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Uveal Melanoma (UM) is a rare and malignant intraocular tumor with dismal prognosis. Even if radiation or surgery permit an efficient control of the primary tumor, up to 50% of patients subsequently develop metastases, mainly in the liver. The treatment of UM metastases is challenging and the patient survival is very poor. The most recurrent event in UM is the activation of Gαq signaling induced by mutations in GNAQ/11. These mutations activate downstream effectors including protein kinase C (PKC) and mitogen-activated protein kinases (MAPK). Clinical trials with inhibitors of these targets have not demonstrated a survival benefit for patients with UM metastasis. Recently, it has been shown that GNAQ promotes YAP activation through the focal adhesion kinase (FAK). Pharmacological inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in UM both in vitro and in vivo. In this study, we have evaluated the synergy of the FAK inhibitor with a series of inhibitors targeting recognized UM deregulated pathways in a panel of cell lines. The combined inhibition of FAK and MEK or PKC had highly synergistic effects by reducing cell viability and inducing apoptosis. Furthermore, we demonstrated that these combinations exert a remarkable in vivo activity in UM patient-derived xenografts. Our study confirms the previously described synergy of the dual inhibition of FAK and MEK and identifies a novel combination of drugs (FAK and PKC inhibitors) as a promising strategy for therapeutic intervention in metastatic UM.

Disruption of splicing patterns due to mutations of genes coding splicing factors in tumors represents a potential source of tumor neoantigens, which would be both public (shared between patients) and tumor-specific (not expressed in normal tissues). In this study, we show that mutations of the splicing factor SF3B1 in uveal melanoma generate such immunogenic neoantigens. Memory CD8+ T cells specific for these neoantigens are preferentially found in 20% of patients with uveal melanoma bearing SF3B1-mutated tumors. Single-cell analyses of neoepitope-specific T cells from the blood identified large clonal T-cell expansions, with distinct effector transcription patterns. Some of these expanded T-cell receptors are also present in the corresponding tumors. CD8+ T-cell clones specific for the neoepitopes specifically recognize and kill SF3B1-mutated tumor cells, supporting the use of this new family of neoantigens as therapeutic targets.Significance:Mutations of the splicing factor SF3B1 in uveal melanoma generate shared neoantigens that are uniquely expressed by tumor cells, leading to recognition and killing by specific CD8 T cells. Mutations in splicing factors can be sources of new therapeutic strategies applicable to diverse tumors.This article is highlighted in the In This Issue feature, p. 1861

Supplementary Figure from Splicing Patterns in SF3B1-Mutated Uveal Melanoma Generate Shared Immunogenic Tumor-Specific Neoepitopes

Supplementary Table from Splicing Patterns in SF3B1-Mutated Uveal Melanoma Generate Shared Immunogenic Tumor-Specific Neoepitopes

Supplementary Data from Splicing Patterns in SF3B1-Mutated Uveal Melanoma Generate Shared Immunogenic Tumor-Specific Neoepitopes

Purpose:Uveal melanomas (UM) are genetically simple tumors carrying few copy number alterations (CNA) and a low mutation burden, except in rare MBD4-deficient, hypermutated cases. The genomics of uveal melanoma metastatic progression has not been described. We assessed the genetic heterogeneity of primary and metastatic MBD4-proficient and -deficient uveal melanomas.Experimental Design: We prospectively collected 75 metastatic and 16 primary samples from 25 consecutive uveal melanoma patients, and performed whole-exome sequencing.Results:MBD4-proficient uveal melanomas contained stable genomes at the nucleotide level, acquiring few new single nucleotide variants (SNVs; 16 vs. 13 in metastases and primary tumors, respectively), and no new driver mutation. Five CNAs were recurrently acquired in metastases (losses of 1p, 6q, gains of 1q, 8q, and isodisomy 3). In contrast, MBD4-deficient uveal melanomas carried more than 266 SNVs per sample, with high genetic heterogeneity and TP53, SMARCA4, and GNAS new driver mutations. SNVs in MBD4-deficient contexts were exploited to unveil the timeline of oncogenic events, revealing that metastatic clones arose early after tumor onset. Surprisingly, metastases were not enriched in monosomy 3, a previously defined metastatic risk genomic feature. Monosomy 3 was associated with shorter metastatic-free interval compared with disomy 3 rather than higher rate of relapse.Conclusions:MBD4-proficient uveal melanomas are stable at the nucleotide level, without new actionable alterations when metastatic. In contrast, MBD4 deficiency is associated with high genetic heterogeneity and acquisition of new driver mutations. Monosomy 3 is associated with time to relapse rather than rate of relapse, thus opening avenues for a new genetic prognostic classification of uveal melanomas.

Supplementary figure 6 - Comparisons of CCFs between samples including inter- and intra-metastatic comparisons in MBD4pro samples.

Supplementary figure 3 - Summary diagrams illustrating the median SNV heterogeneity ratios.

Supplementary figure 7 - Minigene assay of BAP1 intronic insert splicing.

Supplementary figure 1 - Copy number / allelic imbalance profiles of all cases as generated by Facets.

Supplementary figure 5 - Pyclone profiles of all 25 cases

Supplementary Table 1: Patients and tumors characteristics

Surgical management of liver metastases of uveal melanoma (LMUM) is associated with the best survival rates, especially for patients with a low tumor burden in the liver. The aim was to determine whether the tumor growth rate (TGRThis retrospective study included 99 patients with LMUM treated with liver resection between November 2007 and November 2020. TGRDFS and OS had a statistically significant positive linear relationship (Spearman correlation r = 0.68, p0.001). A disease-free interval (DFI)24 months and a TGRThe assessment of TGR

The replacement and desmoplastic histopathological growth patterns (HGP) were studied in patients with uveal melanoma liver metastases (MUM). L1CAM and laminin vascular network were detected at the advancing front of the high-risk replacement HGP but not in the more prognostically favorable desmoplastic HGP. Any percentage of replacement HGP (rHGP), predominant rHGP, or pure rHGP in MUM had a significant adverse effect on metastasis-specific overall survival (p = 0.038; (p = 0.0058); p = 0.0064 The replacement histopathologic growth pattern (rHGP) in melanoma liver metastases connotes an aggressive phenotype (vascular co-option; angiotropic extravascular migratory spread) and adverse prognosis. Herein, replacement and desmoplastic HGP (dHGP) were studied in uveal melanoma liver metastases (MUM). In particular, L1CAM and a "laminin vascular network" were detected at the advancing front of 14/20 cases (p = 0.014) and 16/20 cases (p = 6.4e-05) rHGPs, respectively, but both were absent in the dHGP (8/8 cases) (p = 0.014, and p = 6.3e-05, respectively). L1CAM highlighted progressive extension of angiotropic melanoma cells along sinusoidal vessels in a pericytic location (pericytic mimicry) into the hepatic parenchyma. An inverse relationship between L1CAM expression and melanin index (p = 0.012) suggested differentiation toward an amelanotic embryonic migratory phenotype in rHGP. Laminin labeled the basement membrane zone interposed between sinusoidal vascular channels and angiotropic melanoma cells at the advancing front. Other new findings: any percentage of rHGP and pure rHGP had a significant adverse effect on metastasis-specific overall survival (p = 0.038; p = 0.0064), as well as predominant rHGP (p = 0.0058). Pure rHGP also was associated with diminished metastasis-free survival relative to dHGP (p = 0.040), possibly having important implications for mechanisms of tumor spread. In conclusion, we report for the first time that L1CAM and a laminin vascular network are directly involved in this high-risk replacement phenotype. Further, this study provides more detailed information about the adverse prognostic effect of the rHGP in MUM.

MBD4 mutations have been reported in uveal melanomas, acute myeloid leukemias, colorectal adenocarcinomas, gliomas, and spiradenocarcinomas and cause a hypermutated phenotype. Although metastatic uveal melanomas (mUM) are usually resistant to immune checkpoint inhibitors (ICI), the first reported MBD4-mutated (MBD4m) patient responded to ICI, suggesting that MBD4 mutation may predict response to ICI.Retrospective cohort of mUM patients treated with ICI. MBD4 was sequenced in a subset of these patients.Three hundred mUM patients were included. Median follow-up was 17.3 months. Ten patients with an objective response and 20 cases with stable disease forgt;12 months were observed, corresponding to an objective response rate of 3.3% and a clinical benefit (i.e., responder patients and stable disease) rate of 10%. Of the 131 tumors sequenced for MBD4, five (3.8%) were mutated. MBD4 mutation was associated with a better objective response rate as three out of five MBD4m versus 4% of MBD4 wild-type patients responded (p lt; 0.001). Of these five responders, three presented progressive disease at 2.8, 13.9, and 22.3 months. Median PFS was 4.0 months in MBD4 wild-type and 22.3 months in MBD4m patients (HR = 0.22; p = 0.01). Median OS in MBD4def patients was unreached as compared to 16.6 months in MBD4pro (HR = 0.11; 95% CI: 0.02-0.86; log-rank p-test = 0.04; Fig. 2e).In mUM patients, MBD4 mutation is highly predictive for the response, PFS, and overall survival benefit to ICI. MBD4 could be a tissue-agnostic biomarker and should be sequenced in mUM, and other tumor types where MBD4 mutations are reported.

BACKGROUND: The RECIST-based response variably matches the clinical benefit of systemic therapies for liver metastatic uveal melanoma (LMUM). The aims were to determine whether the tumour growth rate (TGR) can help predict the survival in patients with LMUM and to provide information for the management of first-line systemic treatment. METHODS: This retrospective study included 147 (training: n = 110, validation: n = 37) patients with LMUM treated with first-line systemic treatment between 2010 and 2021. Two TGR-derived parameters were calculated, TGR(0) and TGR(3m). Multivariate Cox analyses identified independent predictors of progression-free survival (PFS) and overall survival (OS). RESULTS: TGR(3m) was a strong independent prognostic factor of PFS and OS (p

Simple Summary Histone Deacetylases (HDACs) have been reportedly associated with tumor development and progression in several types of human malignancy, being currently investigated as potential targets of anti-cancer therapy. The aim of this study is to assess the clinical significance and prognostic role of the of HDAC-1, -2, -4, and -6 immunohistochemical expression, in 75 uveal melanoma (UM) cases. HDACs are differentially expressed in UMs, HDAC-2 being the most frequently expressed isoform, whereas cytoplasmic expression of class I HDAC isoforms is also observed. Additionally, HDAC-1 was associated with increased tumor size, HDAC-6 with mitotic index, and HDAC-2 with epithelioid cell morphology and presence of tumor-infiltrating lymphocytes, both parameters of adverse prognosis. Moreover, our data support a significant association of HDAC-2 with patients’ improved OS. These findings suggest that HDACs, and especially HDAC-2, may be implicated in the formation and progression of UM. Abstract Background: Uveal melanoma (UM) represents the most common primary intraocular malignancy in adults, exerting high metastatic potential and poor prognosis. Histone deacetylases (HDACs) play a key role in carcinogenesis, and HDAC inhibitors (HDACIs) are currently being explored as anti-cancer agents in clinical settings. The aim of this study was to evaluate the clinical significance of HDAC-1, -2, -4, and -6 expression in UM. Methods: HDAC-1, -2, -4, and -6 expression was examined immunohistochemically in 75 UM tissue specimens and was correlated with tumors’ clinicopathological characteristics, the presence of tumor-infiltrating lymphocytes (TILS), as well as with our patients’ overall survival (OS). Results: HDAC-2 was the most frequently expressed isoform (66%), whereas we confirmed in addition to the expected nuclear expression the presence of cytoplasmic expression of class I HDAC isoforms, namely HDAC-1 (33%) and HDAC-2 (9.5%). HDAC-4 and -6 expression was cytoplasmic. HDAC-1 nuclear expression was associated with increased tumor size (p = 0.03), HDAC-6 with higher mitotic index (p = 0.03), and nuclear HDAC-2 with epithelioid cell morphology (p = 0.03) and presence of tumor-infiltrating lymphocytes (p = 0.04). The association with the remaining parameters including Monosomy 3 was not significant. Moreover, the presence as well as the nuclear expression pattern of HDAC-2 were correlated with patients’ improved OS and remained significant in multivariate survival analysis. Conclusions: These findings provide evidence for a potential role of HDACs and especially HDAC-2 in the biological mechanisms governing UM evolution and progression.

Conjunctival melanoma (ConjMel) is a potentially deadly ocular melanoma, originating from partially sunlight-exposed mucosa. We explored the mutational landscape of ConjMel and studied the correlation with etiological factors. We collected 47 primary ConjMel samples and performed next-generation sequencing of 400 genes. Hotspot mutations in BRAF, NRAS, HRAS, and KIT were observed in 16 (34%), 5 (11%), 2, and 2 cases, respectively. Patients with BRAF and CDKN2A-mutated ConjMel tended to be younger while the NF1-mutated one tended to be older. The eight tumors arising from nevi were enriched in CTNNB1 mutations (63% vs. 8%, Fisher’s exact p-test = 0.001) compared to non-nevi ConjMel and five were devoid of BRAF, RAS, NF1, or KIT mutations, suggesting a specific oncogenic process in these tumors. The two KIT-mutated cases carried SF3B1 mutations and were located on sun-protected mucosa, a genotype shared with genital and anorectal mucosal melanomas. Targetable mutations were observed in ERBB2, IDH1, MET, and MAP2K1 (one occurrence each). Mutational landscape of ConjMel characterizes distinct molecular subtypes with oncogenic drivers common with mucosal and skin melanomas. CTNNB1 mutations were associated with nevus-derived ConjMel. Concomitant KIT/SF3B1 mutations in sun-protected cases suggest a common tumorigenic process with genital and anorectal mucosal melanomas.

To evaluate the prognostic implications of melanin quantification assessed by magnetic resonance imaging (MRI) with respect to the clinical, pathological, and genetic features of liver metastases of uveal melanoma (LMUM). This single-center retrospective cohort study included 63 patients eligible for margin-free resection of LMUM between 2007 and 2018. Comparative genomic hybridization of resected liver metastases on microarrays was performed for genetic risk classification. Metastases exhibiting monosomy 3 with any type of gain of chromosome 8 (M3/8g) were considered high-genetic-risk. MRI melanin quantification using the mean T1 signal (mT1s) in liver metastases was assessed quantitatively on preoperative imaging examination and compared to that of gross pathological evaluation. The association between MRI melanin quantification and overall survival (OS) was assessed by multivariate analysis using the Cox proportional hazards model. Gross pathological assessment of melanin content and MRI melanin quantification were strongly correlated (r = 0.8, p &lt, 0.001). Independent prognostic factors associated with OS were disease-free interval ≤ 24 months (HR = 3.1, 95% CI, 1.6–6.0, p &lt, 0.001), high-genetic-risk (HR = 2.2, 95% CI, 1.1–4.8, p = 0.04), mT1s &gt, 1.1 (HR = 2.3, 95% CI, 1.2–4.7, p = 0.019), and complete hepatic resection (HR = 0.3, 95% CI, 0.2–0.7, p = 0.004). In patients with high-genetic-risk, mT1s showed a significant association with OS (HR = 3.7, 95% CI, 1.5–9.3, p = 0.006). The median OS was 17.5 months vs. 27 months for &gt, 1.1 and ≤1.1 mT1s tumors, respectively (p = 0.003). We showed that the level of pigmentation in M3/8g LMUM identified two subsets that were correlated with distinct clinical outcomes.

The outcome of patients with cutaneous melanoma has been strongly modified by recent advances obtained with Immune Checkpoint Inhibitors (ICIs). However, despite this breakthrough, durable response to ICIs is limited to a subset of patients. We investigated whether the expression of TRF2, which preserves telomere integrity, and have an effect on tumor immunosurveillance notably by directly recruiting and activating myeloid-derived suppressor cells (MDSCs), could be a prognostic biomarker in patients with relapsed or metastatic melanoma based on different treatment regimens. We evaluated retrospectively the association of TRF2 expressed in melanoma cells in combination with intratumoral CD33+ CD15+ CD14- MDSCs, as detected by immunohistochemistry and quantified by digital analysis, to clinicopathological features and overall survival (OS) among 48 patients treated with ICIs and 77 patients treated with other treatment options. The densities/mm2 of TRF2+ cells (P=.003) and CD33+ cells (P=.004) were individually significantly related to poor OS. In addition, only the combined expression of CD33+/CD15+/CD14- cells/mm2 was significantly correlated to poor OS (P=.017) in the whole study population as well as in patients treated by ICIs (P=.023). There was no significant difference in OS when analyzing the other markers individually or in combination according to the treatment regimen. The pre-treatment assessment of TRF2 expression and CD33+ cells/mm2 along with the density of CD33+/CD15+/CD14- cells/mm2 could assess OS and better predict clinical response of patients with melanoma treated by ICIs.

e21601 Background: MBD4 encodes a glycosylase implicated in repair of lesions induced by DNA deamination and its inactivation in tumors is associated with a hypermutated phenotype. Uveal melanoma (UM) is a rare but aggressive form of melanoma carrying an extremely low mutation burden. Although metastatic UM (mUM) are usually resistant to immune checkpoint inhibitors (ICI), the first reported MBD4-mutated (MBD4m) patient responded to ICI, suggesting that MBD4 mutation may predict response to ICI. Since then, other MBD4-inactivated UMs, acute myeloid leukemias, colorectal adenocarcinomas, gliomas and spiradenocarcinoma cases have been reported. Methods: Retrospective cohort of mUM patients treated with ICI. MBD4 was sequenced in a subset of these patients. Results: Three hundred mUM patients were analyzed. Median follow-up was 17.3 months. Ten objective responses and 20 stable disease for > 12 months were observed, corresponding to an objective response rate of 3.3% and a clinical benefit (CB; i.e. responder patients and stable disease) rate of 10%. Median progression-free survivals (PFS) in patients without and with CB were 3.1 and 16.3 months, respectively (p < 0.0001). Of the 134 tumors sequenced for MBD4, five (3.7%) were mutated. MBD4 inactivation was associated with higher mutation burden (179 to 643 variants versus a median of 16 in MBD4 wild-type), better objective response rate as 60% of MBD4m versus 4% of MBD4-wild type patients responded (Fisher’s exact p-test = 0.0009). Median PFS was 4.0 months in MBD4-wild type and 22.3 months in MBD4m patients (HR = 0.22; p = 0.01). Median overall survival was 16.6 months in MBD4-wild type and unreached in MBD4m patients (HR = 0.11; p = 0.004). Conclusions: In mUM patients, MBD4 mutation is highly predictive for response to ICI, PFS and overall survival benefit. MBD4 could be a tissue-agnostic biomarker and should be sequenced in mUM and other tumor types where MBD4 mutations are reported.

Disruption of splicing patterns due to mutations of genes coding splicing factors in tumors represents a potential source of tumor neoantigens, which would be both public (shared between patients) and tumor-specific (not expressed in normal tissues). In this study, we show that mutations of the splicing factor SF3B1 in uveal melanoma generate such immunogenic neoantigens. Memory CD8+ T cells specific for these neoantigens are preferentially found in 20% of patients with uveal melanoma bearing SF3B1-mutated tumors. Single-cell analyses of neoepitope-specific T cells from the blood identified large clonal T-cell expansions, with distinct effector transcription patterns. Some of these expanded T-cell receptors are also present in the corresponding tumors. CD8+ T-cell clones specific for the neoepitopes specifically recognize and kill SF3B1-mutated tumor cells, supporting the use of this new family of neoantigens as therapeutic targets. Significance: Mutations of the splicing factor SF3B1 in uveal melanoma generate shared neoantigens that are uniquely expressed by tumor cells, leading to recognition and killing by specific CD8 T cells. Mutations in splicing factors can be sources of new therapeutic strategies applicable to diverse tumors. This article is highlighted in the In This Issue feature, p. 1861

Uveal melanoma is the most common primary intraocular malignancy in adults. The development of distant metastases is associated with a poor prognosis. Ephrine receptors (Eph) are the largest subpopulation of tyrosine kinase receptors. They play an important role in processes related to the formation and progression of cancer. The aim of the study was to evaluate the expression of ephrin receptors EphA1, EphA5, and EphA7 in uveal melanoma and its associations with clinicopathological parameters, overall survival, and disease-free survival. The study included 94 previously untreated patients who underwent enucleation due to uveal melanoma. High expression of EphA1 was positively correlated with a smaller tumor size, less frequent extra-scleral extension, lower mitotic activity, and more frequent vitreous hemorrhage. High expression of EphA5 was associated with less frequent chromosome 3 loss, absence of distant metastases, and more frequent vitreous hemorrhage. High expression of EphA7 was associated with a more frequent primary tumor location in the posterior pole. High EphA5 expression was associated with longer overall survival time. The above findings indicate that high expression of EphA1 and EphA5 can be considered a beneficial prognostic factor in uveal melanoma.

Disruption of splicing patterns due to mutations of genes coding splicing factors in tumors represents a potential source of tumor neoantigens, which would be both public (shared between patients) and tumor-specific (not expressed in normal tissues). In this study, we show that mutations of the splicing factor

Uveal melanomas (UMs) comprise the most common primary intraocular malignancies in adults, with the eye representing the second most common site for melanoma, following the skin. Prognosis remains poor, with approximately half of the cases presenting with metastatic disease at the time of diagnosis. Erythropoietin-producing human hepatocellular receptors (EPHs) comprise the largest known family of tyrosine receptors, in which, along with their ligands, ephrins, play an important role in a plethora of processes in human physiology, and are implicated in key steps of carcinogenesis. In the present study, EPHA2, EPHA4, and EPHA6 immunohistochemical expressions were investigated in UM tissues and further correlated to a multitude of clinicopathological parameters, including disease stage and patients’ overall survival (OS). High levels of EPHA2 expression were significantly associated with increased tumor vertical thickness (p = 0.03) and the presence of intrascleral involvement (p = 0.05), whereas high EPHA6 nuclear expression was associated with older age at diagnosis (p = 0.03) and absence of retinal detachment (p = 0.05). In a multivariate survival analysis, increased EPHA4 expression was associated with shortened OS along with the presence of metastasis (p < 0.001) and monosomy 3 (p = 0.02). In a separate model, the concurrent overexpression of at least two of the investigated EPHs (HR = 14.7, p = 0.03) also proved to be an independent poor prognostic factor. In conclusion, our results implicate these specific members of the EPHA group as potential biomarkers for disease prognosis as well as possible targets for the development of novel therapeutic interventions.

Up to 50% of uveal melanomas (UM) metastasise to the liver within 10 years of diagnosis, and these almost always prove rapidly fatal. As histopathological growth patterns (HGPs) of liver metastases of the replacement and desmoplastic type, particularly from colon and breast carcinoma, may import valuable biological and prognostic information, we have studied HGP in a series of 41 UM liver metastases originating from 41 patients from the period 2006-2017. Twenty patients underwent enucleation while 21 had radiation therapy. Analysis of UM by array comparative genomic hybridisation revealed: 25 (64%) patients with high risk (monosomy3/8q gain); 13 (33%) intermediate risk (M3/8normal or disomy3/8q gain); and 1 low risk (disomy3/8normal). The principal HGP was replacement in 30 (73%) cases and desmoplastic in 11 (27%) cases. Cases with replacement demonstrated striking vascular co-option/angiotropism. With the development of liver metastasis, only the replacement pattern, largest primary tumour diameter, and R2 (incomplete resection) status predicted diminished overall survival (OS; p < 0.041, p < 0.017, p < 0.047, respectively). On multivariate analysis, only HGP (hazard ratio; HR = 6.51, p = 0.008) and resection status remained significant. The genomic high-risk variable had no prognostic value at this stage of liver metastasis. Chi-square test showed no association of HGP with monosomy 3 or 8q gain. Eighteen of 41 (44%) patients are alive with disease and 23 (56%) patients died with follow-up ranging from 12 to 318 months (mean: 70 months, median: 47 months). In conclusion, we report for the first time the frequency of the replacement and desmoplastic HGPs in liver UM metastases resected from living patients, and their potential important prognostic value for UM patients, as in other solid cancers. These results may potentially be utilised to develop radiological correlates and therapeutic targets for following and treating patients with UM metastases.

Purpose: Uveal melanomas (UM) are genetically simple tumors carrying few copy number alterations (CNA) and a low mutation burden, except in rare MBD4-deficient, hypermutated cases. The genomics of uveal melanoma metastatic progression has not been described. We assessed the genetic heterogeneity of primary and metastatic MBD4-proficient and -deficient uveal melanomas. Experimental Design: We prospectively collected 75 metastatic and 16 primary samples from 25 consecutive uveal melanoma patients, and performed whole-exome sequencing. Results: MBD4-proficient uveal melanomas contained stable genomes at the nucleotide level, acquiring few new single nucleotide variants (SNVs; 16 vs. 13 in metastases and primary tumors, respectively), and no new driver mutation. Five CNAs were recurrently acquired in metastases (losses of 1p, 6q, gains of 1q, 8q, and isodisomy 3). In contrast, MBD4-deficient uveal melanomas carried more than 266 SNVs per sample, with high genetic heterogeneity and TP53, SMARCA4, and GNAS new driver mutations. SNVs in MBD4-deficient contexts were exploited to unveil the timeline of oncogenic events, revealing that metastatic clones arose early after tumor onset. Surprisingly, metastases were not enriched in monosomy 3, a previously defined metastatic risk genomic feature. Monosomy 3 was associated with shorter metastatic-free interval compared with disomy 3 rather than higher rate of relapse. Conclusions: MBD4-proficient uveal melanomas are stable at the nucleotide level, without new actionable alterations when metastatic. In contrast, MBD4 deficiency is associated with high genetic heterogeneity and acquisition of new driver mutations. Monosomy 3 is associated with time to relapse rather than rate of relapse, thus opening avenues for a new genetic prognostic classification of uveal melanomas.

Uveal melanomas (UM) are genetically simple tumors carrying few copy number alterations (CNA) and a low mutation burden, except in rare

Ocular medulloepithelioma (ME) is a rare congenital tumor which occurs usually during childhood but is also reported in adults. They have seen an intraocular tumor in an 89 years-old female with a history of small cell lung carcinoma. Transscleral fine needle aspiration was performed. Aspirates were rich and composed of two distinctive populations of cells. The first consisted of epithelioid large cohesive cells with rare rosettes. Nuclei were oval and chromatin was delicate with small nucleoli. The second population consisted of smaller and dispersed cells with regular nuclei and dusty chromatin. Immunohistochemistry performed on paraffin-embedded cell block sections showed that the larger cells and rosettes were cytokeratin AE1/AE3, Synaptophysin, Chromogranin A, CD56, NSE, and EMA positive, whereas the smaller cells were always negative. Interestingly smaller cells expressed only weak nuclear positivity for TTF1, whereas larger cells were TTF1 negative. Melanocytic markers were negative in both populations. Morphological patterns and immunohistochemical staining confirmed ocular ME and allowed to exclude pulmonary metastasis or primary malignant melanoma. The patient was successfully treated by brachytherapy alone and is alive and well 10 months after treatment. Diagn. Cytopathol. 2017;45:561-564. © 2017 Wiley Periodicals, Inc.

Exome sequencing has recently identified highly recurrent MED12 somatic mutations in fibroadenomas (FAs) and phyllodes tumors (PTs). In the present study, based on a large series, we confirmed the presence of MED12 exon 1 and 2 mutations in 49% (41/83) of PTs, 70% (7/10) of FAs and 9.1% (1/11) of fibromatoses. We show that MED12 mutations are associated with benign behavior of phyllodes tumors, as they are detected less frequently in malignant PTs (27.6%) compared to benign (58.3%) and borderline (63.3%) PTs, respectively (p = 0.0036). Phyllodes tumors presented marked temporal heterogeneity of MED12 mutation status, as 50% (3/6) of primary and recurrent phyllodes tumor pairs with MED12 mutation presented different MED12 mutations between the primary and recurrent tumors. There was no correlation between MED12 status and genomic profiles obtained by array-CGH. MED12 mutations are associated with altered expressions of the genes involved in the WNT (PAX3, WNT3A, AXIN2), TGFB (TAGLN, TGFBR2, CTGF) and THRA (RXRA, THRA) signaling pathways. In conclusion, this study confirmed that MED12 plays a central oncogenic role in breast fibroepithelial tumorigenesis and identified a limited number of altered signaling pathways that maybe associated with MED12 mutations. MED12 exon 1 and 2 mutation status and some of the altered genes identified in this study could constitute useful diagnostic or prognostic markers, and form the basis for novel therapeutic strategies for PTs.

Background: Uveal melanoma is the most common primary intraocular malignancy in adults. In advanced cases, the prognosis is very poor. Thus far, no effective methods of pharmacotherapy of this cancer have been found. The aim of the study was to evaluate the expression of PARP-1, the best-known member of the family of poly(ADP-ribose) polymerases, in uveal melanoma and its associations with clinicopathological parameters, overall survival, and disease-free survival. Methods: The study included 91 patients who underwent enucleation due to uveal melanoma. PARP-1 expression was assessed by immunohistochemistry. Results: High PARP-1 expression was associated with more frequent chromosome 3 loss, higher histopathological grade, bigger tumor size, and absence of intrascleral extension. High PARP-1 expression was associated with shorter overall survival time and disease-free survival time. Conclusions: The above findings indicate that high expression of PARP-1 can be considered as an unfavorable prognostic factor in uveal melanoma.

Summary Purpose Our objective was to assess the results of surgical management of palpebral basal cell carcinomas (BCC) followed by a second line treatment discussed during a Multidisciplinary Team Meeting (MTM). Materials and Methods This retrospective single-centred study includes all surgically-treated basal cell carcinomas of the eyelids between January 2005 and January 2015. After initial surgery, the cases were systematically discussed during a multidisciplinary team meeting in order to assess the need for additional treatment. Data relative to the patient, tumor and management were pulled from the medical record. Results A total of 171 patients were included, with a mean age of 74 years. Among the patients, 151 underwent pentagonal resection of the tumor, and 20 patients had a superficial excision. After surgical management, 120 patients (70.2%) were considered to have sufficient free margins. The other 51 patients (29.8%) had insufficient margins due to remaining tumor cells (38 patients) or free margins less than 1 mm. Among these 51 patients with insufficient margins, 19 received a second surgical treatment, 17 patients received adjuvant radiotherapy, and 15 were followed closely with an intensive biannual follow-up program. No patients were lost to follow-up. With a mean follow-up of 42 months (min. 6 months–max. 128 months), 7 out of 171 patients (4.1%) developed a local recurrence. The mean time between surgical management and recurrence was 24 months. The recurrence rate was higher for the group of patients with a recurrent tumor (11.6%) than for the group of patients referred for initial management (2.8%). Incomplete resection was also associated with a higher recurrence rate (3 recurrences out of 51 patients). Discussion The management of basal cell carcinomas of the eyelid is first and foremost surgical with the goal of complete resection confirmed by histopathological analysis. The histological analyses (Mohs micrographic surgery, frozen section technic, paraffin fixation) and recommended sizes of the margins can vary in the literature, with recurrence rates from 1.8% to 9.5%. Conclusion In our experience, multidisciplinary management of BCC of the eyelid, including initial macroscopic surgery, histopathological analysis stating the histological type and size of the margins, along with additional treatment discussed in a MTM, allows for a recurrence rate of 4.1%.

Metastatic uveal melanoma is a deadly disease with no proven standard of care. Here we present a metastatic uveal melanoma patient with an exceptional high sensitivity to a PD-1 inhibitor associated with outlier CpG>TpG mutation burden, MBD4 germline deleterious mutation, and somatic MBD4 inactivation in the tumor. We identify additional tumors in The Cancer Genome Atlas (TCGA) cohorts with similar hypermutator profiles in patients carrying germline deleterious MBD4 mutations and somatic loss of heterozygosity. This MBD4-related hypermutator phenotype may explain unexpected responses to immune checkpoint inhibitors., Hypermutated tumors respond more favorably to checkpoint inhibitor-based immune therapy. Here, the authors describe a new hypermutated phenotype due to germline mutations and subsequent somatic loss of heterozygosity of MBD4, and a dramatic response to the PD-1 inhibitor pembrolizumab in a patient with a MBD4-inactivated hypermutated uveal melanoma.

Background High throughput molecular screening techniques allow the identification of multiple molecular alterations, some of which are actionable and can be targeted by molecularly targeted agents (MTA). We aimed at evaluating the relevance of using this approach in the frame of Institut Curie Molecular Tumor Board (MTB) to guide patients with cancer to clinical trials with MTAs. Patients and methods We included all patients presented at Institut Curie MTB from 4 October 2014 to 31 October 2017. The following information was extracted from the chart: decision to perform tumour profiling, types of molecular analyses, samples used, molecular alterations identified and those which are actionable, and inclusion in a clinical trial with matched MTA. Results 736 patients were presented at the MTB. Molecular analyses were performed in 442 patients (60%). Techniques used included next-generation sequencing, comparative genomic hybridisation array and/or other techniques including immunohistochemistry in 78%, 51% and 58% of patients, respectively. Analyses were performed on a fresh frozen biopsy in 91 patients (21%), on archival tissue (fixed or frozen) in 326 patients (74%) and on both archival and fresh frozen biopsy in 25 patients (6%). At least one molecular alteration was identified in 280 analysed patients (63%). An actionable molecular alteration was identified in 207 analysed patients (47%). Forty-five analysed patients (10%) were enrolled in a clinical trial with matched MTA and 29 additional patients were oriented and included in a clinical trial based on a molecular alteration identified prior to the MTB analysis. Median time between date of specimen reception and molecular results was 28 days (range: 5–168). Conclusions The implementation of an MTB at Institut Curie enabled the inclusion of 10% of patients into a clinical trial with matched therapy.

Summary Introduction Surgical excision of large malignant lower eyelid tumors may cause important full-thickness eyelid defects. The reconstruction of such defects must restore the physiologic function of the eyelid and also re-establish an acceptable aesthetic result. Materials and methods We report the outcomes of full-thickness excision of tumors extending over half of the horizontal lid length, followed by reconstruction using a nasal chondromucosal graft (coming from the ipsilateral ala of the nose) and an upper eyelid myocutaneous flap . Histological analysis of the specimen identified the tumor type and surgical margins for each patient. Results A total of 25 patients were operated using this reconstruction technique between March 2009 and June 2015: 17 basal cell carcinomas , 3 spindle cell carcinomas and 5 conjunctival melanomas (out of which 2 were associated with lentigo maligna). Mean duration of follow-up after surgery was respectively 36, 41 and 17 months for each of these 3 tumor types. We found a single local tumor recurrence and this was a basal cell carcinoma in a xeroderma pigmentosum patient. After surgery, none of the patients had lagophthalmos or ocular surface complications. Only 4 patients had a 1 mm scleral show postoperatively; 3 other patients developed a small retraction of the eyelid after adjuvant radiotherapy and a 1 mm scleral show occurred. Conclusion In malignant tumors, complete surgical excision with histological margins adapted to tumor type prevents local recurrence in most cases. Our repair strategy of nasal chondromucosal graft and skin-muscle flap for large inferior eyelid defects provides good functional and aesthetic results.

A woman in her early fifties had regular follow-up for a medium-sized divided nevus of the eyelids which had undergone several surgical excisions during childhood for functional and esthetic reasons. Malignant transformation of the nevus occurred in the inferior eyelid, with the appearance of a new pigmented flat lesion. Histology showed in situ melanoma, and an NRAS activating mutation was found. A full-thickness excision of the inferior eyelid was performed, followed by reconstruction. Local recurrence of the melanoma occurred 1 year after surgery. Lifelong follow-up of divided nevi of the eyelids is recommended, even if very few cases of malignant transformation have been reported so far.

Background Uveal melanomas (UM) are BRAF-wild type tumors associated with dismal prognosis for which no systemic therapy is active. We report our experience with immune checkpoint inhibitors (ICI) in metastatic UM patients. Methods Ambispective cohort of metastatic UM patients treated with ICI in eight French centers. Response rate, progression-free survival (PFS) and overall survival were retrieved. Results 206 evaluable patients started their first ICI treatment between December 2012 and January 2019. The population consisted of 98 men and 108 women. Median age was 57.5 yo (19; 82). Seventy-four patients had been enucleated, 131 had received proton beam therapy, one had received brachytherapy. The median time to first metastasis was 29.4 months (0-380.4). ICI were first prescribed in first-line in 68 patients, in second-line in 79 patients, in third-line or more in 80 patients. One hundred forty-four patients received pembrolizumab (63%), 58 nivolumab (25%) and 21 ipilimumab (9%). Metastatic sites at initiation of ICI were liver, lung, skin, and bone for 201, three, one and one patients, respectively. Liver was the sole metastatic site in 201 patients. Patients received a median number of four ICI infusions (1-60+). Treatment was suspended because of immune side effects in 21 patients. Nine objective responses were observed (four complete and five partial responses; 4.4%) including one patient with a hypermutated, MBD4-deficient tumor. Fifty-seven patients showed stable disease as best response (27.7%). Median PFS in the whole cohort was 4.1 months (0; 30.4) in 1st line, 4.9 months (0; 42,6) in 2nd line and 3.1 (1; 17.,3) months in 3rd line. Median PFS was 4.1 months in the ICI-resistant population and 27.4 months in the ICI-sensitive population (log-rank p-test Conclusions ICI are associated with a low response rate in UM but with longer PFS and a trend for longer overall survival in ICI-sensitive tumors. Immunotherapies should be investigated in this disease. ICI-sensitive cases are currently being explored to identify biomarkers of response. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Introduction Uveal melanomas (UM) are chemoresistant tumors that carry few copy number variations (CNV) and a low mutation burden. Genomics of UM metastases have been scarcely described. We assessed genetic heterogeneity in UM at primary and metastatic stages, in order to evaluate if tumor heterogeneity may explain this chemoresistance. Methods Whole-exome sequencing of primary and metastatic UM tumors. Results We obtained 97 tumor samples from 26 patients (16 samples from 15 primary tumors and 81 samples from 50 metastases). MBD4 was inactivated in two cases (MBD4-deficient; MBD4def). All samples presented high variant allelic frequencies of UM driver mutations (Gαq pathway mutations, BAP1, SF3B1 and EIF1AX). Indeed, mutational statuses of these genes were identical between primary tumors and metastases, except for one MBD4def case that carried both SF3B1 and BAP1 mutations in the primary tumor, while the metastasis only carried a different BAP1 mutation. MBD4-proficient (MBD4pro) primary tumors presented a median of 11.5 single nucleotide variants (SNV; range 5-22); while MBD4pro metastases carried a median of 14 SNV (range 6-23) with a very high genetic homogeneity between samples. No mutation was recurrently acquired during the metastatic process. Metastases presented a higher number of copy number variations (CNV; median = 11; range 2-25) than matched primary (median = 5; range 2-18). Metastases-associated CNV were similar to that usually associated with UM primary tumors, including 8q gain, 1p loss, 1q gain, 6q loss and isodisomy 3. MBD4def samples were hypermutated with at least 266 mutations/sample (>20-fold increase compared to MBD4pro) with >70% of CpG>TpG. MBD4def cases presented a higher heterogeneity with less than 60% of common SNV concordance between tumors in a given patient. No major difference in CNV profiles was observed between MBD4def and MBD4pro samples. Conclusion MBD4pro are homogeneous diseases that evolve as metastases with few non-recurrent SNV and recurrent UM-typical CNV. These characteristics lead to simple evolutionary trees with a limited number of short clades and branches, supporting a punctuated evolutionary process. Genetic heterogeneity thus cannot explain the resistance to chemotherapy. In contrast, MBD4 inactivation in UM results in genetic heterogeneity, subclonality and risk of secondary resistance to anticancer drugs. Citation Format: Manuel Rodrigues, Lenha Mobuchon, Alexandre Houy, Samar Alsafadi, Sophie Gardrat, Sophie Piperno-Neumann, Nathalie Cassoux, Gaelle Pierron, Sergio Roman-Roman, Pascale Mariani, Marc-Henri Stern. MBD4 inactivation results in an alternative evolutionary route in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2895.

Ocular medulloepithelioma (ME) is a rare congenital tumor which occurs usually during childhood but is also reported in adults. They have seen an intraocular tumor in an 89 years-old female with a history of small cell lung carcinoma. Transscleral fine needle aspiration was performed. Aspirates were rich and composed of two distinctive populations of cells. The first consisted of epithelioid large cohesive cells with rare rosettes. Nuclei were oval and chromatin was delicate with small nucleoli. The second population consisted of smaller and dispersed cells with regular nuclei and dusty chromatin. Immunohistochemistry performed on paraffin-embedded cell block sections showed that the larger cells and rosettes were cytokeratin AE1/AE3, Synaptophysin, Chromogranin A, CD56, NSE, and EMA positive, whereas the smaller cells were always negative. Interestingly smaller cells expressed only weak nuclear positivity for TTF1, whereas larger cells were TTF1 negative. Melanocytic markers were negative in both populations. Morphological patterns and immunohistochemical staining confirmed ocular ME and allowed to exclude pulmonary metastasis or primary malignant melanoma. The patient was successfully treated by brachytherapy alone and is alive and well 10 months after treatment. Diagn. Cytopathol. 2017;45:561-564. © 2017 Wiley Periodicals, Inc.

Background Uveal melanoma (UM) arises from malignant transformation of melanocytes in the uveal tract of the eye. This rare tumor has a poor outcome with frequent chemo-resistant liver metastases. BAP1 is the only known predisposing gene for UM. UMs are generally characterized by low tumor mutation burden, but some UMs display a high level of CpG>TpG mutations associated with MBD4 inactivation. Here, we explored the incidence of germline MBD4 variants in a consecutive series of 1093 primary UM case patients and a series of 192 UM tumors with monosomy 3 (M3). Methods We performed MBD4 targeted sequencing on pooled germline (n = 1093) and tumor (n = 192) DNA samples of UM patients. MBD4 variants (n = 28) were validated by Sanger sequencing. We performed whole-exome sequencing on available tumor samples harboring MBD4 variants (n = 9). Variants of unknown pathogenicity were further functionally assessed. Results We identified 8 deleterious MBD4 mutations in the consecutive UM series, a 9.15-fold (95% confidence interval = 4.24-fold to 19.73-fold) increased incidence compared with the general population (Fisher exact test, P = 2.00 × 10–5, 2-sided), and 4 additional deleterious MBD4 mutations in the M3 cohort, including 3 germline and 1 somatic mutations. Tumors carrying deleterious MBD4 mutations were all associated with high tumor mutation burden and a CpG>TpG hypermutator phenotype. Conclusions We demonstrate that MBD4 is a new predisposing gene for UM associated with hypermutated M3 tumors. The tumor spectrum of this predisposing condition will likely expand with the addition of MBD4 to diagnostic panels. Tumors arising in such a context should be recognized because they may respond to immunotherapy.