Conjunctival Melanoma Targeted Therapy: MAPK and PI3K/mTOR Pathways Inhibition.

Purpose: To analyze the activity of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases/mechanistic target of rapamycin (PI3K/mTOR) pathways in benign and malignant conjunctival melanocytic proliferations and explore whether specific inhibitors can suppress growth of conjunctival melanoma (CJM) cells. Methods: The presence of a BRAF V600E mutation and activation of ERK, MEK, S6, and AKT were assessed with immunohistochemistry in 35 conjunctival nevi and 31 melanomas. Three CJM cell lines were used: CRMM1, carrying the BRAF V600E mutation; CRMM2, harboring the NRAS Q61L mutation; and T1527A, with a BRAF G466E mutation. WST-1 assays were performed with a BRAF inhibitor (vemurafenib), two MEK inhibitors (trametinib, selumetinib), a PI3K inhibitor (pictilisib), and a dual PI3K/mTOR inhibitor (dactolisib). The phosphorylation of ERK, MEK, and S6 were tested with western blots and apoptosis with cleaved caspase-3 immunostaining. Results: A BRAF V600E mutation was detected in 42.6% of nevi and in 35.5% of CJM. MEK and ERK activation were higher in CJM, occurring in 62.9% and 45.7% of the nevi and 90.3% and 96.8% of the CJM, respectively. There was also a significant increase in S6 activation in CJM (90.3%) compared with the nevi (20%). CRMM1 was sensitive to trametinib and the PI3K inhibitors but only marginally to vemurafenib. CRMM2 was moderately sensitive to pictilisib, whereas T1527A was resistant to all drugs tested. Conclusions: The MAPK pathway activity in CJM is increased, not only as a consequence of the BRAF V600E mutation. Targeted therapy may be useful for patients with CJM, especially those with activating BRAF mutations, whereas NRAS-mutated melanomas are relatively resistant.

Conjunctival melanoma: association of cyclooxygenase-2 tumor expression to prognosis.

PURPOSE: Conjunctival melanoma is a rare but potentially lethal tumor. Its biologic profile is still largely unknown, with recent studies aiming at establishing histopathological and genetic tumor profiles. The aim of this study was to analyze the association between clinicopathological characteristics and tumor expression of cyclooxygenase-2 (COX-2) to prognosis, assessing its usefulness as a possible prognostic marker. METHODS: Case series of 50 patients from 1991 to 2008 with pathologically proven conjunctival melanoma. Demographic, clinical, and pathological characteristics were evaluated by reviewing clinical files and pathology. Expression of COX-2 was studied by immunohistochemistry of formalin-fixed paraffin-embedded tissue samples of 20 melanomas. Samples were classified in a score which included intensity of staining and percentage of cells with positive reactivity. RESULTS: Clinicopathological features significantly associated (p < .05) with a poor prognosis (death) included involvement of fornix and tarsal conjunctiva, tumor thickness exceeding 2 mm, local tumor recurrence, lymph node, and systemic metastasis. In the immunohistochemistry study (n = 20), 18 cases expressed COX-2 although with different scores. However, only cases with a high score were associated with a poor outcome. Multivariate association analysis revealed that recurrence rate, metastasis, corneal invasion, and tumor thickness were associated with high score cases and, therefore, with a clinical profile with a higher risk of death. CONCLUSIONS: Results suggest that higher COX-2 expression may be a negative prognostic factor in conjunctival melanoma. Further studies can address the potential use of anti-COX-2 drugs as adjuvant therapy of this disease.

A novel KRAS mutation in metastasic conjunctival melanoma: a case report and literature review.

Conjunctival melanoma is a rare disease, and little is known about its molecular background. Here, we present the case of a 48-year-old patient with conjunctival melanoma and metachronic lymph node and skin metastasis with KRAS p.K117Y mutation in exon 4 in all the lesions. The cancer genome interpreter predicted this mutation to have driver function. To our knowledge, this is the first time this mutation is found in conjunctival melanoma. An important role in the disease development is suggested.

Management of anaplastic lymphoma kinase positive orbito-conjunctival inflammatory myofibroblastic tumor with crizotinib.

Inflammatory myofibroblastic tumor (IMT) is a distinct mesenchymal neoplasm of myofibroblastic spindle cells associated with an inflammatory infiltrate formed by lymphocytes, eosinophils, and plasma cells in a myxoid or collagenous stroma. This tumor has a predilection for children and young adults and most commonly occurs in the lungs, retroperitoneum, abdomen, and pelvis. Ocular and orbital involvement is exceedingly rare. We describe a case of IMT in a 7-year-old girl involving the cornea, conjunctiva, and the anterior orbit treated with crizotinib, resulting in complete tumor remission.

Analysis of SDHD promoter mutations in various types of melanoma.

OBJECTIVES: Recently, recurrent mutations in regulatory DNA regions, such as promoter mutations in the TERT gene were identified in melanoma. Subsequently, Weinhold et al. reported SDHD promoter mutations occurring in 10% of melanomas and being associated with a lower overall survival rate. Our study analyzes the mutation rate and clinico-pathologic associations of SDHD promoter mutations in a large cohort of different melanoma subtypes. METHODS: 451 melanoma samples (incl. 223 non-acral cutaneous, 38 acral, 33 mucosal, 43 occult, 43 conjunctival and 51 uveal melanoma) were analyzed for the presence of SDHD promoter mutations by Sanger-sequencing. Statistical analysis was performed to screen for potential correlations of SDHD promoter mutation status with various clinico-pathologic criteria. RESULTS: The SDHD promoter was successfully sequenced in 451 tumor samples. ETS binding site changing SDHD promoter mutations were identified in 16 (4%) samples, of which 5 mutations had not been described previously. Additionally, 5 point mutations not located in ETS binding elements were identified. Mutations in UV-exposed tumors were frequently C>T. One germline C>A SDHD promoter mutation was identified. No statistically significant associations between SDHD promoter mutation status and various clinico-pathologic variables or overall patient survival were observed. CONCLUSIONS: Melanomas harbor recurrent SDHD promoter mutations, which occur primarily as C>T alterations in UV-exposed melanomas. In contrast to the initial report and promoter mutations in the TERT gene, our analysis suggests that SDHD promoter mutations are a relatively rare event in melanoma (4% of tumors) of unclear clinical and prognostic relevance.

Expression of SIRT1 in ocular surface squamous neoplasia.

BACKGROUND: The class III histone deacetylase SIRT1 is overexpressed in many malignancies and has been implicated in inactivating proteins that are involved in tumor suppression and DNA damage repair. In the current study, we examined the expression of SIRT1 in normal epithelium (NE) compared with ocular surface squamous neoplasia (OSSN) to elucidate a possible role for SIRT1 in the development or progression of this malignancy. METHODS: We examined SIRT1 expression by immunohistochemistry in 47 cases of OSSN and 10 specimens of NE. Our sample included 11 benign lesions (papillomas), 25 cases of conjunctival intraepithelial neoplasia, and 11 malignant lesions of squamous cell carcinoma. The extent of staining and intensity was scored and the combined raw data were then converted to the German Immunoreactive Score. RESULTS: Nuclear and cytoplasmic expression of SIRT1 was observed in all cases of OSSN. For the NE specimens, 50% showed negative expression and 30% weak expression, and 20% were considered significantly immunoreactive. The differential expression of SIRT1 between NE and OSSN was statistically significant (P < 0.0001). Additionally, when the staining pattern in cases of conjunctival intraepithelial neoplasia was evaluated, the staining of the more differentiated surface cells was remarkably weaker compared with the cells located closer to the basal membrane. CONCLUSIONS: SIRT1 may play an important role in the development and progression of epithelial tumors of the conjunctiva. Further research into the potential of SIRT1 as a novel therapeutic target is warranted.

Expression of matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitor of MMP (TIMP)-1 in conjunctival melanomas and clinical implications.

PURPOSE: To investigate the expression of matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in conjunctival melanomas and their correlations with clinicopathologic parameters and prognosis. METHODS: Fourteen conjunctival melanoma tissue samples and nine conjunctival nevus tissue samples were stained immunohistochemically for MMP-2, MMP-9, and TIMP-1. Association of MMP-2, MMP-9, and TIMP-1 expression in melanoma tissues with clinical progression in terms of metastasis, recurrence, mitotic index, thickness, base diameter, and invasion depth was analyzed. RESULTS: In the melanoma group, 78.6% of samples showed a positive reaction for MMP-2, 85.7% for MMP-9, and 100% for TIMP-1. In the nevus group, 11.1% showed a positive reaction for MMP-2, 66.7% for MMP-9, and 100% for TIMP-1. MMP-2 expression was significantly more induced in conjunctival melanoma than in benign nevi (P = 0.002). In conjunctival melanoma, MMP-9 expression was higher in tumors >1.5 mm thick (P = 0.026) and TIMP-1 expression was higher in recurrent cases (P = 0.03). There was no significant correlation between the expression and metastasis during the follow-up period (mean, 5 years). CONCLUSION: MMP-2, MMP-9, and TIMP-1 were expressed in the majority of conjunctival melanomas, and MMP-2 might play a role in the development and clinical behavior of conjunctival melanoma.

HPV infection and EGFR activation/alteration in HIV-infected East African patients with conjunctival carcinoma.

BACKGROUND: There has been substantial growth in the numbers of patients with conjunctival squamous cell carcinoma infected with HIV in East Africa. The natural history of the conjunctival squamous cell carcinoma appears to be unique in this region of the world, but the etiologic mechanism unclear and therapeutic options limited. This research was carried out to determine if conjunctival squamous cell carcinoma harbors human papillomavirus DNA and is associated with activation of the EGFR signaling pathway. Positive findings would identify etiologic causes and provide clinical guidance to improve treatment. METHODS/FINDINGS: Expression of p-MAPK/MAPK, p-Akt/Akt and p-EGFR/EGFR in cell nuclei and cytoplasm of 38 FFPE specimens were assessed by immunohistochemistry; HPV genotype was detected by qPCR assay; EGFR mutation was assessed by DNA sequencing analysis; and EGFR mRNA expression was measured using relative qPCR. Statistical analyses included two-sided Fisher exact test or chi-square test, Spearman correlation coefficient and ANOVA. HPV 18 was found in 61% of samples, with HPV 16 double-genotype in 6 patients (16%). Immunohistochemistry and qPCR data suggest that activation and expression of the EGFR signaling pathway is related to disease progression of conjunctival cancer. The associations between cytoplasmic p-MAPK, cytoplasmic p-Akt and tumor invasiveness were significant (p = 0.05 or 0.028). Nuclear p-EGFR appeared only in invasive tumors. A significant positive association between EGFR expression and disease invasiveness was observed (p = 0.01). A SNP in 10 patients and one missense mutation were found within EGFR tyrosine kinase domain. Statistical analysis indicates that patients with measurable EGFR expression more likely harbor EGFR mutations, compared to those with negative EGFR expression (35.3% vs. 0%). CONCLUSIONS/SIGNIFICANCE: We conclude that HPV types 16/18 infection is frequent in East African patients with AIDS-associated squamous cell carcinoma of the conjunctiva. EGFR activation/alteration may contribute to and sustain the high prevalence of this cancer. Our findings hint that adoption of HPV vaccination strategies may impact the incidence of conjunctival carcinoma. Agents that target the EGFR pathway may have potential therapeutic benefit.

Therapeutic effect of topical 5-fluorouracil in conjunctival squamous carcinoma is associated with changes in matrix metalloproteinases and tissue inhibitor of metalloproteinases expression.

PURPOSE: To evaluate matrix metalloproteinases (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-1 expression in a case of conjunctival intraepithelial squamous cell carcinoma (SCC) treated with topical 5-fluorouracil (5-FU) chemotherapy. METHODS: Clinicopathologic case report. RESULTS: A 71-year-old male patient presented with an intraepithelial conjunctival SCC. Because of a recurrence, he was placed on topical 5-FU for 4 weeks that ultimately led to a complete resolution of the disease. Conjunctival biopsies, impression cytologies, and tear samples were taken from the mass and the contralateral healthy eye. An overexpression of MMP-2, MMP-9, and TIMP-1 was observed in the tumor by immunohistochemistry. Clinical resolution of the neoplasm obtained using topical 5-FU was accompanied by a reduction in the expression of MMP-2, MMP-9, and TIMP-1 in tears and dysplastic conjunctival epithelium. CONCLUSIONS: In our case report, we have shown that gelatinase and TIMP-1 are unregulated in conjunctival SCC and can be monitored as a marker of response to topical chemotherapy. Further studies are required to define the role of MMPs in growth and resolution of ocular tumors.

Ultraviolet radiation and the role of matrix metalloproteinases in the pathogenesis of ocular surface squamous neoplasia.

PURPOSE: Ocular surface squamous neoplasia (OSSN) is an uncommon tumor of the corneal and conjunctival epithelium associated with risk of permanent visual impairment. The purposes of this study were to (1) identify and localize potential mediators in tissue from patients with OSSN and (2) culture human dysplastic conjunctival epithelial cells (DCECs) to determine their responsiveness to ultraviolet (UV)-B radiation compared with normal conjunctival epithelial cells (NCECs). METHODS: Immunohistochemical analysis was performed on OSSN (n = 23) and normal conjunctival (n = 17) tissue to identify matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Cell viability as well as basal and UVB-modulated levels of MMPs and TIMPs from DCECs and NCECs was determined by immunoassays, zymography, and RT-PCR. RESULTS: A higher proportion of diseased specimens stained for MMP-1 (83%), MMP-3 (86%), TIMP-2 (87%), and TIMP-3 (83%) compared with normal conjunctiva (41%, 41%, 47%, and 53%, respectively). UVB radiation induced cell death and apoptosis at doses >/= 50 mJ/cm(2). MMP-1 and -3 mRNA and protein expression in DCECs was induced by UV and was mitogen-activated protein kinase-dependent, although the same enzymes were upregulated in NCECs only at doses that induced apoptosis. TIMP-1 and -2 levels remained relatively unchanged, except for a dose-dependent suppression of TIMP-3. CONCLUSIONS: The results suggest that MMPs and TIMPs play a significant role in the pathogenesis of OSSN and that UVB initiates and perpetuates the development of this lesion on the ocular surface.

Cyclooxygenase-2 expression in primary and recurrent pterygium.

PURPOSE: Pterygium is a proliferative, inflammatory, and invasive ocular surface disease associated with excessive ultraviolet radiation exposure and has several tumor-like characteristics. Cyclooxygenase-2 (COX-2) is an inducible enzyme and recently increased expression of the enzyme was found in many cancers and premalign lesions. This study was conducted to identify the COX-2 expression in pterygium tissues. METHODS: Immunohistochemical staining using a primary antibody for COX-2 was performed on 30 specimens with primary pterygium (20 pterygium without recurrence and 10 pterygium which recurred during a 12-month follow-up), 11 specimens with recurrent pterygium, and 8 specimens of conjunctival tumor. As a control we used 10 specimens of normal conjunctiva. Extent and intensity of cytoplasmic and membranous staining in epithelial cells were evaluated. RESULTS: Higher expression of COX-2 was detected in conjunctival tumor (87.5%) specimens and recurrent pterygium specimens (72.7%) compared to the both normal conjunctiva (30%) and primary pterygium without recurrence (30%). COX-2 expression in primary pterygium tissues with recurrence (60%) was not different from primary pterygium without recurrence (p=0.114) and recurrent pterygium (p=0.537). However, recurrent pterygium tissues were found to express higher COX-2 than primary pterygium without recurrence (p=0.022). CONCLUSIONS: COX-2 expression is increased in recurrent pterygium tissues and COX-2 expression may be a marker for the prediction of recurrence.

Immunohistochemical localization of NAD(P)H:quinone oxidoreductase in conjunctival melanomas and primary acquired melanosis.

PURPOSE: Mitomycin C has been used in the treatment of primary acquired melanosis and melanomas of the conjunctiva. Because there is increasing evidence that NAD(P)H:quinone oxidoreductase (EC 1.6.99.2, NQO1) or DT-diaphorase plays an important role in the bioactivation of mitomycin C, we examined pathologic specimens of these tumors for NQO1 by immunohistochemistry. METHODS: Formalin-fixed, paraffin-embedded sections with histologic diagnoses of primary acquired melanosis or conjunctival melanomas were obtained from the Eye Pathology Laboratory, University of Colorado Health Sciences Center. Detection of NQO1 in tissues was performed using standard immunohistochemical techniques with monoclonal antibodies against NQO1 and immunoperoxidase staining. Samples were examined by two independent reviewers and NQO1 staining was graded from 0 (no staining) to 3+ (intense staining). RESULTS: Eleven of 11 melanomas (95% confidence interval, 72% to 100%) and three of three lesions with primary acquired melanosis with atypia stained positively for NQO1. In the melanomas, staining was relatively uniform, while in primary acquired melanosis there was cell-to-cell variability in the staining. CONCLUSIONS: NQO1 was detected by immunohistochemistry in every examined section of primary acquired melanosis and melanoma of the conjunctiva, suggesting that NQO1 may play a role in the bioactivation of mitomycin C in these tumors.

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.