Uveal melanoma: Towards a molecular understanding

Uveal melanoma is an aggressive malignancy that originates from melanocytes in the eye. Even if the primary tumor has been successfully treated with radiation or surgery, up to half of all UM patients will eventually develop metastatic disease. Despite the common origin from neural crest-derived cells, uveal and cutaneous melanoma have few overlapping genetic signatures and uveal melanoma has been shown to have a lower mutational burden. As a consequence, many therapies that have proven effective in cutaneous melanoma -such as immunotherapy- have little or no success in uveal melanoma. Several independent studies have recently identified the underlying genetic aberrancies in uveal melanoma, which allow improved tumor classification and prognostication of metastatic disease. In most cases, activating mutations in the Gα11/Q pathway drive uveal melanoma oncogenesis, whereas mutations in the BAP1, SF3B1 or EIF1AX genes predict progression towards metastasis. Intriguingly, the composition of chromosomal anomalies of chromosome 3, 6 and 8, shown to correlate with an adverse outcome, are distinctive in the BAP1mut, SF3B1mut and EIF1AXmut uveal melanoma subtypes. Expression profiling and epigenetic studies underline this subdivision in high-, intermediate-, or low-metastatic risk subgroups and suggest a different approach in the future towards prevention and/or treatment based on the specific mutation present in the tumor of the patients. In this review we discuss the current knowledge of the underlying genetic events that lead to uveal melanoma, their implication for the disease course and prognosis, as well as the therapeutic possibilities that arise from targeting these different aberrant pathways

Zr-89-pembrolizumab biodistribution is influenced by PD-1-mediated uptake in lymphoid organs

Background To better predict response to immune checkpoint therapy and toxicity in healthy tissues, insight in the in vivo behavior of immune checkpoint targeting monoclonal antibodies is essential. Therefore, we aimed to study in vivo pharmacokinetics and whole-body distribution of zirconium-89 (Zr-89) labeled programmed cell death protein-1 (PD-1) targeting pembrolizumab with positron-emission tomography (PET) in humanized mice. Methods Humanized (huNOG) and non-humanized NOG mice were xenografted with human A375M melanoma cells. PET imaging was performed on day 7 post(89)Zr-pembrolizumab (10 mu g, 2.5 MBq) administration, followed by ex vivo biodistribution studies. Other huNOG mice bearing A375M tumors received a co-injection of excess (90 mu g) unlabeled pembrolizumab or(89)Zr-IgG(4)control (10 mu g, 2.5 MBq). Tumor and spleen tissue were studied with autoradiography and immunohistochemically including PD-1. Results PET imaging and biodistribution studies showed high(89)Zr-pembrolizumab uptake in tissues containing human immune cells, including spleen, lymph nodes and bone marrow. Tumor uptake of(89)Zr-pembrolizumab was lower than uptake in lymphoid tissues, but higher than uptake in other organs. High uptake in lymphoid tissues could be reduced by excess unlabeled pembrolizumab. Tracer activity in blood pool was increased by addition of unlabeled pembrolizumab, but tumor uptake was not affected. Autoradiography supported PET findings and immunohistochemical staining on spleen and lymph node tissue showed PD-1 positive cells, whereas tumor tissue was PD-1 negative. Conclusion Zr-89-pembrolizumab whole-body biodistribution showed high PD-1-mediated uptake in lymphoid tissues, such as spleen, lymph nodes and bone marrow, and modest tumor uptake. Our data may enable evaluation of(89)Zr-pembrolizumab whole-body distribution in patients

SRSF2 Mutations in Uveal Melanoma: A Preference for In-Frame Deletions?

Background: Uveal melanoma (UM) is the most common primary ocular malignancy in adults in the Western world. UM with a mutation in SF3B1, a spliceosome gene, is characterized by three or more structural changes of chromosome 1, 6, 8, 9, or 11. Also UM without a mutation in SF3B1 harbors similar chromosomal aberrations. Since, in addition to SF3B1, mutations in U2AF1 and SRSF2 have also been observed in hematological malignancies, UM without a SF3B1 mutation—but with the characteristic chromosomal pattern—might harbor mutations in one of these genes. Methods: 42 UMs were selected based on their chromosomal profile and wildtype SF3B1 status. Sanger sequencing covering the U2AF1 (exon 2 and 7) hotspots and SRSF2 (exon 1 and 2) was performed on DNA extracted from tumor tissue. Data of three UM with an SRSF2 mutation was extracted from the The Cancer Genome Atlas (TCGA). Results: Heterozygous in-frame SRSF2 deletions affecting amino acids 92–100 were detected in two UMs (5%) of 42 selected tumors and in three TGCA UM specimens. Both the UM with an SRSF2 mutation from our cohort and the UM samples from the TCGA showed more than four structural chromosomal aberrations including (partial) gain of chromosome 6 and 8, although in two TCGA UMs monosomy 3 was observed. Conclusions: Whereas in myelodysplastic syndrome predominantly missense SRSF2 mutations are described, the observed SRSF2 mutations in UM are all in-frame deletions of 8–9 amino acids. This suggests that the R625 missense SF3B1 mutations and SRSF2 mutations in UM are different compared to the spliceosome gene mutations in hematological cancers, and probably target a different, as yet unknown, set of genes involved in uv

Clinical-grade N-(4-[18F]fluorobenzoyl)-interleukin-2 for PET imaging of activated T-cells in humans

BACKGROUND: Molecular imaging of immune cells might be a potential tool for response prediction, treatment evaluation and patient selection in inflammatory diseases as well as oncology. Targeting interleukin-2 (IL2) receptors on activated T-cells using positron emission tomography (PET) with N-(4-[18F]fluorobenzoyl)-interleukin-2 ([18F]FB-IL2) could be such a strategy. This paper describes the challenging translation of the partly manual labeling of [18F]FB-IL2 for preclinical studies into an automated procedure following Good Manufacturing Practices (GMP), resulting in a radiopharmaceutical suitable for clinical use. METHODS: The preclinical synthesis of [18F]FB-IL2 was the starting point for translation to a clinical production method. To overcome several challenges, major adaptations in the production process were executed. The final analytical methods and production method were validated and documented. All data with regards to the quality and safety of the final drug product were documented in an investigational medicinal product dossier. RESULTS: Restrictions in the [18F]FB-IL2 production were imposed by hardware configuration of the automated synthesis equipment and by use of disposable cassettes. Critical steps in the [18F]FB-IL2 production comprised the purification method, stability of recombinant human IL2 and the final formulation. With the GMP compliant production method, [18F]FB-IL2 could reliably be produced with consistent quality complying to all specifications. CONCLUSIONS: To enable the use of [18F]FB-IL2 in clinical studies, a fully automated GMP compliant production process was developed. [18F]FB-IL2 is now produced consistently for use in clinical studies

Metastatic disease in polyploid uveal melanoma patients is associated with BAP1 mutations

PURPOSE. Most of the uvea melanoma (UM) display a near-diploid (normal, ~2N) karyotype with only a few chromosomal changes. In contrast to these simple aberrations 18% of the UM samples show a polyploid character (>2N) and this was associated with an unfavorable prognosis. This study attempts to gain insight in the prognostic value of polyploidy in UM. METHODS. In 202 patients the ploidy status of the UM was determined using cytogenetic analysis, fluorescence-in-situ-hybridization (FISH), multiplex ligation dependent probe amplification (MLPA), and/or single nucleotide polymorphism (SNP) array analysis. Immunohistochemistry was used to determine the BAP1 expression and mutation analyses of BAP1 (coding regions) and the mutation hotspots for the SF3B1, EIF1AX, GNAQ, and GNA11 genes was carried out using Sanger sequencing or whole-exome sequencing. RESULTS. Twenty-three patients had a polyploid UM karyotype (11.4%). Patients with a polyploid tumor had larger tumors (15.61 vs. 13.13 mm, P = 0.004), and more often loss of heterozygosity of chromosome 3 (P ¼ 0.003). No difference in occurrence of mutations between polyploid and diploid tumors was observed for BAP1, SF3B1, EIF1AX, GNAQ, and GNA11. Polyploidy did not affect survival (P = 0.143). BAP1 deficiency was the only significant independent prognostic predictor for patients with polyploid tumors, with a 16- fold increased hazard ratio (HR 15.90, P = 0.009). CONCLUSIONS. The prevalence of mutations in the UM related genes is not different in polyploid UM compared with diploid UM. Moreover, similar to patients with diploid UM, BAP1 mutation is the most significant prognostic predictor of metastasis in patients with polyploid UM

Chromosomal rearrangements in uveal melanoma: Chromothripsis

Uveal melanoma (UM) is the most common primary intraocular malignancy in the Western world. Recurrent mutations in GNAQ, GNA11, CYSLTR2, PLCB4, BAP1, EIF1AX, and SF3B1 are described as well as non-random chromosomal aberrations. Chromothripsis is a rare event in which chromosomes are shattered and rearranged and has been reported in a variety of cancers including UM. SNP arrays of 249 UM from patients who underwent enucleation, biopsy or endoresection were reviewed for the presence of chromothripsis. Chromothripsis was defined as ten or more breakpoints per chromosome involved. Genetic analysis of GNAQ, GNA11, BAP1, SF3B1, and EIF1AX was conducted using Sanger and next-generation sequencing. In addition, immunohistochemistry for BAP1 was performed. Chromothripsis was detected in 7 out of 249 tumors and the affected chromosomes were chromosomes 3, 5, 6, 8, 12, and 13. The mean total of fragments per chromosome was 39.8 (range 12-116). In 1 UM, chromothripsis was present in 2 different chromosomes. GNAQ, GNA11 or CYSLTR2 mutations were present in 6 of these tumors and 5 tumors harbored a BAP1 mutation and/or lacked BAP1 protein expression by immunohistochemistry. Four of these tumors metastasized and for the fifth only short follow-up data are available. One of these metastatic tumors harbored an SF3B1 mutation. No EIF1AX mutations were detected in any of the tumors. To conclude, chromothripsis is a rare event in UM, occurring in 2.8% of samples and without significant association with mutations in any of the common UM driver genes

Potential red-flag identification of colorectal adenomas with wide-field fluorescence molecular endoscopy

Adenoma miss rates in colonoscopy are unacceptably high, especially for sessile serrated adenomas / polyps (SSA/Ps) and in high-risk populations, such as patients with Lynch syndrome. Detection rates may be improved by fluorescence molecular endoscopy (FME), which allows morphological visualization of lesions with high-definition white-light imaging as well as fluorescence-guided identification of lesions with a specific molecular marker. In a clinical proof-of-principal study, we investigated FME for colorectal adenoma detection, using a fluorescently labelled antibody (bevacizumab-800CW) against vascular endothelial growth factor A (VEGFA), which is highly upregulated in colorectal adenomas. Methods: Patients with familial adenomatous polyposis (n = 17), received an intravenous injection with 4.5, 10 or 25 mg of bevacizumab-800CW. Three days later, they received NIR-FME. Results: VEGFA-targeted NIR-FME detected colorectal adenomas at all doses. Best results were achieved in the highest (25 mg) cohort, which even detected small adenomas ( < 3 mm). Spectroscopy analyses of freshly excised specimen demonstrated the highest adenoma-to-normal ratio of 1.84 for the 25 mg cohort, with a calculated median tracer concentration in adenomas of 6.43 nmol/mL. Ex vivo signal analyses demonstrated NIR fluorescence within the dysplastic areas of the adenomas. Conclusion: These results suggest that NIR-FME is clinically feasible as a real-time, red-flag technique for detection of colorectal adenomas

The development of a novel diagnostic PCR for Madurella mycetomatis using a comparative genome approach

BACKGROUND: Eumycetoma is a neglected tropical disease most commonly caused by the fungus Madurella mycetomatis. Identification of eumycetoma causative agents can only be reliably performed by molecular identification, most commonly by species-specific PCR. The current M. mycetomatis specific PCR primers were recently discovered to cross-react with Madurella pseudomycetomatis. Here, we used a comparative genome approach to develop a new M. mycetomatis specific PCR for species identification. METHODOLOGY: Predicted-protein coding sequences unique to M. mycetomatis were first identified in BLASTCLUST based on E-value, size and presence of orthologues. Primers were then developed for 16 unique sequences and evaluated against 60 M. mycetomatis isolates and other eumycetoma causing agents including the Madurella sibling species. Out of the 16, only one was found to be specific to M. mycetomatis. CONCLUSION: We have discovered a predicted-protein coding sequence unique to M. mycetomatis and have developed a new species-specific PCR to be used as a novel diagnostic marker for M. mycetomatis

Aberrant microRNA expression and its implications for uveal melanoma metastasis

Uveal melanoma (UM) is the most frequently found primary intra-ocular tumor in adults. It is a highly aggressive cancer that causes metastasis-related mortality in up to half of the patients. Many independent studies have reported somatic genetic changes associated with high metastatic risk, such as monosomy of chromosome 3 and mutations in BAP1. Still, the mechanisms that drive metastatic spread are largely unknown