M Versluis

Prognostic parameters in uveal melanoma and their association with BAP1 expression

Aim To determine whether BAP1 gene and protein expression associates with different prognostic parameters in uveal melanoma and whether BAP1 expression correctly identifies patients as being at risk for metastases, following enucleation of the primary tumour. Methods Thirty cases of uveal melanoma obtained by enucleation between 1999 and 2004 were analysed for a variety of prognostic markers, including histological characteristics, chromosome aberrations obtained by fluorescence in situ hybridisation (FISH) and single nucleotide polymorphism (SNP) analysis and gene expression profiling. These parameters were compared with BAP1 gene expression and BAP1 immunostaining. Results The presence of monosomy of chromosome 3 as identified by the different chromosome 3 tests showed significantly increased HRs (FISH on isolated nuclei cut-off 30%: HR 11.6, p=0.002; SNP analysis: HR 20.3, p=0.004) for death due to metastasis. The gene expression profile class 2, based on the 15-gene expression profile, similarly provided a significantly increased HR for a poor outcome (HR 8.5, p=0.005). Lower BAP1 gene expression and negative BAP1 immunostaining (50% of 28 tumours were immunonegative) were both associated with these markers for prognostication: FISH cut-off 30% monosomy 3 (BAP1 gene expression: p=0.037; BAP1 immunostaining: p=0.001), SNP-monosomy 3 (BAP1 gene expression: p=0.008; BAP1 immunostaining: p=0.002) and class 2 profile (BAP1 gene expression: p<0.001; BAP1 immunostaining: p=0.001) and were themselves associated with an increased risk of death due to metastasis (BAP1 gene expression dichotomised: HR 8.7, p=0.006; BAP1 immunostaining: HR 4.0, p=0.010). Conclusions Loss of BAP1 expression associated well with all of the methods currently used for prognostication and was itself predictive of death due to metastasis in uveal melanoma after enucleation, thereby emphasising the importance of further research on the role of BAP1 in uveal melanoma.

Inhibition of CSF-1R Supports T-Cell Mediated Melanoma Therapy

Tumor associated macrophages (TAM) can promote angiogenesis, invasiveness and immunosuppression. The cytokine CSF-1 (or M-CSF) is an important factor of TAM recruitment and differentiation and several pharmacological agents targeting the CSF-1 receptor (CSF-1R) have been developed to regulate TAM in solid cancers. We show that the kinase inhibitor PLX3397 strongly dampened the systemic and local accumulation of macrophages driven by B16F10 melanomas, without affecting Gr-1+ myeloid derived suppressor cells. Removal of intratumoral macrophages was remarkably efficient and a modest, but statistically significant, delay in melanoma outgrowth was observed. Importantly, CSF-1R inhibition strongly enhanced tumor control by immunotherapy using tumor-specific CD8 T cells. Elevated IFNγ production by T cells was observed in mice treated with the combination of PLX3397 and immunotherapy. These results support the combined use of CSF-1R inhibition with CD8 T cell immunotherapy, especially for macrophage-stimulating tumors.

Digital PCR Validates 8q Dosage as Prognostic Tool in Uveal Melanoma

Background Uveal melanoma (UM) development and progression is correlated with specific molecular changes. Recurrent mutations in GNAQ and GNA11 initiate UM development while tumour progression is correlated with monosomy of chromosome 3 and gain of chromosome 8q. Hence, molecular analysis of UM is useful for diagnosis and prognosis. The aim of this study is to evaluate the use of digital PCR (dPCR) for molecular analysis of UM. Methods A series of 66 UM was analysed with dPCR for three hotspot mutations in GNAQ/GNA11 with mutation specific probes. The status of chromosomes 3 and 8 were analysed with genomic probes. The results of dPCR analysis were cross-validated with Sanger sequencing, SNP array analysis, and karyotyping. Results Using dPCR, we were able to reconstitute the molecular profile of 66 enucleated UM. With digital PCR, GNAQ/GNA11 mutations were detected in 60 of the 66 UM. Sanger sequencing revealed three rare variants, and, combined, these assays revealed GNAQ/GNA11 mutations in 95% of UM. Monosomy 3 was present in 43 and chromosome 8 aberrations in 52 of the 66 UM. Survival analysis showed that increasing 8q copy numbers were positively correlated with metastasis risk. Conclusion Molecular analysis with dPCR is fast and sensitive. Just like the recurrent genomic aberrations of chromosome 3 and 8, hotspot mutations in GNAQ and GNA11 are effectively detected in heterogeneous samples. Increased sensitivity contributes to the number of mutations and chromosomal aberrations detected. Moreover, quantification of copy number with dPCR validated 8q dosage as a sensitive prognostic tool in UM, of which implementation in disease prediction models will further improve prognostication.

Radiation Treatment Affects Chromosome Testing in Uveal Melanoma.

PURPOSE The purpose of this study was to determine whether radiation treatment induces chromosomal aberrations in uveal melanoma (UM) and to evaluate which tumor features determine success of karyotyping and FISH. METHODS Material from 327 UM-containing enucleated eyes was submitted for karyotyping, while FISH for chromosome 3 was performed in 248 samples. Thirty-six UMs had previously undergone irradiation. Karyotypes were analyzed, and the success rate of karyotyping/FISH was evaluated and compared with clinicopathologic tumor characteristics and prior irradiation. RESULTS Aberrations were observed in all chromosomes, with chromosomes 1, 3, 6, 8, 13, 15, 16, and Y being altered in at least 15% of the tumors. Aberrations were more common and more complex in previously irradiated tumors (significant for chromosomes 5 [P = 0.004] and 13 [P = 0.04]). Karyotyping and FISH failed significantly more often in irradiated tumors (both P < 0.001). In nonirradiated cases, successful karyotyping was related to a large tumor prominence (P = 0.004) and a high mitotic count (P = 0.007). The success of FISH in these tumors was not associated with any of the studied parameters. In irradiated tumors, karyotyping succeeded more frequently in cases with a high mitotic count (P = 0.03), whereas FISH was more often successful in tumors with a high mitotic count (P = 0.001), a large diameter (P = 0.009) and large prominence (P = 0.008). CONCLUSIONS Karyotyping and FISH are more often successful in UMs with features characteristic of high tumor aggressiveness, whereas prior irradiation leads to multiple chromosome aberrations and to unsuccessful tests. It will be interesting to determine whether other techniques can provide reliable information on the chromosome status of previously irradiated UMs.

Distribution of GNAQ and GNA11 Mutation Signatures in Uveal Melanoma Points to a Light Dependent Mutation Mechanism

Uveal melanomas (UM) originate from melanocytes in the interior wall of the eye, namely from the iris, ciliary body and the choroid with marked differences in light exposure (from dark anterior to illuminated posterior). In contrast to UV radiation, focused or converging visible light readily reaches the retina and can damage DNA which possibly contributes to UM development. In this report choroidal, ciliochoroidal and iridociliary melanomas were analyzed for GNAQ and GNA11 mutations which were subsequently correlated to the location of tumor origin. Hotspot mutations in GNAQ and GNA11 can be divided in A>T and in A>C mutation signatures. The GNAQ A626C mutation (Q209P) was almost exclusively observed in choroidal melanomas from the illuminated posterior side. On the other hand, ciliochoroidal UM from the dark anterior side with mostly A>T mutations were clearly associated with light-colored eyes. Combined these data suggest a light and a pigment dependent etiology in UM development.

GNAQ and GNA11 mutations and downstream YAP activation in choroidal nevi

Background:Mutations in GNAQ/11 genes are considered an early event in the development of uveal melanoma that may derive from a pre-existing nevus. The Hippo pathway, by way of YAP activation, rather than MAP kinase, has a role in the oncogenic capacity of GNAQ/11 mutations.Methods:We investigated 16 nevi from 13 human eyes for driver GNAQ/11 mutations using droplet digital PCR and determined whether nevi are clonal by quantifying mutant nevus cell fractions. Immunohistochemistry was performed on 15 nevi to analyse YAP activation.Results:For 15 out of 16 nevi, a GNAQ/11 mutation was detected in the nevus cells albeit at a low frequency with a median of 13%. Nuclear YAP, a transcriptional co-activator in the Hippo tumour-suppressor pathway, was detected in 14/15 nevi.Conclusions:Our analysis suggests that a mutation in GNAQ/11 occurs in a subset of choroidal nevus cells. We hypothesise that GNAQ/11 mutant-driven extracellular mitogenic signalling involving YAP activation leads to accumulation of wild-type nevus cells.

Prediction model for regional or distant recurrence in endometrial cancer based on classical pathological and immunological parameters

Background:Adjuvant therapy increases disease-free survival in endometrial cancer (EC), but has no impact on overall survival and negatively influences the quality of life. We investigated the discriminatory power of classical and immunological predictors of recurrence in a cohort of EC patients and confirmed the findings in an independent validation cohort.Methods:We reanalysed the data from 355 EC patients and tested our findings in an independent validation cohort of 72 patients with EC. Predictors were selected and Harrell’s C-index for concordance was used to determine discriminatory power for disease-free survival in the total group and stratified for histological subtype.Results:Predictors for recurrence were FIGO stage, lymphovascular space invasion and numbers of cytotoxic and memory T-cells. For high risk cancer, cytotoxic or memory T-cells predicted recurrence as well as a combination of FIGO stage and lymphovascular space invasion (C-index 0.67 and 0.71 vs 0.70). Recurrence was best predicted when FIGO stage, lymphovascular space invasion and numbers of cytotoxic cells were used in combination (C-index 0.82). Findings were confirmed in the validation cohort.Conclusions:In high-risk EC, clinicopathological or immunological variables can predict regional or distant recurrence with equal accuracy, but the use of these variables in combination is more powerful.

Accurate Quantification of T Cells by Measuring Loss of Germline T-Cell Receptor Loci with Generic Single Duplex Droplet Digital PCR Assays

Quantifying T cells accurately in a variety of tissues of benign, inflammatory, or malignant origin can be of great importance in a variety of clinical applications. Flow cytometry and immunohistochemistry are considered to be gold-standard methods for T-cell quantification. However, these methods require fresh, frozen, or fixated cells and tissue of a certain quality. In addition, conventional and droplet digital PCR (ddPCR), whether followed by deep sequencing techniques, have been used to elucidate T-cell content by focusing on rearranged T-cell receptor (TCR) genes. These approaches typically target the whole TCR repertoire, thereby supplying additional information about TCR use. We alternatively developed and validated two novel generic single duplex ddPCR assays to quantify T cells accurately by measuring loss of specific germline TCR loci and compared them with flow cytometry-based quantification. These assays target sequences between the Dδ2 and Dδ3 genes (TRD locus) and Dβ1 and Jβ1.1 genes (TRB locus) that become deleted systematically early during lymphoid differentiation. Because these ddPCR assays require small amounts of DNA instead of freshly isolated, frozen, or fixated material, initially unanalyzable (scarce) specimens can be assayed from now on, supplying valuable information about T-cell content. Our ddPCR method provides a novel and sensitive way for quantifying T cells relatively fast, accurate, and independent of the cellular context.

Epigenetic regulation of the epithelial phenotype in uveal melanoma

Purpose The presence of epithelial cells is a bad prognostic factor in uveal melanoma (UM) and epithelial tumors are characterized by high expression of E-cadherin. However, CDH1, the gene encoding for E-cadherin, is located at chromosome 16q which is lost in UM with a bad prognosis. In order to solve this paradox we studied the underlying epigenetic mechanism of the spindle to epithelial transition and the accompanying E-cadherin expression. Methods UM cell lines were in vitro treated with methylation and deacetylation inhibitors, or were cultured under hypoxic conditions to mimic in vivo UM conditions to induce epigenetic reprogramming. RNA and DNA was isolated for gene expression, DNA methylation, and chromosome structure analysis, while cell lysates were used for E-cadherin analysis. Results Treatment with methylation and deacetylation inhibitors resulted in a phenotypic change from a spindle to more epithelial cell type. Furthermore, inhibition of methylation and hypoxia treatment both upregulated E-cadherin expression in vitro and induced euchromatin formation at 16q22, where CDH1 is located. Conclusion By revealing epigenetic regulation of CDH1 in UM we resolve a step in the dynamic process of spindle to epitheloid celltype switch in UM. Since this switch could be induced by hypoxia we establish a link between environmental stress and progression. With these data we provide evidence for epigenetic regulation of UM progression as well as an option for future treatment.

Gain of chromosome 6 status does not influence HLA‐expression in uveal melanoma

Purpose Chromosomal aberrations and the inflammatory phenotype have been identified as predictive factors for survival of uveal melanoma (UM). The mechanism by which these factors are linked remains obscure. We took the human leukocyte antigen (HLA), located on chromosome 6p as marker for inflammation and studied whether aberrations of chromosome 6p influenced the HLA expression in UM. Methods SNP-array copy number analysis and gene expression profiling were performed on 28 uveal melanomas of patients who underwent enucleation between 1999 and 2004 at the Leiden University Medical Center, Leiden, in The Netherlands. UM protein expression of HLA-A, -B and –DR was measured with immunohistochemistry. The status of chromosome 3, 6p, and gene expression of HLA and HLA regulators were analyzed, as well as protein expression of HLA. Results Gain of 6p was present in 8 cases (29%) and not related with survival. An increased gene expression was seen in the group who died due to metastatic UM for HLA-A and –B, and B2M. Gene expression correlated with protein expression for HLA-A, HLA-B, but not with HLA-DR. Tumors did not differ in level of HLA protein expression, HLA gene expression, and HLA regulator gene expression with regard to chromosome 6p status. Conclusion High HLA gene- and protein expression in UM are not influenced by gain of 6p. Yet for the first time we demonstrated that in UM an increased expression of HLA class I genes correlated with the elevated protein expression of HLA class I.