CRISPR/Cas9-mediated demethylation of FOXP3-TSDR toward Treg-characteristic programming of Jurkat T cells.

Demethylation of FOXP3-TSDR (Treg specific demethylated region) is a hallmark of stable differentiation and suppressive function of regulatory T (Treg) cells. Previous protocols aiming at human naïve T cell differentiation failed to implement a Treg cell specific epigenetic signature. Ten-eleven translocation (TET) enzymes catalyze DNA demethylation. Plasmids towardexpression of a fusion protein encompassing nonfunctional Cas9, the catalytic domain of TET1, blue fluorescent protein, and encoding single guide RNAs (sgRNAs) targeting specific segments of the FOXP3-TSDR were engineered and transfected into Jurkat T cells. FOXP3-TSDR methylation was analyzed by deep-amplicon bisulfite sequencing while cellular Foxp3, Tbet, Gata3, and Rorgt mRNA levels were determined by real-time PCR. Overexpression of dCas9TET1 significantly decreased Jurkat cell FOXP3-TSDR methylation and increased Foxp3 mRNA expression while expressions of master transcription factor mRNAs of other major T cell lineages remained largely unaffected. dCas9-TET1 construct transfection mediated Treg programming of patients' primary T cells might be feasible.

Chromosome 3 is a valid marker for prognostic testing of biopsy material from uveal melanoma later treated by brachytherapy.

PURPOSE: Monosomy 3 (M3) in uveal melanoma (UM) obtained after enucleation is significantly associated with metastatic death. With improved biopsy techniques, samples from patients treated with eye-preserving methods have become available. As the choice of treatment depends on tumour size, patients treated with eye-preserving brachytherapy tend to have smaller tumours. It has to be determined if M3 is a valid marker for prognosis of these patients. METHODS: Follow-up and clinical data were collected from a total of 451 UM patients: 291 patients were treated by brachytherapy. Tumour tissue was sampled by transretinal biopsy using the 23-gauge Essen biopsy forceps prior to therapy in 114 of them. Chromosome 3 status was determined by microsatellite analysis. Data were compared to those from 160 patients treated by enucleation. RESULTS: Chromosome 3 status correlates significantly with disease-related survival in both patient groups. The proportion of tumours with M3 is lower in the brachytherapy group compared to patients treated with enucleation (25/77 32% and 102/144 71%, respectively). CONCLUSIONS: M3 is a valid marker for poor prognosis in uveal melanoma later treated by brachytherapy. The higher proportion of D3 tumours might explain, at least in part, the more favourable prognosis of patients treated by brachytherapy.

Compound heterozygosity of low-frequency promoter deletions and rare loss-of-function mutations in TXNL4A causes Burn-McKeown syndrome.

Mutations in components of the major spliceosome have been described in disorders with craniofacial anomalies, e.g., Nager syndrome and mandibulofacial dysostosis type Guion-Almeida. The U5 spliceosomal complex of eight highly conserved proteins is critical for pre-mRNA splicing. We identified biallelic mutations in TXNL4A, a member of this complex, in individuals with Burn-McKeown syndrome (BMKS). This rare condition is characterized by bilateral choanal atresia, hearing loss, cleft lip and/or palate, and other craniofacial dysmorphisms. Mutations were found in 9 of 11 affected families. In 8 families, affected individuals carried a rare loss-of-function mutation (nonsense, frameshift, or microdeletion) on one allele and a low-frequency 34 bp deletion (allele frequency 0.76%) in the core promoter region on the other allele. In a single highly consanguineous family, formerly diagnosed as oculo-oto-facial dysplasia, the four affected individuals were homozygous for a 34 bp promoter deletion, which differed from the promoter deletion in the other families. Reporter gene and in vivo assays showed that the promoter deletions led to reduced expression of TXNL4A. Depletion of TXNL4A (Dib1) in yeast demonstrated reduced assembly of the tri-snRNP complex. Our results indicate that BMKS is an autosomal-recessive condition, which is frequently caused by compound heterozygosity of low-frequency promoter deletions in combination with very rare loss-of-function mutations.

Relevance of Foxp3⁺ regulatory T cells for early and late phases of murine sepsis.

The role of Foxp3(+) regulatory T (Treg) cells in the course of the early hyper-inflammatory and subsequent hypo-inflammatory phases of sepsis is ambiguous. Whereas Nrp1 expression has been reported to discriminate natural Treg cells from induced Treg cells, the Treg cell stability depends on the methylation status of foxp3-TSDR. To specifically evaluate the role of Foxp3(+) Treg cells in the early and late phases of sepsis, we induced sepsis by caecal ligation and puncture and subsequent Pseudomonas aeruginosa lung infection in a DEREG (DEpletion of REGulatory T cells) mouse model. We found an increase of Foxp3(+) Treg cells to all CD4(+) T cells during murine sepsis. Using a new methylation-sensitive quantitative RT-PCR method and deep amplicon sequencing, we demonstrated that natural (Nrp1(+) Foxp3(+) ) Treg cells and most induced (Nrp1(-) Foxp3(+) ) Treg cells are stable and exhibit unmethylated foxp3-TSDR, and that both Treg populations are functionally suppressive in healthy and septic mice. DEREG mice depleted of Foxp3(+) Treg cells exhibit higher disease scores, mortality rates and interleukin-6 expression levels than do non-depleted DEREG mice in early-phase sepsis, a finding indicating that Foxp3(+) Treg cells limit the hyper-inflammatory response and accelerate recovery. Treg cell depletion before secondary infection with P. aeruginosa 1 week after caecal ligation and puncture does not influence cytokine levels or the course of secondary infection. However, a moderate Treg cell recurrence, which we observed in DEREG mice during secondary infection, may interfere with these results. In summary, Treg cells contribute to a positive outcome after early-phase sepsis, but the data do not support a significant role of Treg cells in immune paralysis during late-phase sepsis.

Impact of 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate for induction of human regulatory T cells.

The epigenetic regulation of transcription factor genes is critical for T-cell lineage specification. A specific methylation pattern within a conserved region of the lineage specifying transcription factor gene FOXP3, the Treg-specific demethylated region (TSDR), is restricted to regulatory T (Treg) cells and is required for stable expression of FOXP3 and suppressive function. We analysed the impact of hypomethylating agents 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate on human CD4(+)  CD25(-) T cells for generating demethylation within FOXP3-TSDR and inducing functional Treg cells. Gene expression, including lineage-specifying transcription factors of the major T-cell lineages and their leading cytokines, functional properties and global transcriptome changes were analysed. The FOXP3-TSDR methylation pattern was determined by using deep amplicon bisulphite sequencing. 5-aza-2'-deoxycytidine induced FOXP3-TSDR hypomethylation and expression of the Treg-cell-specific genes FOXP3 and LRRC32. Proliferation of 5-aza-2'-deoxycytidine-treated cells was reduced, but the cells did not show suppressive function. Hypomethylation was not restricted to FOXP3-TSDR and expression of master transcription factors and leading cytokines of T helper type 1 and type 17 cells were induced. Epigallocatechin-3-gallate induced global DNA hypomethylation to a lesser extent than 5-aza-2'-deoxycitidine, but no relevant hypomethylation within FOXP3-TSDR or expression of Treg-cell-specific genes. Neither of the DNA methyltransferase inhibitors induced fully functional human Treg cells. 5-aza-2'-deoxycitidine-treated cells resembled Treg cells, but they did not suppress proliferation of responder cells, which is an essential capability to be used for Treg cell transfer therapy. Using a recently developed targeted demethylation technology might be a more promising approach for the generation of functional Treg cells.

A new face of Borjeson-Forssman-Lehmann syndrome? De novo mutations in PHF6 in seven females with a distinct phenotype.

BACKGROUND: Borjeson-Forssman-Lehmann syndrome (BFLS) is an X-linked recessive intellectual disability (ID) disorder caused by mutations in the PHF6 gene and characterised by variable cognitive impairment, a distinct facial gestalt, obesity, and hypogonadism. Female carriers are usually not affected or only mildly affected, and so far only two females with de novo mutations or deletions in PHF6 have been reported. METHODS AND RESULTS: We performed PHF6 mutational analysis and screening for intragenic deletions and duplications by quantitative real-time PCR and multiplex ligation dependent probe amplification (MLPA) in female patients with variable ID and a distinct appearance of sparse hair, remarkable facial features, hypoplastic nails, and teeth anomalies. We detected two truncating mutations and two duplications of exons 4 and 5. Furthermore, two female patients with PHF6 deletions and a similar phenotype were identified by routine molecular karyotyping. Recently, two patients with a clinical diagnosis of Coffin-Siris syndrome in early infancy had been found to harbour mutations in PHF6, and their phenotype in advanced ages is now described. Further studies revealed skewed X-inactivation in blood lymphocytes, while it was normal in fibroblasts, thus indicating functional mosaicism. CONCLUSIONS: Our findings indicate that de novo defects in PHF6 in females result in a recognisable phenotype which might have been under-recognised so far and which comprises variable ID, a characteristic facial gestalt, hypoplastic nails, brachydactyly, clinodactyly mainly of fingers IV and V, dental anomalies, and linear skin hyperpigmentation. It shows overlap with BFLS but also additional distinct features, thus adding a new facet to this disorder.

Mass Spectrometry-Based Profiling of Histone Post-Translational Modifications in Uveal Melanoma Tissues, Human Melanocytes, and Uveal Melanoma Cell Lines - A Pilot Study.

Purpose: Epigenetic alterations in uveal melanoma (UM) are still neither well characterized, nor understood. In this pilot study, we sought to provide a deeper insight into the possible role of epigenetic alterations in the pathogenesis of UM and their potential prognostic relevance. To this aim, we comprehensively profiled histone post-translational modifications (PTMs), which represent epigenetic features regulating chromatin accessibility and gene transcription, in UM formalin-fixed paraffin-embedded (FFPE) tissues, control tissues, UM cell lines, and healthy melanocytes. Methods: FFPE tissues of UM (n = 24), normal choroid (n = 4), human UM cell lines (n = 7), skin melanocytes (n = 6), and uveal melanocytes (n = 2) were analyzed through a quantitative liquid chromatography-mass spectrometry (LC-MS) approach. Results: Hierarchical clustering showed a clear separation with several histone PTMs that changed significantly in a tumor compared to normal samples, in both tissues and cell lines. In addition, several acetylations and H4K20me1 showed lower levels in BAP1 mutant tumors. Some of these changes were also observed when we compared GNA11 mutant tumors with GNAQ tumors. The epigenetic profiling of cell lines revealed that the UM cell lines MP65 and UPMM1 have a histone PTM pattern closer to the primary tissues than the other cell lines analyzed. Conclusions: Our results suggest the existence of different histone PTM patterns that may be important for diagnosis and prognosis in UM. However, further analyses are needed to confirm these findings in a larger cohort. The epigenetic characterization of a panel of UM cell lines suggested which cellular models are more suitable for epigenetic investigations.

Pancreatic cancer acquires resistance to MAPK pathway inhibition by clonal expansion and adaptive DNA hypermethylation.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. It is marked by extraordinary resistance to conventional therapies including chemotherapy and radiation, as well as to essentially all targeted therapies evaluated so far. More than 90% of PDAC cases harbor an activating KRAS mutation. As the most common KRAS variants in PDAC remain undruggable so far, it seemed promising to inhibit a downstream target in the MAPK pathway such as MEK1/2, but up to now preclinical and clinical evaluation of MEK inhibitors (MEKi) failed due to inherent and acquired resistance mechanisms. To gain insights into molecular changes during the formation of resistance to oncogenic MAPK pathway inhibition, we utilized short-term passaged primary tumor cells from ten PDACs of genetically engineered mice. We followed gain and loss of resistance upon MEKi exposure and withdrawal by longitudinal integrative analysis of whole genome sequencing, whole genome bisulfite sequencing, RNA-sequencing and mass spectrometry data. RESULTS: We found that resistant cell populations under increasing MEKi treatment evolved by the expansion of a single clone but were not a direct consequence of known resistance-conferring mutations. Rather, resistant cells showed adaptive DNA hypermethylation of 209 and hypomethylation of 8 genomic sites, most of which overlap with regulatory elements known to be active in murine PDAC cells. Both DNA methylation changes and MEKi resistance were transient and reversible upon drug withdrawal. Furthermore, MEKi resistance could be reversed by DNA methyltransferase inhibition with remarkable sensitivity exclusively in the resistant cells. CONCLUSION: Overall, the concept of acquired therapy resistance as a result of the expansion of a single cell clone with epigenetic plasticity sheds light on genetic, epigenetic and phenotypic patterns during evolvement of treatment resistance in a tumor with high adaptive capabilities and provides potential for reversion through epigenetic targeting.

Analysis of uveal melanomas and paired constitutional DNA for exclusion of a BAP1-tumor predisposition syndrome.

Uveal melanoma (UM) is a rare tumor originating from melanocytic cells in the eye. Familial aggregation of UM is rare and can occur as part of the tumor predisposition syndrome BAP1-TPDS. However, family history alone will only identify a subset of patients with BAP1-TPDS. In the present study, we used sequential testing of tumor and blood DNA from UM patients for differential diagnosis of BAP1-TPDS. The study group was an unselected prospective cohort of patients from whom UM tissue was available. First, chromosome 3 status in tumor DNA was determined in all 140 patients who consented to participate. As tumors with disomy 3 rarely show BAP1 alterations, sequence analysis of this gene was performed in the 72 tumors with monosomy 3 (M3) or partial M3 only. We identified oncogenic BAP1 alterations in 52 of these tumors (72%). Targeted sequencing of DNA from matched peripheral blood showed pathogenic variants in two patients (3.8%) thus proving BAP1-TPDS. Only one of these two patients also had a medical history suggestive of this syndrome. Conversely, in three patients known to have had additional tumors before diagnosis of UM, constitutional heterozygosity for a BAP1 mutation was excluded. Altogether, in 50 patients we could exclude BAP1-TPDS with high diagnostic certainty. The results of our study support that genetic testing for BAP1-TPDS should be offered to all patients with UM. Moreover, as genetic information from the tumor can help exclude heritable risk, the strategy for analysis should include efforts to obtain tumor samples for testing.

Release of Cell-Free Tumor DNA in the Plasma of Uveal Melanoma Patients Under Radiotherapy.

Purpose: Uveal melanoma (UM) is a tumor of the eye that metastasizes in approximately half of cases. Prognostic testing requires accessibility to tumor tissue, which is usually not available with eye-preserving therapies. Noninvasive approaches to prognostic testing that provide valuable information for patient care are therefore needed. The aim of this study was to evaluate the use of circulating cell-free plasma DNA analysis in UM patients undergoing brachytherapy. Methods: The study recruited 26 uveal melanoma patients referred to the department between February and October 2020. Blood samples were collected at various time points before, during, and after treatment, and deep amplicon sequencing was used to identify oncogenic variant alleles of the GNAQ and GNA11 genes, which serve as indicators for the presence of circulating tumor DNA (ctDNA). Results: The results showed that all patients were ctDNA negative before brachytherapy. In 31% of patients, ctDNA was detected during therapy. The variant allele fraction of GNAQ or GNA11 alleles in ctDNA positive samples ranged from 0.24% to 2% and correlates with the largest basal diameter and thickness of the tumor. Conclusions: The findings suggest that brachytherapy increases the presence of tumor DNA in the plasma of UM patients. Thus ctDNA analysis may offer a noninvasive approach for prognostic testing. However, efforts are still required to lower the limit of detection for tumor-specific genetic alterations.

Reduced Staphylococcus Abundance Characterizes the Lesional Microbiome of Actinic Keratosis Patients after Field-Directed Therapies.

Skin microbiome dysbiosis with a Staphylococcus overabundance is a feature of actinic keratosis (AK) and squamous skin carcinoma (SCC) patients. The impact of lesion-directed treatments for AK lesions such as diclofenac (DIC) and cold atmospheric plasma (CAP) on the lesional microbiome is not established. We studied 321 skin microbiome samples of 59 AK patients treated with DIC 3% gel versus CAP. Microbial DNA from skin swabs taken before start of treatment (week 0), at the end of the treatment period (week 24), and 3 months after end of treatment (week 36) was analyzed after sequencing the V3/V4 region of the 16S rRNA gene. The relative abundance of S. aureus was scrutinized by a tuf gene specific TaqMan PCR assay. The total bacterial load and both, relative and absolute abundance of Staphylococcus genus was reduced upon both therapies at week 24 and 36 compared to week 0. Notably, the lesional microbiome of patients responding to CAP therapy at week 24 was characterized by an increased relative abundance of Corynebacterium genus compared to nonresponders. A higher relative abundance of Staphylococcus aureus at week 36 was a feature of patients classified as nonresponders for both treatments 12 weeks after therapy completion. The reduction of the Staphylococcus abundance after treatment of AK lesions and alterations linked to treatment response encourage further studies for investigation of the role of the skin microbiome for both, the carcinogenesis of epithelial skin cancer and its function as predictive therapeutic biomarker in AK. IMPORTANCE The relevance of the skin microbiome for development of actinic keratosis (AK), its progression into squamous skin cancer, and for field-directed treatment response is unknown. An overabundance of staphylococci characterizes the skin microbiome of AK lesions. In this study, analyses of the lesional microbiome from 321 samples of 59 AK patients treated with diclophenac gel versus cold atmospheric plasma (CAP) revealed a reduced total bacterial load and reduced relative and absolute Staphylococcus genus abundance upon both treatments. A higher relative Corynebacterium abundance was a feature of patients classified as responders at the end of CAP-treatment period (week 24) compared with nonresponders and the Staphylococcus aureus abundance of patients classified as responders 3 months after treatment completion was significantly lower than in nonresponders. The alterations of the skin microbiome upon AK treatment encourage further investigations for establishing its role for carcinogenesis and its function as predictive biomarker in AK.

No evidence for intervention-associated DNA methylation changes in monocytes of patients with posttraumatic stress disorder.

DNA methylation patterns can be responsive to environmental influences. This observation has sparked interest in the potential for psychological interventions to influence epigenetic processes. Recent studies have observed correlations between DNA methylation changes and therapy outcome. However, most did not control for changes in cell composition. This study had two aims: first, we sought to replicate therapy-associated changes in DNA methylation of commonly assessed candidate genes in isolated monocytes from 60 female patients with post-traumatic stress disorder (PTSD). Our second, exploratory goal was to identify novel genomic regions with substantial pre-to-post intervention DNA methylation changes by performing whole-genome bisulfite sequencing (WGBS) in two patients with PTSD. Equivalence testing and Bayesian analyses provided evidence against physiologically meaningful intervention-associated DNA methylation changes in monocytes of PTSD patients in commonly investigated target genes (NR3C1, FKBP5, SLC6A4, OXTR). Furthermore, WGBS yielded only a limited set of candidate regions with suggestive evidence of differential DNA methylation pre- to post-therapy. These differential DNA methylation patterns did not prove replicable when investigated in the entire cohort. We conclude that there is no evidence for major, recurrent intervention-associated DNA methylation changes in the investigated genes in monocytes of patients with PTSD.

Massive parallel bisulfite sequencing of CG-rich DNA fragments reveals that methylation of many X-chromosomal CpG islands in female blood DNA is incomplete.

Methylation of CpG islands (CGIs) plays an important role in gene silencing. For genome-wide methylation analysis of CGIs in female white blood cells and in sperm, we used four restriction enzymes and a size selection step to prepare DNA libraries enriched with CGIs. The DNA libraries were treated with sodium bisulfite and subjected to a modified 454/Roche Genome Sequencer protocol. We obtained 163 034 and 129 620 reads from blood and sperm, respectively, with an average read length of 133 bp. Bioinformatic analysis revealed that 12 358 (7.6%) blood library reads and 10 216 (7.9%) sperm library reads map to 6167 and 5796 different CGIs, respectively. In blood and sperm DNA, we identified 824 (13.7%) and 482 (8.5%) fully methylated autosomal CGIs, respectively. Differential methylation, which is characterized by the presence of methylated and unmethylated reads of the same CGI, was observed in 53 and 52 autosomal CGIs in blood and sperm DNA, respectively. Remarkably, methylation of X-chromosomal CGIs in female blood cells was most often incomplete (25-75%). Such incomplete methylation was mainly found on the X-chromosome, suggesting that it is linked to X-chromosome inactivation.

EFS shows biallelic methylation in uveal melanoma with poor prognosis as well as tissue-specific methylation.

BACKGROUND: Uveal melanoma (UM) is a rare eye tumor. There are two classes of UM, which can be discriminated by the chromosome 3 status or global mRNA expression profile. Metastatic progression is predominantly originated from class II tumors or from tumors showing loss of an entire chromosome 3 (monosomy 3). We performed detailed EFS (embryonal Fyn-associated substrate) methylation analyses in UM, cultured uveal melanocytes and normal tissues, to explore the role of the differentially methylated EFS promoter region CpG island in tumor classification and metastatic progression. METHODS: EFS methylation was determined by direct sequencing of PCR products from bisulfite-treated DNA or by sequence analysis of individual cloned PCR products. The results were associated with clinical features of tumors and tumor-related death of patients. RESULTS: Analysis of 16 UM showed full methylation of the EFS CpG island in 8 (50%), no methylation in 5 (31%) and partial methylation in 3 (19%) tumors. Kaplan-Meier analysis revealed a higher risk of metastatic progression for tumors with EFS methylation (p = 0.02). This correlation was confirmed in an independent set of 24 randomly chosen tumors. Notably, only UM with EFS methylation gave rise to metastases. Real-time quantitative RT-PCR expression analysis revealed a significant inverse correlation of EFS mRNA expression with EFS methylation in UM. We further found that EFS methylation is tissue-specific with full methylation in peripheral blood cells, and no methylation in sperm, cultured primary fibroblasts and fetal muscle, kidney and brain. Adult brain samples, cultured melanocytes from the uveal tract, fetal liver and 3 of 4 buccal swab samples showed partial methylation. EFS methylation always affects both alleles in normal and tumor samples. CONCLUSIONS: Biallelic EFS methylation is likely to be the result of a site-directed methylation mechanism. Based on partial methylation as observed in cultured melanocytes we hypothesize that there might be methylated and unmethylated precursor cells located in the uveal tract. The EFS methylation of a UM may depend on which type of precursor cell the tumor originated from.

FOXP3 Expression in GARP-Transduced Helper T Cells Is Not Associated with FOXP3 TSDR Demethylation.

AIM: Glycoprotein A repetitions predominant (GARP or LRRC32) represents a human regulatory CD4+ CD25(hi) FOXP3+ T (T(reg)) cell-specific receptor that controls FOXP3. Ectopic expression of GARP in helper T (T(h)) cells has been shown to be sufficient for the induction of FOXP3 and generation of a stable regulatory phenotype. Since expression of FOXP3 in Treg cells is epigenetically controlled by a conserved motif, the so-called T(reg)-specific demethylated region (TSDR), we asked whether GARP-mediated upregulation of FOXP3 in Th cells is similarly accompanied by demethylation of the TSDR. METHODS: DNA methylation of the FOXP3 TSDR was analyzed by direct sequencing of polymerase chain reaction (PCR) products from bisulfite-treated genomic DNA. RESULTS: Although GARP-transduced T(h) cells exhibit constitutive FOXP3 expression and a regulatory phenotype, the FOXP3 TSDR is completely methylated as in naive T(h) cells. GARP-mediated FOXP3 upregulation in T(h) cells is not associated with T(reg)-specific demethylation of the FOXP3 TSDR. CONCLUSION: Although GARP-engineered T(h) cells exhibit stable FOXP3 expression and a phenotypic reprogramming towards T(reg) cells in vitro, these cells do not completely mimic the epigenotype of natural T(reg) cells. Thus, concepts based on the genetic modification of T(h) cells as cellular therapies to treat autoimmune diseases or to control transplantation tolerance should be critically tested before any clinical application.

Early detection of metastatic uveal melanoma by the analysis of tumor-specific mutations in cell-free plasma DNA.

BACKGROUND: Eye salvaging therapy of malignant melanomas of the uvea can preserve the eye in most cases, but still about half of patients die from metastatic disease. Previous analyses of cell-free DNA from plasma had shown detectable levels of tumor-specific GNAQ/GNA11 mutations in patients with the clinical diagnosis of progressive disease. However, data on the time span that elapses from the detection of ctDNA in plasma to the clinical detection of metastases (diagnostic lead time) are missing. METHODS: We examined 135 patients with uveal melanoma. Cell-free DNA was isolated from a total of 807 blood samples which were taken over a period of up to 41 months and analyzed for the presence of GNAQ/GNA11 mutations by deep amplicon sequencing. RESULTS: Twenty-one of the 135 patients developed metastases or recurrence. A ctDNA signal was identified in the plasma of 17 of the 21 patients. In 10 patients, this ctDNA signal preceded the clinical diagnosis of metastasis by 2-10 months. In 10 other patients, a ctDNA signal was only detected in samples obtained shortly before or after radiotherapy. The presence of a ctDNA signal in 16 of the remaining 125 patients was linked to clinical manifestation of metastases (n = 14) or tumor recurrence (n = 2) with a sensitivity and specificity of 80% and 96%, respectively. CONCLUSION: Detection of ctDNA in plasma can provide a diagnostic lead time over the clinical diagnosis of metastases or tumor recurrence. Longer lead times are to be expected if intervals between sampling are shortened.

Eye Tumors in Childhood as First Sign of Tumor Predisposition Syndromes: Insights from an Observational Study Conducted in Germany and Austria.

Retinoblastoma and other eye tumors in childhood are rare diseases. Many eye tumors are the first signs of a genetic tumor predisposition syndrome and the affected children carry a higher risk of developing other cancers later in life. Clinical and genetic data of all children with eye tumors diagnosed between 2013-2018 in Germany and Austria were collected in a multicenter prospective observational study. In five years, 300 children were recruited into the study: 287 with retinoblastoma, 7 uveal melanoma, 3 ciliary body medulloepithelioma, 2 retinal astrocytoma, 1 meningioma of the optic nerve extending into the eye. Heritable retinoblastoma was diagnosed in 44% of children with retinoblastoma. One child with meningioma of the optic nerve extending into the eye was diagnosed with neurofibromatosis 2. No pathogenic constitutional variant in DICER1 was detected in a child with medulloepithelioma while two children did not receive genetic analysis. Because of the known association with tumor predisposition syndromes, genetic counseling should be offered to all children with eye tumors. Children with a genetic predisposition to cancer should receive a tailored surveillance including detailed history, physical examinations and, if indicated, imaging to screen for other cancer. Early detection of cancers may reduce mortality.

Novel cell lines derived by long-term culture of primary uveal melanomas.

AIMS: Uveal melanomas with or without monosomy 3 have different metastatic potentials. Therefore, it would be of great experimental importance to obtain cell lines of both groups - established and characterized under the same conditions. METHODS: Long-term culture of biopsies derived from untreated primary uveal melanomas was performed, and the status of chromosome 3 in the tumour was determined. Characterization by immunocytochemistry and in vitro assays for cell migration, vasculogenic cord formation and proliferation were performed. RESULTS: Establishment of cell lines succeeded in 6 out of 128 uveal melanomas. No histopathological feature was predictive for cultivation success. Two out of 4 cell lines derived from tumours with monosomy 3 were invasive in vitro. Both cell lines with disomy 3 showed higher proliferation levels (p = 0.002). CONCLUSIONS: The in vitro tendency for invasion did not correlate with the proliferative behaviour of uveal melanoma cell lines, but was partly associated with monosomy of chromosome 3.

GNAQ Q209R Mutations Are Highly Specific for Circumscribed Choroidal Hemangioma.

Several tumors, including uveal melanoma, show somatic mutations of GNAQ/GNA11. Circumscribed choroidal hemangioma is a benign tumor that becomes symptomatic in adulthood. In some patients, morphologic examination of biopsies is required for differential diagnosis between amelanotic choroidal melanoma and circumscribed choroidal hemangioma. Here, we report the results of GNAQ/GNA11 mutation analysis in samples from circumscribed choroidal hemangioma. Deep amplicon sequencing (Illumina MiSeq, San Diego, CA, USA) of positions R183 and Q209 of GNAQ and GNA11 in tissue samples from 33 patients with histologically diagnosed circumscribed choroidal hemangioma. All patients underwent biopsy or enucleation at our clinic between 2008 and 2018. To enable detection of variant alleles at low fractions, read depth exceeded 15,000-fold. DNA for genetic analysis was prepared from either snap-frozen (n = 22) or FFPE (n = 11) tissue samples. Samples from 28/33 patients (85%) showed a somatic missense mutation of GNAQ (c.626 A > G) predicted to result in p.Q209R. Variant allele fraction was variable (range 2.3% to 28%). Variants of GNAQ resulting in p.Q209 are characteristic for circumscribed choroidal hemangiomas. It appears that the GNAQ mutation spectrum in this tumor is narrow, possibly restricted to p.Q209R. Moreover, the spectrum is distinct from that of uveal melanoma, in which alterations resulting in p.Q209R are very rare.

Secondary Somatic Mutations in G-Protein-Related Pathways and Mutation Signatures in Uveal Melanoma.

BACKGROUND: Uveal melanoma (UM), a rare cancer of the eye, is characterized by initiating mutations in the genes G-protein subunit alpha Q (GNAQ), G-protein subunit alpha 11 (GNA11), cysteinyl leukotriene receptor 2 (CYSLTR2), and phospholipase C beta 4 (PLCB4) and by metastasis-promoting mutations in the genes splicing factor 3B1 (SF3B1), serine and arginine rich splicing factor 2 (SRSF2), and BRCA1-associated protein 1 (BAP1). Here, we tested the hypothesis that additional mutations, though occurring in only a few cases ("secondary drivers"), might influence tumor development. METHODS: We analyzed all the 4125 mutations detected in exome sequencing datasets, comprising a total of 139 Ums, and tested the enrichment of secondary drivers in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that also contained the initiating mutations. We searched for additional mutations in the putative secondary driver gene protein tyrosine kinase 2 beta (PTK2B) and we developed new mutational signatures that explain the mutational pattern observed in UM. RESULTS: Secondary drivers were significantly enriched in KEGG pathways that also contained GNAQ and GNA11, such as the calcium-signaling pathway. Many of the secondary drivers were known cancer driver genes and were strongly associated with metastasis and survival. We identified additional mutations in PTK2B. Sparse dictionary learning allowed for the identification of mutational signatures specific for UM. CONCLUSIONS: A considerable part of rare mutations that occur in addition to known driver mutations are likely to affect tumor development and progression.