Monoclonal antibodies to uveal melanoma

This thesis concerns the immunology of uveal melanoma It is introduced by a critical review of the literature on this neoplasm, and on tumour immunology with special reference to melanoma Two main conclusions could be drawn from this review Firstly, further progress in the immunology of uveal melanoma is dependent on a better understanding of the antigenicity of this tumour and on the availability of suitable antigens for testing the anti-tumour immune response Secondly, the best chance of achieving these objectives, at present, is by the application of monoclonal antibody technology Accordingly, the main aims of this study were to determine the feasibility and scope of producing monoclonal antibodies to uveal melanoma Monoclonal antibodies were prepared using the rat hybridoma system Tissue specimens from over 80 uveal melanomas were used to immunise rats and for the necessary immuno-assays Hybridomas were screened by ELISA Eleven monoclonal antibodies were produced Most of these were of the IgM isotype and all reacted with intracellular antigens The most specific was mAb 4A3 Nevertheless, immunohistochemistry on frozen and fixed sections and immunofluorescence microscopy using cell suspensions showed that this antibody reacted with several types of normal and malignant cells Western blotting demonstrated that the 4A3 antigen had a molecular weight of 55-62 kD The 4A3 antigen was detected in the subretinal fluid of five patients with uveal melanoma with the use of this technique, and in one of two patients with rhegmatogenous retinal detachment ELISA reactivity of the rat monoclonal antibodies with uveal melanoma cells was inhibited by serum from 10 out of 12 patients with uveal melanoma and from two out of eight healthy individuals B Lymphocytes of patients with uveal melanoma were EBV transformed in vitro and tested by ELISA for reactivity with autologous tumour The results were similar to those obtained with lymphocytes from healthy individuals This suggested that such techniques were inadequate for analysing humoral immunity to melanoma and unsuitable for the production of human monoclonal antibodies to this tumour In conclusion, specific monoclonal antibodies to uveal melanoma could be very useful, but such antibodies are unlikely to be produced using conventional methods Better results will probably be achieved if uncultured tissue from a single tumour were used for all immunisation and assay procedures required for one fusion, and if more efficient techniques for selecting hybridomas were available

Is tumour thickness measurement required for MOLES scoring of melanocytic choroidal tumours?

Introduction: It can be challenging to distinguish between choroidal naevi and melanomas in the community setting, particularly without access to ultrasonography, required to measure the thickness of melanocytic choroidal tumours. We aimed to determine whether thickness measurement is required for MOLES scoring of melanocytic choroidal tumours. Methods: The dataset of a recent MOLES evaluation was reviewed. Patients were selected for the present study if their MOLES tumour size category was determined by tumour thickness measured with ultrasonography (US). The largest basal tumour diameter and tumour thickness were then measured from ultra-widefield fundus images and optical coherence tomography (OCT) images, respectively. Results: The tumour size category was determined by tumour diameter in 203/222 (91.4%) with no influence of tumour thickness. The tumour thickness influenced the MOLES score in 19/222 (8.6%) patients. In 11/19 patients with OCT measurements of tumour thickness, the US measurement exceeded the OCT by more than 25% in 5 patients, more than 50% in 2 patients and more than 75% in 1 patient. As a result, the revised tumour thickness based on OCT determined the size category in 4/216 (1.8%) patients. The Optos measurements increased the diameter score by 1 in 5 patients. As a result, the revised tumour thickness determined the size category in 4/216 (1.8%) patients. If both the revised diameter and thickness scores were considered, the MOLES score reduced in 4 patients. If both the diameter and thickness scores were considered, the MOLES score reduced in 5 and increased in 1. Only 0.94% (2/211) of melanocytic choroidal tumours assessed with MOLES when using Optos ultra-widefield fundus images diameter and OCT to measure tumour diameter and thickness, respectively, required a change in management from a reduction in MOLES score from 1 to 0. Discussion/Conclusion: This study suggests that the MOLES category for size is influenced more by the tumour diameter, if it can be measured accurately, than by the thickness. This study suggests ignoring tumour thickness if this cannot be measured accurately with OCT, unless the tumour has a mushroom shape

Cluster analysis of multiplex ligation-dependent probe amplification data in choroidal melanoma.

PurposeTo determine underlying correlations in multiplex ligation-dependent probe amplification (MLPA) data and their significance regarding survival following treatment of choroidal melanoma (CM).MethodsMLPA data were available for 31 loci across four chromosomes (1p, 3, 6, and 8) in tumor material obtained from 602 patients with CM treated at the Liverpool Ocular Oncology Center (LOOC) between 1993 and 2012. Data representing chromosomes 3 and 8q were analyzed in depth since their association with CM patient survival is well-known. Unsupervised k-means cluster analysis was performed to detect latent structure in the data set. Principal component analysis (PCA) was also performed to determine the intrinsic dimensionality of the data. Survival analyses of the identified clusters were performed using Kaplan-Meier (KM) and log-rank statistical tests. Correlation with largest basal tumor diameter (LTD) was investigated.ResultsChromosome 3: A two-cluster (bimodal) solution was found in chromosome 3, characterized by centroids at unilaterally normal probe values and unilateral deletion. There was a large, significant difference in the survival characteristics of the two clusters (log-rank, p<0.001; 5-year survival: 80% versus 40%). Both clusters had a broad distribution in LTD, although larger tumors were characteristically in the poorer outcome group (Mann-Whitney, p<0.001). Threshold values of 0.85 for deletion and 1.15 for gain optimized the classification of the clusters. PCA showed that the first principal component (PC1) contained more than 80% of the data set variance and all of the bimodality, with uniform coefficients (0.28±0.03). Chromosome 8q: No clusters were found in chromosome 8q. Using a conventional threshold-based definition of 8q gain, and in conjunction with the chromosome 3 clusters, three prognostic groups were identified: chromosomes 3 and 8q both normal, either chromosome 3 or 8q abnormal, and both chromosomes 3 and 8q abnormal. KM analysis showed 5-year survival figures of approximately 97%, 80%, and 30% for these prognostic groups, respectively (log-rank, p<0.001). All MLPA probes within both chromosomes were significantly correlated with each other (Spearman, p<0.001).ConclusionsWithin chromosome 3, the strong correlation between the MLPA variables and the uniform coefficients from the PCA indicates a lack of evidence for a signature gene that might account for the bimodality we observed. We hypothesize that the two clusters we found correspond to binary underlying states of complete monosomy or disomy 3 and that these states are sampled by the complete ensemble of probes. Consequently, we would expect a similar pattern to emerge in higher-resolution MLPA data sets. LTD may be a significant confounding factor. Considering chromosome 8q, we found that chromosome 3 cluster membership and 8q gain as traditionally defined have an indistinguishable impact on patient outcome

Baerveldt implant for secondary glaucoma due to iris melanoma

Annelie N Tan1, Juliette GMM Hoevenaars1, Carroll AB Webers1, Bertil Damato2, Henny JM Beckers11University Eye Clinic, Maastricht, The Netherlands; 2Ocular Oncology Service Royal Liverpool University Hospital, Liverpool, United KingdomBackground: Proton beam therapy (PBT) is effective in the treatment of iris melanoma. Reported complications after PBT are radiation-induced cataract and raised intraocular pressure (IOP). Filtering glaucoma surgery has generally been avoided because of fears of seeding.Case report: A 37-year-old man presented with a self-discovered, pigmented lesion on his right iris. Four years later, the pigmented lesion was diagnosed as an iris melanoma, because of documented growth. The patient was treated with PBT but developed secondary glaucoma one month later. The IOP could not be controlled despite maximal medical therapy and selective laser trabeculoplasty (SLT). Finally, Baerveldt implant surgery was performed, resulting in an IOP lowering to 10 mmHg and stabilization of the glaucomatous visual field loss.Conclusion: Our case demonstrates that Baerveldt implant surgery is a reasonable therapy for glaucoma following successful radiotherapy of iris melanoma.Keywords: iris melanoma, proton beam therapy, secondary glaucoma, Baerveldt implant surger

Clinical and Epidemiologic Research Devising Two-Stage and Multistage Phase II Studies on Systemic Adjuvant Therapy for Uveal Melanoma

PURPOSE. Almost all uveal melanomas showing chromosome 3 loss (i.e., monosomy 3) are fatal. Randomized clinical trials are therefore needed to evaluate various systemic adjuvant therapies. Conventional trial designs require large numbers of patients, which are difficult to achieve in a rare disease. The aim of this study was to use existing data to estimate how sample size and study duration could be reduced by selecting high-risk patients and adopting multistage trial designs. METHODS. We identified 217 patients with a monosomy 3 melanoma exceeding 15 mm in basal diameter; these patients had a median survival of 3.27 years. Several trial designs comparing overall survival were explored for such a population. A power of 0.90 to detect a hazard ratio of 0.737 was set, and recruitment of 16 patients per month was assumed. RESULTS. A suitable single-stage study would require 960 patients and a duration of 76 months. A two-stage design with an interim analysis based on 852 patients after 53.3 months would have a 50% probability of stopping because no statistically significant treatment effect is seen. Encouraging but inconclusive results would require a further 108 patients and prolongation of the study to 77.2 months. A multistage design would have a 43% probability of stopping before 47 months having recruited 759 patients. CONCLUSIONS. Prospects for clinical studies of systemic adjuvant therapy for uveal melanoma are enhanced by multistage trial designs enrolling only high-risk patients. (Invest Ophthalmol Vis Sci. 2012;53:4986-4989

A Case of Metastatic Atypical Neuroendocrine Tumor with ALK Translocation and Diffuse Brain Metastases.

A challenge in precision medicine requires identification of actionable driver mutations. Critical to such effort is the deployment of sensitive and well-validated assays for mutation detection. Although identification of such alterations within the tumor tissue remains the gold standard, many advanced non-small cell lung cancer cases have only limited tissue samples, derived from small biopsies or fine-needle aspirates, available for testing. More recently, noninvasive methods using either circulating tumor cells or tumor DNA (ctDNA) have become an alternative method for identifying molecular biomarkers and screening patients eligible for targeted therapies. In this article, we present a case of a 52-year-old never-smoking male who presented with widely metastatic atypical neuroendocrine tumor to the bones and the brain. Molecular genotyping using DNA harvested from a bone metastasis was unsuccessful due to limited material. Subsequent ctDNA analysis revealed an ALK translocation. The clinical significance of the mutation in this particular cancer type and therapeutic strategies are discussed.Key pointsTo our knowledge, this index case represents the first reported ALK translocation identified in an atypical carcinoid tumor.Liquid biopsy such as circulating tumor DNA is a feasible alternative platform for identifying sensitizing genomic alterations.Second-generation ALK inhibitors represent a new paradigm for treating ALK-positive patients with brain metastases

Multicenter, International Assessment of the Eighth Edition of the American Joint Committee on Cancer Cancer Staging Manual for Conjunctival Melanoma

This case series assesses whether the eighth edition of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual can be used to accurately estimate mortality rates of conjunctival melanoma. Key PointsQuestionCan the eighth edition of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual for conjunctival melanoma be used to accurately estimate metastasis and mortality rates? FindingsIn this case series of 288 patients, there was a significantly higher cumulative mortality rate among patients with cT2 and cT3 conjunctival melanoma compared with those presenting with cT1 conjunctival melanoma. MeaningHigher T-staged tumors were associated with both an earlier and greater incidence of metastasis; therefore, the results from this multicenter, international registry study support the use of the eighth edition AJCC staging system for conjunctival melanoma. ImportanceEye cancer staging systems used for standardizing patient care and research need to be validated. ObjectiveTo evaluate the accuracy of the eighth edition of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual in estimating metastatis and mortality rates of conjunctival melanoma. Design, Setting, and ParticipantsThis international, multicenter, registry-based case series pooled data from 10 ophthalmic oncology centers from 9 countries on 4 continents. A total of 288 patients diagnosed with conjunctival melanoma from January 1, 2001, to December 31, 2013, were studied. Data analysis was performed from July 7, 2018, to September 11, 2018. InterventionsTreatments included excision biopsy, cryotherapy, topical chemotherapy, radiation therapy, enucleation, and exenteration. Main Outcomes and MeasuresMetastasis rates and 5-year and 10-year Kaplan-Meier mortality rates according to the clinical T categories and subcategories of the eighth edition of the AJCC Cancer Staging Manual. ResultsA total of 288 eyes from 288 patients (mean [SD] age, 59.7 [16.8] years; 147 [51.0%] male) with conjunctival melanoma were studied. Clinical primary tumors (cT) were staged at presentation as cT1 in 218 patients (75.7%), cT2 in 34 (11.8%), cT3 in 15 (5.2%), and cTx in 21 (7.3%). There were no T4 tumors. Pathological T categories (pT) were pTis in 43 patients (14.9%), pT1 in 169 (58.7%), pT2 in 33 (11.5%), pT3 in 12 (4.2%), and pTx in 31 (10.8%). Metastasis at presentation was seen in 5 patients (1.7%). Metastasis during follow-up developed in 24 patients (8.5%) after a median time of 4.3 years (interquartile range, 2.9-6.0 years). Of the 288 patients, 29 died (melanoma-related mortality, 10.1%) at a median time of 5.3 years (interquartile range, 1.8-7.0 years). The cumulative rates of mortality among patients with cT1 tumors were 0% at 1 year, 2.5% (95% CI, 0.7%-7.7%) at 5 years, and 15.2% (95% CI, 8.1%-27.4%) at 10 years of follow-up; among patients with cT2 tumors, 0% at 1 year, 28.6% (95% CI, 12.9%-58.4%) at 5 years, and 43.6% (95% CI, 19.6%-77.9%) at 10 years of follow-up; and among patients with cT3 tumors, 21.1% (95% CI, 8.1%-52.7%) at 1 year of follow-up and 31.6% (95% CI, 13.5%-64.9%) at 5 years of follow-up. Patients with cT2 and cT3 tumors had a significantly higher cumulative mortality rate compared with those presenting with cT1 tumors (log-rank PPeer reviewe

Choroidal melanoma in a 7-year-old child treated by trans-scleral local resection

Abstract Purpose To report a choroidal melanoma in a 7-year-old child treated by trans-scleral local resection and adjuvant brachytherapy with a family history of neurofibromatosis type I (NF1) and cutaneous melanoma. Patient and methods A 7-year-old child was referred for treatment of a choroidal tumor in her left eye with a differential diagnosis of melanoma, neurilemmoma, leiomyoma, and neurofibroma. Trans-scleral local resection and, subsequently, adjuvant brachytherapy were performed. Results Histopathology and immunohistochemistry of the specimen diagnosed an amelanotic melanoma of spindle cell type, with a moderately high number of mitoses (7/40 HPF). Multiplex ligation-dependent probe amplification (MLPA) analysis showed two copies of chromosome 3, three copies of the short arm of chromosome 6, and two copies of chromosome 8, strongly suggesting a good prognosis. Postoperative ophthalmic evaluation at 6 months showed no visible tumor and flat retina with visual acuity (VA) of 6/60. Conclusions Trans-scleral local resection with adjuvant brachytherapy in children is possible using the same techniques as for adults. Although the follow-up is short, our patient retained the eye with good vision and our cytogenetic studies allowed us to reassure the mother