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Treatment of uveal melanoma (UM) patients with metastatic disease is unfortunately limited. Twenty percent of UM harbor a mutation in splicing factor gene SF3B1, suggesting that aberrant spliceosome functioning plays a vital role in tumorigenesis. Splicing inhibitors exploit the preferential sensitivity of spliceosome compromised leukemic cells to these compounds. We have studied the effect of splicing inhibitor E7107 using two UM cell lines and ex vivo cultured SF3B1 and BAP1 mutated primary UM tumor slices. These UM cell lines and ex vivo tumor slices were exposed for 24h to different concentrations of E7107. Tumor slices were stained with H&E and incubated with BAP1, MelanA, MIB-1 and caspase-3 antisera. E7107 exposed UM cell lines showed decreased cell viability and increased apoptosis with the largest effect sizes in SF3B1-mutated UM. A similar effect was observed upon exposure of E7107 on UM tumor slices. Additionally, RNA was isolated for transcriptome analysis and the type and number of alternative and aberrant transcripts was evaluated. Ninety-seven transcripts had a decrease in aberrant transcripts in all samples after E7107 exposure, and this effect was mostly in intron retention. This study indicates / suggests that mutated SF3B1 UM cells are more sensitive to splicing inhibitor E7107 compared to wild-type SF3B1 UM cells.Simple SummaryUveal melanoma (UM) is an aggressive malignancy of the eye. UM prognosis varies and depends on mutational status. Mutations in spliceosome gene Splicing Factor 3b subunit 1 (SF3B1) are in UM associated with late onset metastasis. Also in other malignancies, such as breast cancer or leukemia, SF3B1 mutations have been implied as drivers for tumor formation. Targeting the spliceosome with inhibitors is possible and would make SF3B1-mutated UM amenable for treatment. We investigated and demonstrated using two cell lines and ex-vivo culturing of 3 SF3B1-mutated tumors and 17 SF3B1-wildtype tumors that spliceosome inhibitor E7107 decreased the cell viability of both SF3B1-mutated and SF3B1-wildtype UM cell line, but significantly more in SF3B1-mutated UM cell line. Therefore, inhibiting the spliceosome has the highest therapeutic potential in SF3B1-mutated UM, but further research is recommended to determine the best suited strategy to minimize risk and maximize efficacy in a therapeutic setting.

Approximately 25% of all uveal melanoma (UM) contain driver mutations in the gene encoding the spliceosome factor SF3B1, and whilst patients with such SF3B1 mutations generally have an intermediate risk on developing metastatic disease, a third of these patients develop early metastasis within 5 years after diagnosis. We therefore investigated whether clinical and/or genetic variables could be indicative of short progression-free survival (PFS < 60 months) or long PFS (PFS ≥ 60 months) for SF3B1-mutated (SF3B1mut) UM patients. We collected 146 SF3B1mut UM from our Rotterdam Ocular Melanoma Studygroup (ROMS) database and external published datasets. After stratification of all SF3B1mut UM using short PFS vs. long PFS, only largest tumor diameter (LTD) was significantly larger (mean: 17.7 mm (±2.8 SD) in the short PFS SF3B1mut group vs. the long PFS group (mean: 14.7 (±3.7 SD, p = 0.001). Combined ROMS and The Cancer Genome Atlas (TCGA) transcriptomic data were evaluated, and we identified SF3B1mut-specific canonical transcripts (e.g., a low expression of ABHD6 indicative for early-onset metastatic disease) or distinct expression of SF3B1mut UM aberrant transcripts, indicative of early- or late-onset or no metastatic SF3B1mut UM.

Simple Summary We investigated the increased prevalence of Barrett's esophagus in adults with esophageal atresia. A higher polygenic risk score and disturbances in inflammatory, stress response and oncological pathways upon acid exposure suggest a genetic susceptibility and increased induction of inflammatory processes. Although further research is required to explore this hypothesis, this could be a first-step into selecting patients that are more at risk to develop Barrett's esophagus and/or esophageal carcinoma. Currently, an endoscopic screening and surveillance program is in practice in our institution for patients born with esophageal atresia, to early detect (pre)malignant lesions. Since recurrent endoscopies can be a burden for the patient, selecting patients by for example genetic susceptibility would allow to only include those at risk in future practice. The prevalence of Barrett's esophagus (BE) in adults born with esophageal atresia (EA) is four times higher than in the general population and presents at a younger age (34 vs. 60 years). This is (partly) a consequence of chronic gastroesophageal reflux. Given the overlap between genes and pathways involved in foregut and BE development, we hypothesized that EA patients have an intrinsic predisposition to develop BE. Transcriptomes of Esophageal biopsies of EA patients with BE (n = 19, EA/BE); EA patients without BE (n = 44, EA-only) and BE patients without EA (n = 10, BE-only) were compared by RNA expression profiling. Subsequently, we simulated a reflux episode by exposing fibroblasts of 3 EA patients and 3 controls to acidic conditions. Transcriptome responses were compared to the differential expressed transcripts in the biopsies. Predisposing single nucleotide polymorphisms, associated with BE, were slightly increased in EA/BE versus BE-only patients. RNA expression profiling and pathway enrichment analysis revealed differences in retinoic acid metabolism and downstream signaling pathways and inflammatory, stress response and oncological processes. There was a similar effect on retinoic acid signaling and immune response in EA patients upon acid exposure. These results indicate that epithelial tissue homeostasis in EA patients is more prone to acidic disturbances.

__Background:__ Patients born with esophageal atresia (EA) have a higher incidence of infantile hypertrophic pyloric stenosis (IHPS), suggestive of a relationship. A shared etiology makes sense from a developmental perspective as both affected structures are foregut derived. A genetic component has been described for both conditions as single entities and EA and IHPS are variable components in several monogenetic syndromes. We hypothesized that defects disturbing foregut morphogenesis are responsible for this combination of malformations. __Methods:__ We investigated the genetic variation of 15 patients with both EA and IHPS with unaffected parents using exome sequencing and SNP arraybased genotyping, and compared the results to mouse transcriptome data of the developing foregut. __Results:__ We did not identify putatively deleterious de novo mutations or recessive variants. However, we detected rare inherited variants in EA or IHPS disease genes or in genes important in foregut morphogenesis, expressed at the proper developmental time-points. Two pathways were significantly enriched (p < 1 × 10−5): proliferation and differentiation of smooth muscle cells and self-renewal of satellite cells. __Conclusions:__ None of our findings could fully explain the combination of abnormalities on its own, which makes complex inheritance the most plausible genetic explanation, most likely in combination with mechanical and/or environmental factors. As we did not find one defining monogenetic cause for the EA/IHPS phenotype, the impact of the corrective surgery could should be further investigated.

The aim of this study was exploration of the genetic background of conjunctival melanoma (CM) and correlation with recurrent and metastatic disease. Twenty-eight CM from the Rotterdam Ocular Melanoma Study group were collected and DNA was isolated from the formalin-fixed paraffin embedded tissue. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1, and TERT genes. Recurrences and metastasis were present in eight (29%) and nine (32%) CM cases, respectively. TERT promoter mutations were most common (54%), but BRAF (46%), NRAS (21%), BAP1 (18%), PTEN (14%), c-KIT (7%), and SF3B1 (4%) mutations were also observed. No mutations in GNAQ, GNA11, and EIF1AX were found. None of the mutations was significantly associated with recurrent disease. Presence of a TERT promoter mutation was associated with metastatic disease (p-value = 0.008). Based on our molecular findings, CM comprises a separate entity within melanoma, although there are overlapping molecular features with uveal melanoma, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data, in the light of clinical findings. About three quarter of CM contain drug-targetable mutations, and TERT promoter mutations are correlated to metastatic disease in CM.

The aim of this study was exploration of the genetic background of conjunctival melanoma (CM) and correlation with recurrent and metastatic disease. Twenty-eight CM from the Rotterdam Ocular Melanoma Study group were collected and DNA was isolated from the formalin-fixed paraffin embedded tissue. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1, and TERT genes. Recurrences and metastasis were present in eight (29%) and nine (32%) CM cases, respectively. TERT promoter mutations were most common (54%), but BRAF (46%), NRAS (21%), BAP1 (18%), PTEN (14%), c-KIT (7%), and SF3B1 (4%) mutations were also observed. No mutations in GNAQ, GNA11, and EIF1AX were found. None of the mutations was significantly associated with recurrent disease. Presence of a TERT promoter mutation was associated with metastatic disease (p-value = 0.008). Based on our molecular findings, CM comprises a separate entity within melanoma, although there are overlapping molecular features with uveal melanoma, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data, in the light of clinical findings. About three quarter of CM contain drug-targetable mutations, and TERT promoter mutations are correlated to metastatic disease in CM.

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.

Huntington disease (HD) is an autosomal dominant, neurodegenerative disease caused by a CAG repeat expansion within the coding sequence of the HTT gene, resulting in a highly toxic protein with an expanded polyglutamine stretch that forms typical protein aggregates throughout the brain. We generated human induced pluripotent stem cells (hiPSCs) from two HD patients using non-integrating Sendai virus (SeV). The hiPSCs display a normal karyotype, express all pluripotency markers, have the same CAG repeat expansion as the original fibroblasts and are able to differentiate into the three germ layers in vitro.

Aim of the Study Patients born with esophageal atresia (EA) appear to have a 30 times higher prevalence of infantile hypertrophic pyloric stenosis (IHPS). This makes sense from a developmental perspective as both the esophagus and the pyloric sphincter are foregut derived structures. We hypothesized that genetic defects, disturbing foregut morphogenesis, are responsible for the specific combination of EA and IHPS. Methods Patients with both EA and IHPS born between 1970 and 2017 and where possible their parents were included. This study was approved by the internal review board of the Erasmus MC. After parental written informed consent, we determined genetic profiles with whole exome sequencing and SNP-array-based copy number variation analysis. We focused on (1) genetic variation in known EA and IHPS disease genes, (2) pathways important for foregut morphogenesis, (3) shared rare genetic variation, and (4) ultrarare variants in variant-intolerant genes, which have a high chance of being de novo. Segregation analysis of possible candidate variants was performed. Results Twenty-seven out of 664 patients (4.1%) born with EA during the study period developed IHPS, of which 15 cases were analyzed. As none of the parents were affected, we considered dominant (de novo) or recessive and X-linked inheritance models. Unfortunately, we could neither identify rare de novo mutations, nor variants fitting a recessive model. We did however identify inherited putative deleterious heterozygous variants in genes either known to be involved in EA or IHPS (e.g., COL7A1, TNXB, WDR11, GDF6) or important in foregut morphogenesis (e.g., GLI3, NKX2.1) in all patients. Moreover, seven genes were affected by rare variation in ≥2 patients (ADAMTSL4, ANKRD26, CNTN2, HSPG2, KCNN3, LDB3, SEC16B) and expressed in the developing foregut, esophagus or pyloric sphincter in mice between E8.25 and E18.5. However, burden analysis did not show a significant difference with unaffected controls. Conclusions Although the presence of genetic variation in likely candidate genes suggests a genetic component, genetic factors alone could not explain the abnormalities seen in these patients. Therefore, we propose a multifactorial model in which the combination of high impact genetic, mechanical, and environmental factors together can shift the balance from normal to abnormal development.

Patients born with esophageal atresia (EA) have a 30 times higher prevalence of infantile hypertrophic pyloric stenosis (IHPS). This makes sense from a developmental perspective as both the esophagus and the pyloric sphincter are foregut derived structures. EA and IHPS are variable features in several (monogenetic) syndromes. This, and twin and familial studies, indicates a genetic component for both conditions as single entities. We hypothesized that genetic defects, disturbing foregut morphogenesis, are responsible for this combination of malformations. Non-genetic factors could also contribute, as mice exposed to Adriamycin develop EA andin uterodiethylstilbestrol exposure is associated with EA.We investigated the copy number profiles and protein coding variants of 15 patients with both EA and IHPS. As all parents were unaffected, we first considered dominant(de novo)or recessive inheritance models but could not identify putatively deleterious mutations or recessive variants. We did identify inherited variants in genes either known to be involved in EA or IHPS or important in foregut morphogenesis in all patients. Unfortunately, variant burden analysis did not show a significant difference with unaffected controls. However, the IHPS associated risk SNP rs1933683 had a significantly higher incidence (OR 3.29, p=0.009).Although the genetic variation in likely candidate genes as well as the predisposing locus nearBARX1(rs1933683) suggest a genetic component, it does not fully explain the abnormalities seen in these patients. Therefore, we hypothesize that a combination of high impact genetic, mechanical and environmental factors together can shift the balance to abnormal development.Summary statementInstead of one affected gene, the higher incidence of IHPS in EA patients is more likely the result of multiple (epi)genetic and environmental factors together shifting the balance to disease development.

Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch type (HCHWA-D) is an autosomal dominant hereditary disease caused by a point mutation in exon 17 of the APP gene. We generated human induced pluripotent stem cells (hiPSCs) from a symptomatic HCHWA-D patient by using non-integrating Sendai virus (SeV). The newly generated hiPSCs express all pluripotency markers, have a normal karyotype, carry the Dutch mutation, can differentiate in the three germ layers in vitro and are SeV free.

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 uveal melanoma etiology.

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.

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. This study aimed to elucidate the potential role of microRNAs in the metastasis of UM. Using a next-generation sequencing approach in 26 UM samples we identified thirteen differentially expressed microRNAs between high-risk UM and low/intermediate-risk UM, including the known oncomirs microRNA-17-5p, microRNA-21-5p, and miR-151a-3p. Integration of the differentially expressed microRNAs with expression data of predicted target genes revealed 106 genes likely to be affected by aberrant microRNA expression. These genes were involved in pathways such as cell cycle regulation, EGF signaling and EIF2 signaling. Our findings demonstrate that aberrant microRNA expression in UM may affect the expression of genes in a variety of cancer-related pathways. This implies that some microRNAs can be responsible for UM metastasis and are promising potential targets for future treatment.