Usefulness of PAX8 Immunohistochemistry in Adult Intraocular Tumor Diagnosis.

PURPOSE: To evaluate the distribution of the PAX8 transcription factor protein in ocular tissues and to investigate if immunohistochemical stains for this biomarker are useful in the diagnosis of intraocular tumors. DESIGN: Observational case series. PARTICIPANTS: Excision and cytologic analysis specimens of 6 ciliary body epithelial neoplasms, 2 iris epithelial neoplasms, 3 retinal pigment epithelial neoplasms, 3 intraocular medulloepitheliomas, 15 uveal melanomas, and 5 uveal melanocytomas. METHODS: Hematoxylin-eosin and PAX8 immunohistochemical stains were performed on all specimens. In appropriate cases, bleached preparations and other immunohistochemical stains, including AE1/AE3 cytokeratin, Lin28A, and CD45, were performed. MAIN OUTCOME MEASURES: Distribution of PAX8 expression in normal and neoplastic tissue. RESULTS: Strong nuclear PAX8 expression was observed in the normal corneal epithelium, iris sphincter pupillae muscle, iris pigment epithelium and dilator muscle complex, nonpigmented and pigmented epithelia of the ciliary body, lens epithelium, and a subset of retinal neurons. The normal retinal pigment epithelium and uveal melanocytes did not stain for PAX8. The ciliary body epithelial and neuroepithelial tumors (adenoma, adenocarcinoma, and medulloepithelioma) showed uniform strong nuclear PAX8 immunoreactivity. All melanocytic tumors (iris melanoma, ciliary-choroidal melanoma, and melanocytoma) and retinal pigment epithelial neoplasms showed negative results for PAX8. A subset of tumor-associated lymphocytes, most prominent in uveal melanoma, showed positive results for PAX8. The uniformity of the PAX8 staining was superior to the variable cytokeratin staining in the ciliary epithelial neoplasms and the variable Lin28A staining in malignant medulloepithelioma. The veracity of PAX8 staining was equally as robust on cytologic analysis and open-flap biopsy specimens of ciliary epithelial and iris epithelial neoplasms, melanocytoma, and melanoma. CONCLUSIONS: PAX8 has proven to be a very useful diagnostic marker in a select group of adult intraocular tumors, and we highly recommend its inclusion in diagnostic antibody panels of morphologically challenging intraocular neoplasms.

Case Report: Tapioca Iris Melanoma: An Uncommon Presentation of an Uncommon Tumor.

SIGNIFICANCE: Iris tumors are rare conditions, and there is a relative paucity of recent published data on its broad clinical spectrum. Tapioca iris melanoma is a rarer yet devastating form with wide and challenging differential diagnoses because of its amelanotic nodular appearance. PURPOSE: This study aimed to report the challenging presentation of an uncommon iris melanoma, describing the clinical and histological findings and comparing them with the existing published data. CASE REPORT: An uncommon clinicopathological report on the tumor unusual localization, patient age, absence of elevated IOP and heterochromia, and negative S-100 stain that caused diagnostic uncertainty is presented. The patient remains free of metastatic disease 7 years after a complete tumor full-thickness excision. CONCLUSIONS: Tapioca iris melanomas are uncommon tumors with a presentation/surgical management that differs from other malignant tumors. Ophthalmologists should consider it among the vast differential diagnoses when observing amelanotic lesions, even without the hallmark signs being evident.

Protein expression of KIT, BRAF, GNA11, GNAQ and RASSF1 in feline diffuse iris melanomas.

Feline iris melanoma, the most common feline intraocular tumour, has a reported metastatic rate of 19-63%. However, there is a lack of knowledge about its molecular biology. Previous studies have reported that feline iris melanomas do not harbour mutations comparable to common mutations found in their human counterpart. Nevertheless, there are differences in the gene expression patterns. The aim of this study was to investigate the protein expression of B-RAF oncogene serine/threonine kinase (BRAF), G protein subunit alpha q (GNAQ) and 11 (GNA11), KIT proto-oncogene receptor tyrosine kinase (KIT), and Ras association family member 1 (RASSF1) in feline iris melanomas. Fifty-seven formalin-fixed paraffin embedded (FFPE) iris melanomas and 25 FFPE eyes without ocular abnormalities were stained with antibodies against the respective proteins using immunofluorescence. Averaged pixel intensities/µm2 and percentage of stained area from total tissue area were measured and the results were compared. Compared to the control group, iris melanomas showed overexpression of BRAF, GNAQ, GNA11 and KIT. The higher expression of BRAF, GNAQ, GNA11 and KIT in feline iris melanomas suggest that these proteins may play a key role in the development of feline iris melanomas and KIT may present a possible target for future therapies in cats with feline iris melanomas.

Frequent GNAQ, GNA11, and EIF1AX Mutations in Iris Melanoma.

Purpose: The most common malignant intraocular tumors with a high mortality in adults are uveal melanomas. Uveal melanomas arise most frequently in the choroid or ciliary body (97%) and rarely in the iris (3%). Whereas conjunctival and posterior uveal (ciliary body and choroidal) melanomas have been studied in more detail genetically, little data exist regarding iris melanomas. Methods: In our study, we genetically analyzed 19 iris melanomas, 8 ciliary body melanomas, 3 ring melanomas, and 4 iris nevi. A targeted next-generation sequencing approach was applied, covering the mutational hotspot regions of nine genes known to be mutated in conjunctival and uveal melanoma (BRAF, NRAS, KIT, GNAQ, GNA11, CYSLTR2, SF3B1, EIF1AX, and BAP1). Results: Activating GNAQ or GNA11 hotspot mutations were detected in a mutually exclusive fashion in 84% (16/19) of iris melanomas. EIF1AX gene mutations also were frequent, detected in 42% (8/19) of iris melanomas. In 4 iris nevi, one GNAQ mutation was identified. GNAQ, GNA11, EIF1AX, and BAP1 mutations were identified at varying frequencies in ciliary body and ring melanomas. Conclusions: In this most comprehensive genetic analysis of iris melanomas published to date, we find iris melanomas to be related genetically to choroidal and ciliary body melanomas, frequently harboring GNAQ, GNA11, and EIF1AX mutations. Future studies will need to assess if screening mutation profiles in iris melanomas may be of diagnostic or prognostic value.

Small incision iris tumour biopsy using a cavernous sampling forceps.

BACKGROUND: The aim of this retrospective report is to describe our experience with the Essen-23G biopsy forceps (Akgül forceps) for biopsies of pigmented iris tumours. METHODS: In this retrospective study of cases between October 2012 and September 2013, patients with iris tumours and clinical signs for malignancy underwent biopsy to secure the diagnosis. The Essen-23G-forceps was used to grasp and extract tissue through a clear corneal incision. Eventual entry and bimanual manipulation with a 23G mini-scissors was achieved through a second incision. Tissue samples were fixed in a sterile tube for further histopathological and immunohistochemical evaluation. RESULTS: Seven eyes of seven patients underwent biopsy using the forceps. The average thickness of the iris tumours was 1.07±0.79 mm. A second corneal incision for scissoring in a bimanual technique was necessary in 5 cases (71%). In 6 cases (85%), a precise histological and immunohistochemical diagnosis was achieved. Complications were limited to minute bleeding at the biopsy site and one case of relative pupil enlargement (anisocoria) without further refractive issues. CONCLUSIONS: Iris tumour biopsies can be successfully approached using a cavernous 23G intraocular forceps with a low risk for procedure-related complications. The conical interior design allows for removal of whole tissue pieces with minimal manipulative artefacts. An optional bimanual access through a second corneal incision and use of a 23G scissors provides better efficacy.

Three cases of intraocular mesectodermal leiomyoma expressing progesterone and androgen receptors.

BACKGROUND/AIMS: A prospective study identified three patients between 2004 and 2010 with mesectodermal leiomyoma. The study was conducted to analyse the presence or absence of sex steroid hormone receptors in mesectodermal leiomyomas. METHODS: The clinical features were collated. All three patients had operative procedures to either remove or sample the mesectodermal leiomyomas. The tissue was fixed in formalin and exposed to conventional histological processing. Immunohistochemistry using antibodies to androgen (AR), oestrogen (ER), and progesterone (PR) receptors was performed, followed by stain scoring to assess for expression status. RESULTS: All three cases were confirmed by histology to be examples of mesectodermal leiomyomas. All three expressed sex steroid hormone receptors. One case expressed both PR and AR, one case PR only and another case AR only. None of the cases expressed ER receptors. CONCLUSION: All three cases displayed some sex steroid hormone receptor expression. This is supportive evidence that sex steroid hormones may have a role in the pathogenesis of this tumour and suggest that it may be amenable to hormonal manipulation therapy, in a manner similar to conventional uterine leiomyomas.

Expression of vascular endothelial growth factor a, matrix metalloproteinase 9 and extravascular matrix patterns in iris and ciliary body melanomas.

PURPOSE: It was the aim of this study to assess the expression of vascular endothelial growth factor (VEGF)-A, matrix metalloproteinase (MMP)-9 and extravascular matrix patterns (EMPs) in iris and ciliary body melanomas and their correlations with histopathologic parameters. METHODS: The study was conducted on 3 iris and 15 ciliary body melanomas. All tumors were subjected to immunohistochemical techniques for VEGF-A and MMP-9 expressions, the presence of EMPs was assessed, and routine paraffin sections were stained with hematoxylin-eosin. Cell type, tumor localization, degree of pigmentation, necrosis, mitotic index, lymphocytic infiltration and sclera invasion were analyzed using light microscopy. RESULTS: The mean patient age at the time of treatment was 43 years (range 19-69, median 39.5); 10 (55.6%) patients were males and 8 (44.4%) females. Histopathological cell types were spindle cells in 55.6%, mixed cells in 16.7%, and epithelioid cell types in 27.8% of tumors. Positive reaction for VEGF-A and MMP-9 was present in 66.7 and 72.3% of the tumors, respectively. Microvascular loops and/or networks were seen in 33.4% of the tumors, with the remaining 66.7% of tumors displaying one or more of the other patterns. Metastatic disease developed in only 1 patient during follow-up. Tumor cell type, tumor size, mitotic rate, degree of pigmentation and EMPs were not correlated with metastasis. CONCLUSIONS: This study suggests that VEGF-A and MMP-9 were positive in the majority of iris and ciliary body melanomas. No correlation was found between VEGF-A and MMP-9 immunoreactivity and EMPs and occurrence of metastases in cases of anterior uveal melanoma.

Increased interleukin-6 in aqueous humor of a patient with lung-metastatic intraocular adenocarcinoma.

PURPOSE: We report increased interleukin (IL)-6 level in aqueous humor of a case of lung adenocarcinoma with metastasis to iris and anterior chamber. DESIGN: Interventional case report. METHODS: In one case and 11 healthy cataract patients as controls, we checked the levels of IL-6, IL-2, and tumor necrosis factor (TNF)-alpha in aqueous humor and serum by enzyme-linked immunosorbent assay. RESULTS: Levels of these three cytokines in serum were almost the same as with the levels in aqueous humor in the control group. The IL-6 concentrations ranged from 12.6 to 27.0 pg/ml (mean, 23.5 pg/ml) in serum and 15.0 to 43.1 pg/ml (mean, 22.4 pg/ml) in aqueous humor. There was no statistically significant difference between them. Compared with the control, the IL-6 concentration in aqueous humor of this patient, 282.0 pg/ml, increased more than 10-fold. CONCLUSION: This is a report describing elevated IL-6 level in the aqueous humor of a patient with intraocular metastasis from lung adenocarcinoma.

p53 Immunoreactivity, Ki-67 expression, and microcirculation patterns in melanoma of the iris, ciliary body, and choroid.

PURPOSE: The major role of p53 is to modulate cell proliferation, but recently, it has been found that p53 also modulates angiogenesis through several pathways. Because both cellular proliferation and microcirculation patterns are important prognostic markers in uveal melanoma, we tested the relationships between p53 expression, the expression of the cell proliferation marker Ki-67, and the presence of various microcirculation patterns in uveal melanoma. METHODS: Immunostaining of p53 and Ki-67 using the bp53.12 and the MIB-1 antibodies, respectively, were preformed in 98 uveal melanomas (18 melanomas confined to the iris, 30 ciliary body melanomas, and 50 choroidal melanomas). Percent of p53 positive cells, and the mean MIB-1 positive cell count per high power field were calculated in each section by two observers. Microcirculation patterns were assessed in adjacent PAS stained sections. RESULTS: p53 immunoreactivity was found in 14 of the 98 melanomas. High proliferative activity and epithelioid cell type were associated with p53 immunoreactivity. However, p53 immunoreactivity was not associated with any of the microcirculation patterns or with tumor location. CONCLUSIONS: The findings suggest that alterations in p53 expression are associated with the expression of the cellular proliferation marker, Ki-67, but are not associated with the presence of microcirculation patterns.

Uveal melanomas express vascular endothelial growth factor and basic fibroblast growth factor and support endothelial cell growth.

BACKGROUND: Tumour microvascularity is a significant determinant of prognosis for a large number of different tumours, including uveal melanoma. The development of blood vessels within these and other tumours is partly controlled by soluble pro-angiogenic cytokines, of which basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF) are the best described. METHODS: Because VEGF has been inconsistently found within uveal melanomas and bFGF is described as an autocrine growth factor in cutaneous melanoma, the authors looked at the expression of these cytokines in uveal melanomas using immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). The cross talk between uveal melanoma cells and endothelial cells was then assessed in an in vitro co-culture model. RESULTS: While most tumour cells expressed bFGF at the protein level by immunohistochemistry (89%), relatively few (22%) expressed VEGF, and this was of limited extent. All 20 tumours tested by RT-PCR contained mRNA for both bFGF and VEGF. Co-culture experiments using an ATP based bioassay showed that uveal melanomas could support the growth of a rat brain endothelial cell line (GPNT) and human umbilical vein endothelial cells (HUVEC), and that this could be modulated by cytokines and anti-cytokine antibodies. CONCLUSION: These results suggest that angiogenesis within uveal melanoma may be the result of a complex interplay between endothelial and tumour cells, and that bFGF and VEGF could play a part.

Iridocorneal melanoma associated with type 1 neurofibromatosis: a clinicopathologic study.

OBJECTIVE: A clinicopathologic study of an iridocorneal melanoma associated with type 1 (peripheral) neurofibromatosis is presented. DESIGN: Case report with clinicopathologic correlation. PARTICIPANT: A 32-year-old white woman with type 1 neurofibromatosis presented with long-standing blindness of her right eye due to diffuse intrastromal brown corneal discoloration. INTERVENTION: The patient underwent penetrating keratoplasty and the corneal button was inspected. RESULTS: Histopathologic evaluation of the corneal button after penetrating keratoplasty revealed an intrastromal mixed-type malignant melanoma, which stained positively with HMB-45 and S-100 protein and spared the corneal epithelium and limbus. The corneal graft remained transparent, with best-corrected visual acuity of 20/30. Twenty-two months after surgery, the tumor involved the anterior chamber angle and the iris. Three years later, it caused refractory glaucoma necessitating enucleation. The iris tumor did not extend beyond the iris-lens diaphragm and showed the same cytologic features as the corneal stromal tumor. CONCLUSION: To our best knowledge, this is the first report of iridocorneal melanoma associated with peripheral neurofibromatosis. The location of the tumor in the deep corneal stroma, without initial macroscopic involvement of the angle or iris, may suggest that the corneal portion of the tumor may have developed "in situ" rather than as an extension of iris melanoma. The common origin of melanoma cells and Schwann cells from the neural crest and the proliferation of the Schwann cells in neurofibromatosis provides additional support for this hypothesis.

A leiomyoma of the iris documented by immunohistochemistry and electron microscopy.

PURPOSE: To report an iris leiomyoma documented in accordance with today's stricter diagnostic criteria. METHODS: Light microscopy, immunohistochemistry and electron microscopy were performed on the 3 mm ball-like, greyish-white vascularized tumour of the iris, close to the sphincter pupillae. RESULTS: Light microscopy of the extirpated neoplasm showed the characteristic appearance of a leiomyoma with densely packed spindle-shaped cells, with oval nuclei and granular cytoplasm. On electron microscopic examination the tumour exhibited the characteristic features of a smooth muscle neoplasm. The immunohistochemistry was consistent with a myogenic tumour because the tumour cells were positive for smooth muscle actin and desmin, but negative for S-100 and melanin. CONCLUSION: The case illustrated the necessity of performing ancillary procedures such as electron microscopy and immunohistochemistry to substantiate a correct, although rare diagnosis.

Aspiration cytopathology of malignant melanoma in children. A morphologic spectrum.

Malignant melanoma (MM) is an uncommon neoplasm in the practice of pediatric cytopathology. The clinical and morphologic features of three white children with this neoplasm diagnosed by fine needle aspiration biopsy are described. All cytologic diagnoses were subsequently confirmed histologically. Two children were 16 years old and one was 9 years old. Two patients had metastatic MM to head and neck lymph nodes. In one of these children, a prior diagnosis of MM was known, whereas in the other it was unsuspected. A primary melanoma of the iris developed in the third child. The cytopathology of these children are similar to that described in adults. With the cytologic similarities, some striking differences were seen. Principal among these was the abundance of melanin in one case, its uneveness in another, and its absence in a third. The variation in individual cell morphology among the three cases is also described. Malignant melanoma is a rare neoplasm of children that can be recognized by fine-needle aspiration cytopathology.

S100 immunophenotypes of uveal melanomas.

To determine whether ocular melanomas are immunophenotypically identical to cutaneous melanomas, 34 primary and metastatic choroidal melanomas representing all major histotypes defined by the Callender's classification, plus one melanoma of the iris and one conjunctival melanoma, were subjected to a panel of immunostains designed to distinguish anaplastic biopsies of cutaneous melanomas from carcinomas and lymphomas. All ocular melanomas were found to express the intermediate filament vimentin but not keratin, and all but 2 were melanotic by immunostaining. Thirty-three of 34 (97%) choroidal melanomas were strongly stained with a rabbit polyclonal antibody (P-S100) developed against the S100 protein family. In contrast, none of 14 spindle cell type primary lesions was stained with a monoclonal antibody (MAB-079) specific for both S100 alpha and S100 beta, the best-characterized S100 polypeptides. Furthermore, only 2 of 5 epithelioid and 3 of 10 mixed-cell-type melanomas were weakly reactive. Overall, 14.7% (5 of 29) were stained. In comparison, MAB079 stained 85% of all cutaneous melanomas. Five metastases of choroidal melanomas (spindle B, epithelioid, and mixed cell types) from different organ sites also were stained by P-S100 but not by MAB079. These findings were corroborated by immunostaining with another monoclonal antibody (MAB4D4) specific for S100 beta. Differential staining by the polyclonal but not the monoclonal antibodies suggests the possible presence of a variant S100 polypeptide(s) in choroidal melanomas. Since S100 alpha, S100 beta, and related proteins appear to be physiologically important, additional studies of these S100 proteins may shed light on the etiology or pathology of choroidal melanomas.