Iris melanoma outcomes based on the Cancer Genome Atlas (TCGA) classification in 78 consecutive patients.

BACKGROUND: The Cancer Genome Atlas (TCGA) classification of genetic alterations in uveal melanoma is widely used for prognostication. We present novel observations on the impact of TCGA Group specifically for iris melanoma. METHODS: This was a retrospective cohort study at a tertiary referral ocular oncology center. All patients with a diagnosis of iris melanoma who underwent genetic evaluation and assessment for TCGA classification between 20 November 1995 and 5 April 2021 were included. The main outcome measures were visual acuity, secondary glaucoma, tumor recurrence, melanoma-related metastasis and death per TCGA group. RESULTS: There were a total of 78 patients included. The mean patient age was 49.6 years (median 53.0, range 3.0-85.0), mean tumor basal diameter was 6.7 mm (median 6.0, range 1.5-22.0), and mean tumor thickness was 2.6 mm (median 2.5, range 0.5-8.5). Cytology results confirmed iris melanoma (93%) or were inconclusive (7%). The TCGA groups included Group A (n = 36, 46%), Group B (n = 7, 9%), Group C (n = 34, 44%), and Group D (n = 1, 1%). There was no statistically significant difference in outcomes of visual acuity, tumor thickness reduction, secondary glaucoma, tumor recurrence, melanoma-related metastasis or death per individual TCGA group (A vs. B vs. C vs. D) and per bimodal comparison (A/B vs. C/D). CONCLUSIONS: In this analysis, iris melanoma was classified as TCGA group A or B in 55% and as C or D in 45%. The TCGA classification was not predictive of melanoma-related metastasis or death.

Molecular profiling of vitreous fluid by massively parallel sequencing: a case series.

INTRODUCTION: In cases of suspected intraocular malignancy, vitreous may be the preferred pathologic sample; however, cellularity may be insufficient for definitive cytopathological diagnosis. Ancillary methodology to study vitreous fluid aspiration for mutational analysis may assist in treatment decisions. MATERIALS AND METHODS: Three individual patient vitreous humor samples were received in the laboratory for mutation testing. The samples were collected during standard of care and analyzed for routine cytopathology. In each case, cytopathology was inconclusive and mutational analyses to support diagnostic suspicions were clinically requested. Based on the clinically and pathologically suspected diagnoses, an appropriate massively parallel sequencing assay previously validated for clinical use was performed using DNA extracted from vitreous samples that had previously undergone various processing. Nucleic acid yield was assessed by fluorometric or spectrophotometric methods, with yield ranging from 2.7 to 86.5 ng. Library preparations were performed using standard laboratory protocols. RESULTS: Two of the cases were suspicious for melanoma and a 50-gene solid tumor panel was performed. The third case was worrisome for vitreoretinal lymphoma and a 49-gene myeloid panel was performed. CONCLUSIONS: In all cases, the molecular profiling assisted with the clinical assessment and/or management of each patient.

Whole genome landscapes of uveal melanoma show an ultraviolet radiation signature in iris tumours.

Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease. Therapeutic options for metastatic UM are limited, with clinical trials having little impact. Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris). While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM. All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX). We identify three other significantly mutated genes (TP53, RPL5 and CENPE).

Iris pigmented lesions as a marker of cutaneous melanoma risk: an Australian case-control study.

BACKGROUND: Iris naevi and iris freckles have a frequency of 4% and 50% in the European population, respectively. They are associated with dysplastic naevi, but few studies have examined their link to cutaneous melanoma. OBJECTIVES: To assess whether iris pigmented lesions are a predictive indicator for cutaneous melanoma. METHODS: This is a melanoma case-control study of 1254 European-background Australians. Sun exposure and melanoma history, a saliva sample for DNA analysis and eye photographs taken with a digital camera were collected from 1117 participants. Iris images were assessed by up to four trained observers for the number of iris pigmented lesions. The data were analysed for correlations between iris pigmented lesions and melanoma history. RESULTS: Case participants over the age of 40 had similar numbers of iris pigmented lesions to age matched controls (mean 5·7 vs. 5·2, P = 0·02), but in younger case and control participants there was a greater difference (mean 3·96 vs. 2·19, P = 0·004). A logistic regression adjusted for age, sex, skin, hair and eye colour, skin freckling and naevus count found that the presence of three or more iris pigmented lesions increases the melanoma risk 1·45-fold [95% confidence interval (CI) 1·07-1·95]. HERC2/OCA2 rs12913832 and IRF4 rs12203592 influenced both eye colour and the number of iris pigmented lesions. On the HERC2/OCA2 A/A and A/G genotype background there was an increasing proportion of blue eye colour when carrying the IRF4 T allele (P = 3 × 10-4 ) and a higher number of iris pigmented lesions with the IRF4 T/T homozygote (P = 3 × 10-9 ). CONCLUSIONS: Iris pigmented lesion count provides additional predictive information for melanoma risk above that from conventional risk factors.

Genetic Background of Iris Melanomas and Iris Melanocytic Tumors of Uncertain Malignant Potential.

PURPOSE: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Iris melanoma comprises 4% to 10% of all UMs and has a lower mortality rate. The genetic changes in iris melanoma are not as well characterized as ciliary body or choroidal melanoma. The aim of this study was to gain more insight into the genetic background of iris melanoma and iris nevi. DESIGN: Multicenter, retrospective case series. PARTICIPANTS: Patients diagnosed with iris melanoma or iris nevi who underwent surgical intervention as primary or secondary treatment. METHODS: Next-generation sequencing of GNAQ, GNA11, EIF1AX, SF3B1, BAP1, NRAS, BRAF, PTEN, c-Kit, TP53, and TERT was performed on 30 iris melanomas and 7 iris nevi. Copy number status was detected using single nucleotide polymorphisms (SNPs) included in the next-generation sequencing (NGS) panel, SNP array, or fluorescent in situ hybridization. BAP1 immunohistochemistry was performed on all samples. MAIN OUTCOME MEASURES: Mutation and copy number status were analyzed. Results of BAP1 immunohistochemistry were used for survival analysis. RESULTS: In 26 of the 30 iris melanoma and all iris nevi, at least 1 mutation was identified. Multiple mutations were detected in 23 iris melanoma and 5 nevi, as well as mutations in GNAQ and GNA11. Furthermore, 13 of 30 BAP1, 5 of 30 EIF1AX, and 2 of 30 SF3B1 mutations were identified in iris melanoma. No correlation between BAP1 status and disease-free survival was found. The iris nevi showed 1 EIF1AX and 3 BAP1 mutations. Two of the nevi, with a BAP1 mutation, were histologically borderline malignant. Mutations in NRAS, BRAF, PTEN, c-KIT, and TP53 were detected in 6 iris melanomas and 4 iris nevi. CONCLUSIONS: Mutations that are often found in uveal and cutaneous melanoma were identified in this cohort of iris melanomas and iris nevi. Therefore, iris melanomas harbor a molecular profile comparable to both choroidal melanoma and cutaneous melanoma. These findings may offer adjuvant targeted therapies for iris melanoma. There was no prognostic significance of BAP1 expression as seen in choroidal melanoma. Consequently, iris melanoma is a distinct molecular subgroup of UM. Histologic borderline malignant iris nevi can harbor BAP1 mutations and may be designated iris melanocytic tumors of uncertain malignant potential.

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.

Genetic findings in treatment-naïve and proton-beam-radiated iris melanomas.

BACKGROUND/AIMS: Iris melanomas (IM) are rare and have a lower mortality than posterior uveal melanomas (UM). Our aims were to determine the prevalence of genetic changes associated with prognosis of posterior UM, in both treated and non-treated IM. METHODS: Retrospective database review and molecular analysis of all patients diagnosed with IM at the Liverpool Ocular Oncology Centre (LOOC) between 1993 and 2015. Archival pathology specimens of confirmed IM cases were analysed for chromosomal alterations, using multiplex ligation-dependent probe amplification (MLPA) or microsatellite analysis (MSA) depending on DNA yield, and BRAF mutation status. RESULTS: 5189 patients were diagnosed with intraocular melanoma at LOOC from 1993 to 2015. Of these, 303 (5.8%) patients were diagnosed with IM. Tissue samples were available for 26 IM cases. Twelve of these cases had biopsies taken post-proton beam radiotherapy (PBR). Histological subtyping showed 14 IM being spindle, 2 epithelioid and 10 were of mixed cell type. Twenty of the 26 IM cases (77%) analysed genetically were classified as either disomy 3 (n=16) or monosomy 3 (n=4). Chromosome 6p gain was detected in 4/18 (22%) IM, and polysomy 8q in 6%. BRAF mutations were not detected in any of the four IM cases examined. One patient with IM died from metastatic disease: this tumour was disomy 3 with 6p and 8q gains. All other patients were alive with no evidence of metastases at study closure. CONCLUSIONS: Chromosomal aberrations seen in posterior UM can also be demonstrated using MLPA or MSA in both treatment naïve and PBR-treated IM. Most IM display a low-metastatic risk chromosomal profile.

Uveal melanoma: the inflammatory microenvironment.

Uveal melanoma is a highly malignant intraocular tumor with quite homogeneous tumor tissue and a diffuse leukocytic infiltration. In contrast with many other malignancies, the presence of infiltrating macrophages and T cells is associated with a poor prognosis rather than a good one. The clear link between inflammation and cancer in this malignancy provides a paradigm for macrophage plasticity and function. Macrophages in uveal melanoma have an M2-like phenotype and are associated with the loss of one specific chromosome - monosomy 3. The central players involved in this process and discussed in this review include macrophages, T lymphocytes, chemokines and cytokines, including the macrophage-attraction molecules. When a tumor acquires the ability to release significant amounts of macrophage-attraction molecules it causes the expansion of a population of myeloid immature cells that may not only help the tumor to suppress immune reactions but also aid in the construction of new blood vessels for tumor growth. A better understanding of the molecular basis of a local myelomonocytic cell population will bring a better understanding of the immunopathology of this disease and will lead to therapeutic interventions in uveal melanoma. This review focuses on the roles of the local inflammatory microenvironment in the development and progression of uveal melanoma.

Cytogenetic testing of iris melanoma using fine needle aspiration biopsy in 17 patients.

PURPOSE: To determine the incidence of Chromosome 3 monosomy in iris melanoma using fine needle aspiration biopsy. METHODS: Noncomparative case series of 17 patients. Fine needle aspiration biopsy was performed intraoperatively immediately before treatment of iris melanoma. Genetic analysis using DNA amplification and microsatellite assay was performed in the specimen. RESULTS: Clinical features and outcomes related to Chromosome 3 monosomy were reviewed. Disomy 3 was found in 5 melanomas (29%), partial Monosomy 3 in 7 melanomas (41%), and complete Monosomy 3 in 5 melanomas (29%). The only feature statistically associated with partial/complete Monosomy 3 (vs. Disomy 3) was older patients' age (median, 60 vs. 46 years, P = 0.03). A comparison of clinical features showed Monosomy 3 (vs. Disomy 3) tumors to be thinner (median, 2.8 vs. 4.2 mm) and with smaller base (median, 5.1 vs. 10 mm) but with greater iris seeding (mean, 5.7 vs. 2.4 clock hours) and greater angle seeding (mean, 3.2 vs. 0 clock hours), producing elevated intraocular pressure <22 mmHg (17 vs. 0%). Monosomy 3 tumors showed mixed/epithelioid cell type in 80% versus 0% in Disomy 3 (P = 0.14). No patients developed local melanoma recurrence or melanoma-related metastasis or death in the short 16-month mean follow-up. CONCLUSION: Using fine needle aspiration biopsy, cytogenetic analysis can be achieved in iris melanoma. Iris melanoma demonstrated partial or complete Monosomy 3 in 71%, and this statistically correlated with increasing patients' age. Mixed/epithelioid cell type was far more commonly seen in patients with Monosomy 3, although this did not reach statistical significance.

Chromosomal aberrations in iris melanomas.

BACKGROUND: Uveal melanomas can develop in the choroid, ciliary body and iris. In choroidal and ciliary body melanomas, specific chromosomal changes correlate with metastatic disease. Iris melanomas have a better prognosis than choroidal melanomas, and it would be interesting to know if they share chromosomal changes. In addition, iris melanomas might harbour UV-induced mutations of tumour suppressor genes, such as PTEN and CDKN2A. METHODS: Twenty iris melanomas were analysed for chromosome 1p, 3, 6, 8, 9p and 10q abnormalities using fluorescence in situ hybridisation. These results were correlated to clinical follow-up data using statistical analyses. RESULTS: (Partial) loss of chromosome 3 was observed in nine iris melanomas, and gain of 8q was present in seven tumours. Loss of chromosome 9p was demonstrated in seven tumours, but no deletions of the PTEN region on chromosome 10 were found. Three patients died of metastatic disease, and one patient developed liver metastases, but is still alive. Univariate analysis indicated a lower disease-free survival for patients with diffuse growing melanomas (p=0.01), melanomas that lost a copy of chromosome 3 (p=0.03), or invading the ciliary body (p=0.01). In a multivariate analysis, none of the correlations were significant. CONCLUSION: Loss of chromosome 3 as well as loss of chromosomal region 9p21 (that entails tumour suppressor gene CDKN2A) plays a role in iris melanoma. A firm correlation with disease-free survival could not be established, possibly due to the small sample size.

Mixed-phenotype acute leukemia relapse in the iris.

Mixed-phenotype acute leukemia is a rare condition with no previously reported intraocular involvement. We present clinical, radiologic, and cytologic findings of leukemic intraocular relapse in a 23-month-old girl, with lineage switch presenting as conjunctivitis after allogeneic bone marrow transplantation. A diagnostic approach using fine needle aspiration is described.

A quantitative assessment of the burden and distribution of Lisch nodules in adults with neurofibromatosis type 1.

PURPOSE: The presence of two or more Lisch nodules (melanocytic hamartomas of the iris) is one of seven diagnostic criteria for neurofibromatosis type 1 (NF1), a common monogenic disorder of dysregulated neurocutaneous growth. The hypothesis that Lisch nodules arise secondary to exposure to ultraviolet (UV) radiation from sunlight was investigated. METHODS: Lisch nodule burden was mapped and quantified in the irides of 77 adults with NF1. Lifetime sunlight (UV radiation) exposure was inventoried, NF1 neurocutaneous severity determined, and two NF1 mutations predictive of severity selectively genotyped. RESULTS: There was high interindividual variability in Lisch nodule burden. Lisch nodules were primarily located in the inferior hemifield (half) of the iris, regardless of its color (P = 3.0 x 10(-20)). Light irides harbored significantly more Lisch nodules than dark irides (P = 4.8 x 10(-5)). There was no statistically significant correlation of Lisch nodule burden to lifetime sunlight exposure "dose" or NF1 neurocutaneous severity. CONCLUSIONS: The difference in Lisch nodule burden between the superior and inferior iris hemifields is most likely due to the sunlight-shielding effects on the superior half by periocular structures. The difference in Lisch nodule burden between light and dark irides is probably due to the photoprotective effects of pigmentation. The genes underlying the control of iris color may thus be viewed as modifiers of severity of Lisch nodule burden in NF1. Given the role of UV radiation and, presumably, DNA damage in Lisch nodule pathogenesis, "benign tumor of the iris," not "hamartoma," may be a better descriptor.

[Lisch nodules. Markers for a non-invasive diagnosis in Recklinghausen neurofibromatosis]. / Nodulii LISCH. Markeri de diagnostic noninvaziv in neurofibromatoza Recklinghausen.

INTRODUCTION: Von Recklinghausen's neurofibromatosis is an autosomal dominant disorder that affects both sexes at the same rate. The diagnosis is suggested by the family history, the presence of multiple neurofibromas and cutaneous pigmentary macules but is only confirmed by the presence of the Lisch nodules at the iris level and by histological exam. The disease is associated with different nervous system, bone, endocrine, blood vascular abnormalities. PURPOSE: Evaluation of Lisch nodules in patients with Recklinghausen disease or immediate relatives, that do not present characteristic cutaneous lesions. MATERIAL AND METHOD: The study involves three cases of Recklinghausen neurofibromatosis and also a part of their immediate relatives. The diagnosis of NF1 was based on clinical aspects, family pedigree analysis and ophthalmological exam. The histological exam was only performed for complicated cutaneous lesions. RESULTS AND DISCUSSIONS: The clinical exam revealed characteristic cutaneous lesions but also Lisch nodules in all three studied cases. According to the pedigrees, Recklinghausen disease is inherited as an autosomal dominant trait (in 2 patients), but it may also arise as de novo mutations (1 patient). The ophthalmological exam performed for the patients' relatives, identified Lisch nodules in all subjects presenting cutaneous lesions (neurofibromas), and also in children without characteristic cutaneous features. Both pedigree analysis and ophthalmological examination are compulsory, noninvasive methods for precocious diagnosis of the disease and the identification of high-risk patients. CONCLUSIONS: Lisch nodules are ophthalmological and pathognomonic markers of Recklinghausen neurofibromatosis. Lisch nodules facilitate a precocious, noninvasive diagnosis inclusively in children without cutaneous features (neurofibromas) but positive antecedents. Ophthalmological examination and pedigree analysis are compulsory both in patients and their immediate relatives.

The T1799A BRAF mutation is present in iris melanoma.

PURPOSE: An activating mutation in exon 15 of the BRAF gene has been found in a high proportion of cutaneous pigmented lesions, but only in one case of uveal melanoma. Iris melanoma is the least common uveal melanoma and displays a less aggressive clinical course compared with posterior uveal melanoma. To date, no study has been conducted to investigate the T1799A mutation in iris melanoma. The purpose of this study was to determine whether the T1799A BRAF mutation is present in iris melanoma. METHODS: DNA was extracted from 19 archival, paraffin-embedded tissue sections of iris melanomas. Nested PCR was used to amplify exon 15 of the BRAF gene, and the product was purified, cloned into a sequencing vector, and sequenced. The sequences obtained were compared with the wild-type sequence of the BRAF gene. The presence or absence of the BRAF mutation was also compared with the clinicopathological features. RESULTS: The T1799A mutation was identified by sequencing in 9 of 19 iris melanomas. Six of the 9 cases with the BRAF mutation were recurrent tumors. All other tumors were resections for primary tumors. There was a statistically significant association between the BRAF mutation and recurrent tumor (P = 0.003). There was no association between the presence of the BRAF mutation and other clinicopathological characteristics. CONCLUSIONS: In this small study, the T1799A BRAF mutation was identified in almost half of the iris melanoma tissues samples examined. This finding suggests that there may be genetic as well as clinical differences between iris and posterior uveal melanomas.

Unilateral Lisch nodules in the absence of other features of neurofibromatosis 1.

PURPOSE: To report a 14-year-old boy with unilateral Lisch nodules without any other diagnostic features of neurofibromatosis type 1. DESIGN: Observational case report. METHODS: A complete ophthalmologic examination and medical genetics examination was performed. RESULTS: By clinical examination, multiple Lisch nodules were identified in the left eye. CONCLUSIONS: Lisch nodules, in the absence of other diagnostic features of neurofibromatosis type 1, are an atypical finding.

Cytogenetics of iris melanomas: disparity with other uveal tract melanomas.

The chromosomal alterations of iris melanomas are poorly characterized, only one report has been detailed. Cytogenetic analysis was performed on the tumors and heparinized blood samples of three patients with iris melanomas; in one case a primary tumor and its related seedling were examined. On analysis of lymphocytes, two of the patients were found to experience a low level fragility of chromosome 9, in the region of a cutaneous melanoma susceptibility gene. All iris melanoma lesions were karyotyped. Clonal abnormalities of chromosomes 3, 5, 6, 7, 8, 9, 12, 15, 17, 18, 19, and Y were found, and in one case a large number of marker chromosomes were observed. No specific chromosomal change was common to the iris melanomas, but two cases had different abnormalities of chromosomes 5 and 18. Variations between the primary tumor and its related seedling were the acquisition of an additional chromosome 15, and a polyploid form of the cell line in the seedling. This study suggests that the most common chromosomal changes of posterior uveal melanomas are less frequent in iris melanomas. Iris melanomas also appear to experience relatively high levels of chromosomal alterations, including the formation of marker chromosomes, which is perhaps reminiscent of cutaneous melanoma.

[Lisch nodules. Markers of neurofibromatosis 1 and immunohistochemical references for neuroectodermal differentiation]. / Lisch-Knötchen. Marker der Neurofibromatose I und immunhistochemische Hinweise für eine neuroektodermale Differenzierung.

Iris nodules in neurofibromatosis I have become an important tool in the differential diagnosis of phakomatoses. The clinical appearance and importance of these nodules first recognized by Karl Lisch in Munich in 1937. The diagnosis and differential diagnosis of Lisch nodules are illustrated. The importance of iris nodules in genetic counselling of patients and their relatives is discussed, with emphasis on monosymptomatic cases. Histologically Lisch nodules are formed by aggregations of oval to round cells that form dome-shaped papules on the anterior layer of the iris. Immunohistochemically these cells are characterized by positive staining with antibodies against vimentin and S-100 protein. This proves their ectodermal differentiation. Thus Lisch nodules can be seen as a direct manifestation of neuroectodermal disturbances in neurofibromatosis I.