenomas and the number of adenomas/lung were observed in Cyp2e1-/- compared to Cyp2e1+/+ mice. In the Harderian gland, the incidences of hyperplasia and adenomas were significantly lower in Cyp2e1-/- compared to Cyp2e+/+ mice at the 10 mg/kg dose, with no significant differences observed at the high or low doses. In conclusion, this work demonstrated a significant reduction of urethane-induced carcinogenicity in Cyp2e1-/- compared to Cyp2e1+/+ mice and proved that CYP2E1-mediated oxidation plays an essential role in urethane-induced carcinogenicity.

Oxidants and ocular tumors.

Serum superoxide dismutase and catalase assays were performed using spectrophotometry in 60 adults and children with benign or malignant tumors and in controls. There was a statistically significant difference in the antioxidative status of children with intraocular tumors (primary retinoblastoma) compared with children without tumors. The difference was not significant in adults. These enzymes may be of value in the early diagnosis of malignant intraocular tumor, especially retinoblastoma.

Telomerase activity with concurrent loss of cell cycle regulation in feline post-traumatic ocular sarcomas.

Paraffin wax-embedded ocular globes of cats with post-traumatic ocular sarcomas were examined for the presence of TERT, the active subunit of telomerase. The latter is a ribonucleoprotein complex essential for immortalization and expressed by most malignant tumours, germ line cells, lens epithelial cells, and some stem cells. Due to the frequent loss of cell cycle control with the increased expression of telomerase activity, post-traumatic ocular sarcomas were also examined for loss of p16 expression and alterations in p53, the findings being related to mitotic score, tumour grade, and proliferating cell nuclear antigen. These sarcomas expressed telomerase at a high frequency (62.5%); in addition, the majority showed alterations in cell cycle control, as evaluated by lack of p16 immunolabelling (66.7%). Alterations in p53 were the sole mechanism by which cell cycle control was dysregulated in only two tumours expressing TERT (13%). These findings suggest that p16, and not p53, represents the primary mechanism by which post-traumatic ocular sarcomas that express telomerase activity escape cell cycle control.

Characterization of 3',5' cyclic nucleotide phosphodiesterase activity in Y79 retinoblastoma cells: absence of functional PDE6.

PURPOSE: Previous studies identified rod photoreceptor cyclic GMP phosphodiesterase (PDE6) transcripts in the human Y79 retinoblastoma cell line. To assess the potential to utilize this cell line for structure/function studies of PDE6, we analyzed 3',5' cyclic nucleotide phosphodiesterase activity focusing on expression of PDE6. METHODS: DEAE-chromatography was used to fractionate PDE activity from Y79 cell homogenates. PCR was performed on cDNA generated from Y79 cells and retina with PDE isoform specific primers. Western blots were performed with antibodies to PDE1, PDE4, or rod PDE6. DNA sequencing and protein truncation tests were performed with plasmids containing the entire coding region of Y79 rod PDE6 transcripts. Proteasome mediated degradation of PDE6 subunits was analyzed with a pathway specific inhibitor. Polysome isolation was performed by fractionation on sucrose gradients followed by RT-PCR for the PDE6 transcripts. RESULTS: Of three peaks of PDE activity, peaks 1 and 2 were activated by Ca2+/calmodulin, inhibited by dipyridamole and zaprinast, and were reactive with a PDE1 antibody. Peak 3 hydrolyzed only cAMP and was rolipram sensitive, indicative of PDE4. Transcripts for rod and cone PDE6 isoforms were detected in Y79 total RNA, however PDE6 antibodies recognized only a single 99 kDa polypeptide from immunoprecipitated 35S labeled Y79 extracts. DNA sequencing of PDE6 alpha, beta, gamma, and PDE6 associated delta-subunit cDNA revealed some polymorphism, but no apparent mutations. Each of the PDE6 transcripts could be translated into protein of the correct length. The concentration of cGMP in the cells was greatly reduced in comparison to that reported in the photoreceptor cell. Addition of cyclic nucleotide analogues, zinc, or butyrate did not enhance the expression of PDE6. Transduction into Y79 cells of adenovirus expressing PDE6 subunits failed to produce functional enzyme CONCLUSIONS: PDE1 and PDE4 enzyme activities predominate in Y79 cells. Despite the presence of PDE6 transcripts and the ability to translate each into protein in vitro, a functional PDE6 enzyme could not be detected. Attempts to enhance expression with cell culture or with introduction of virus expressing PDE6 were not successful. The results indicate that expression of a fully active stable PDE6 enzyme requires other post-transcriptional events that do not occur or are inhibited in Y79 cells.

Intraocular inflammatory myofibroblastic tumor with ALK overexpression.

We report a case of an intraocular inflammatory myofibroblastic tumor nearly filling the vitreous cavity of the eye of a 50-year-old man. The tumor was composed of a mixture of spindle cells and mixed inflammatory elements, including numerous plasma cells. The differential diagnosis included inflammatory pseudotumor and neoplastic mimics of this condition. Further investigation with immunohistochemistry revealed the mass to be composed of myofibroblasts, positive for smooth muscle actin stains and with weak anaplastic lymphoma kinase (ALK) expression in some tumor cells. Evaluation by fluorescence in situ hybridization revealed the tumor cells to have multiple copies of chromosome 2 and ALK but no rearrangement of the ALK gene. The authors propose that multiple copies of the ALK gene may be involved in inflammatory myofibroblastic tumor tumorigenesis, in addition to ALK gene rearrangements.

Case report: carcinoma of the extraorbital lacrimal gland in a female Fischer 344 rat.

A large subcutaneous mass was observed at necropsy in the right neck area of a 95-week-old female Fischer 344 rat that served as an untreated control animal in a 2-year carcinogenicity study. Formalin-fixed, paraffin-embedded sections of the mass were stained with hematoxylin and eosin along with the immunohistochemical biomarkers lactoperoxidase, catalase, and amylase. Based on its histomorphological and immunohistochemical features, the lesion was diagnosed as a carcinoma of the extraorbital lacrimal gland.

Immunohistochemical localization of NQO1 in epithelial dysplasia and neoplasia and in donor eyes.

PURPOSE: To examine the expression of NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase), a potential bioactivating enzyme for mitomycin C in corneal and conjunctival epithelial dysplasia and neoplasia and in normal tissues from human donor eyes, by immunohistochemistry. METHODS: Formalin-fixed, paraffin-embedded sections of human donor eyes and tissue sections with histologic diagnoses of corneal and conjunctival epithelial dysplasia and neoplasia from the Eye Pathology Laboratory, University of Colorado Health Sciences Center were analyzed. Detection of NQO1 in tissues was performed using standard immunohistochemical techniques with monoclonal antibodies against NQO1 and immunoperoxidase staining. RESULTS: All 20 tumors stained positive for NQO1. In seven eyes from four donors, positive staining for NQO1 was detected in all epithelial and endothelial layers, in fibroblasts, in all retinal layers except the photoreceptor outer segments, and in the fascicles and arachnoid of the optic nerve. Only minimal staining was detected in the photoreceptor outer segments and the optic nerve pia and dura. Immunostaining was markedly reduced in all tissues in both eyes from donor 5. Genetic analysis confirmed that this individual was homozygous for a polymorphism in NQO1 (NQO1*2). CONCLUSIONS: NQO1 was detected by immunohistochemistry in every examined section of corneal and conjunctival epithelial dysplasia and neoplasia, suggesting that NQO1 may play a role in the bioactivation of mitomycin C in these tumors. However, the presence of NQO1 in the corneal, conjunctival, and ciliary epithelium; the retinas; and the optic nerves of donor eyes may indicate the potential for mitomycin C toxicity, particularly at higher doses.