Local environmental influences on uveal melanoma: vitreous humor promotes uveal melanoma invasion, whereas the aqueous can be inhibitory

BACKGROUND: Uveal melanomas of the choroid and ciliary body are aggressive tumors causing the death of approximately 50% of patients. In contrast, iris melanomas only infrequently metastasize; why these differences exist is not known. The local environment can regulate cancer growth and development, and it is probable the aqueous and vitreous humors have an important role in regulating uveal melanoma behavior. METHODS: To explore this possibility cultures of uveal melanoma were exposed to aqueous and vitreous and the effects investigated using invasion and proliferation assays. ChemiArrays (Chemicon International, Temecula, Calif) were performed to determine which regulatory factors might influence the process. RESULTS: The vitreous universally promoted uveal melanoma invasion, whereas the aqueous mainly had no effect or was inhibitory. Tumor location, and the baseline invasion of the melanoma, affected the ability of aqueous and vitreous from different patients to regulate invasive behavior. Proliferation was not significantly altered as a result of exposure to the aqueous or vitreous. The ability of the humors to regulate uveal melanomas may involve TIMP-2, TIMP-3, and TGF-beta2, as high expression was found by ChemiArray analysis and there were differences in the levels of the regulators in the aqueous compared with the vitreous. CONCLUSIONS: The findings suggest that in situ uveal melanoma development reflects an interaction between the tumor and the environment of the eye. Exposure to the aqueous would therefore contribute to the benign nature of iris melanomas, whereas potential interaction with the vitreous appears to promote the aggressive behavior of posterior uveal melanomas.     

Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma

Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have been recently investigated in several cancer types, but their respective clinical significance remains to be determined. In our prospective study, we compared the detection rate and the prognostic value of these two circulating biomarkers in patients with metastatic uveal melanoma. GNAQ/GNA11 mutations were characterized in archived tumor tissue. Using a highly sensitive and mutation‐specific bidirectional pyrophosphorolysis‐activated polymerization (bi‐PAP) technique, GNAQ c.626A>T, GNAQ c.626A>C and GNA11 c.626A>T copy numbers were quantified in plasma from 12 mL of blood. CTCs were detected at the same time in 7.5 mL of blood by the CellSearch® technique. Patient characteristics and outcome were prospectively collected. CTCs (≥1) were detected in 12 of the 40 included patients (30%, range 1–20). Among the 26 patients with known detectable mutations, ctDNA was detected and quantified in 22 (84%, range 4–11,421 copies/mL). CTC count and ctDNA levels were associated with the presence of miliary hepatic metastasis (p = 0.004 and 0.03, respectively), with metastasis volume (p = 0.005 and 0.004) and with each other (p < 0.0001). CTC count and ctDNA levels were both strongly associated with progression‐free survival (p = 0.003 and 0.001) and overall survival (p = 0.0009 and <0.0001). In multivariate analyses, ctDNA appeared to be a better prognostic marker than CTC. In conclusion, ctDNA and CTC are correlated and both have poor prognostic significance. CTC detection can be performed in every patient but, in patients with detectable mutations, ctDNA was more frequently detected than CTC and has possibly more prognostic value.     

International uveal melanoma incidence trends in view of a decreasing proportion of morphological verification

The introduction of eye-preserving therapies for uveal melanoma in the 1970s complicates time trend analyses of the uveal melanoma incidence because the proportion of morphologically verified uveal melanoma has been decreasing over the decades. We carried out incidence trend analyses, based on data from internationally accredited population-based cancer registries throughout the world that take missing data on topography, morphology and basis of diagnosis of eye tumours into account. We selected incidence data of cancer registries that were included in Cancer Incidence In 5 Continents, Volumes VI-VIII covering a registration period of at least 15 years (usually 1983 to 1997) and classified each eye cancer as morphologically verified uveal melanoma, clinically diagnosed uveal melanoma, uveal melanoma identified as DCO case (Death certificate only), possibly uveal melanoma, other eye tumour or unclassifiable eye tumour and calculated age-standardized incidence rates by 3-year calendar periods using the World Standard Population as the reference. The uveal melanoma incidence decline in the United States SEER Caucasian population is due mainly to an incidence decline in the early registration period (from 1974-76 to 1986-88). The data from France and Italy suggest a recent increase in incidence. Uveal melanoma diagnosed clinically increasingly contribute to the overall uveal melanoma incidence over time. Combining all registries, the proportion of morphologically verified uveal melanoma decreased from 82% in 1983-87 to 75% in 1993-97. Uveal melanoma incidence rates remained quite stable during the period 1983-97. The interpretation of uveal melanoma incidence trends is complicated by missing data on topography within the eye, morphology and basis of diagnosis.     

人葡萄膜黑色素瘤小鼠肝内种植模型生长行为研究

目的：采用肝内注射人眼葡萄膜恶性黑色素瘤株（MuM2B细胞）方法制备该肿瘤的小鼠肝内种植模型,研究肿瘤在其体内的生物学行为。方法：选用BALB／C-nu裸鼠,将5×10^4个MUM2B细胞注入裸鼠肝脏包膜下。连续观察6周,每周随机处死3只裸鼠以观察成瘤率及侵袭率。采集裸鼠的荷瘤肝脏、心脏、脾脏、肺脏、肾脏、胃和肠等组织器官,常规石蜡切片,HE染色后镜检肿瘤在肝脏内的生长行为及其向其他脏器侵袭和转移的情况。结果：该模型的成瘤率可达83．3％。接种后的前4周,肿瘤在肝脏内浸润性生长,未侵犯周围脏器;接种后第5周,肿瘤开始侵袭肾脏、脾脏、胃和十二指肠的包膜,并可突破胃及肠道的包膜,在其浆膜层中侵袭生长;接种后第6周,肿瘤对肾脏、脾脏、十二指肠和胃的侵袭加剧。肿瘤细胞可侵入裸鼠十二指肠的肌层内生长,亦可侵入裸鼠胃肌层及胃黏膜下层,甚至向胃黏膜肌层内侵袭生长;接种后第6周,肿瘤开始向裸鼠肺脏转移,所有受检裸鼠的肺门组织及肺实质内均可查见转移性瘤灶,转移率达100％;除肺脏外,其余脏器内均未查见转移性瘤灶,提示肺脏是该肝内种植瘤的主要转移靶器官。此外,荷瘤2周后,裸鼠脾脏红髓增生明显,髓内可见大量巨核细胞及髓系来源的粒细胞,呈现白血病样反应;荷瘤4周后,脾脏内星空样现象明显,巨噬细胞活跃,提示裸鼠脾脏组织学改变与荷瘤生长状态密切相关。结论：裸鼠肝内注射MUM2B细胞能够建立起稳定的人葡萄膜黑色素瘤小鼠肝内种植模型。种植的肿瘤细胞在裸鼠肝脏内呈浸润性生长,侵袭包括胃、十二指肠、脾脏及肾脏在内的多个器官,并可远端转移至肺脏。     

Cooking and uveal melanoma risk: results from two German case-control studies

Objective: Two recent studies indicated that cooks may have an increased risk of uveal melanoma. Here we report findings of two German case–control studies regarding cooking and uveal melanoma risk. Methods: We conducted a hospital and population-based case–control study of uveal melanoma and occupational exposures. We then pooled these results. Overall, 118 cases and 475 controls matching on age, sex and study regions were interviewed. We classified subjects as exposed to an occupational category (i.e. cooks) if they had ever worked within this category for at least six months or more. Subjects who had worked as cooks were rated as either (a) having prepared food without having cooked and therefore unexposed to cooking or (b) having cooked. We used conditional logistic regression models to calculate pooled odds ratios (OR) and 95% confidence intervals (95% CI). Results: Subjects who had ever cooked had an OR of 6.1 (95% CI: 1.7–22.2). Cooking was associated with an OR of 4.0 (95% CI: 0.8–20.1) for a job duration of 0.5–2 years and with an OR of 11.4 (95% CI: 1.6–81.9) for a job duration more than 2 years. Conclusions: In light of the similar finding in other studies, the association deserves further attention.     

Lack of BAP1 protein expression in uveal melanoma is associated with increased metastatic risk and has utility in routine prognostic testing

Background:

The absence of BRCA1-associated protein 1 (BAP1) expression in uveal melanoma (UM) is associated with metastatic progression and reduced survival. In this study, we examine nuclear BAP1 (nBAP1) protein expression in primary UMs (PUMs) that show both ‘typical'' and ‘atypical'' clinical courses according to their chromosome 3 status, and secondary hepatic metastatic UM (MUM), correlating the results with histological, clinical and survival data.

Methods:

Nuclear BAP1 expression was immunohistochemically assessed in tissue microarrays (TMAs) of: (a) 68 PUM patients, who had been treated surgically; and (b) 13 MUM patients, with 5 cases being paired with primary tumour tissue. All cases were fully annotated. The percentage of tumour cell nuclei staining positively for BAP1 was scored by independent observers.

Results:

Nuclear BAP1 protein expression was absent in 35 out of 68 (51%) PUM patients, correlating strongly with poor prognostic clinicopathological and genetic parameters and reduced survival (Log rank, P<0.001). Lack of nBAP1 expression importantly identified a subset of ‘atypical'' PUM patients with disomy of chromosome 3 but with unexpected metastatic relapse. Nuclear BAP1 expression was absent in 10 out of 13 (77%) MUM and expression was concordant in all paired PUM and MUM patients.

Conclusions:

Absent nBAP1 protein expression is an independent survival predictor for UM patients, easily examined using immunohistochemistry.     

Gene expression profiling identifies tumour markers potentially playing a role in uveal melanoma development

Microarray is a powerful tool to compare the gene expression of different tumour specimens and cell lines simultaneously and quantitatively. To get a better insight into genes that are involved in uveal melanoma tumorigenesis, we compared the gene expression profiles of 12 different uveal melanoma cell lines with three melanocyte cell cultures obtained from healthy donor eyes. Gene expression profiles were obtained by nylon filter arrays, containing 1176 gene spots related to cancer development. The expression levels of selected genes were validated on cell lines and primary uveal melanomas by real time RT-PCR, and were subsequently included in cluster analysis. Four candidate tumour markers, Laminin Receptor 1, Endothelin 2, Von Hippel Lindau Binding protein 1 and Cullin 2, have been selected from genes that were differentially expressed in the uveal melanoma cell lines compared to the normal uveal melanocytes. In primary uveal melanomas, these four markers could discriminate between two classes of uveal melanoma, which may be indicative of a differential disease process.     

Prognostic significance of chromosome 3 alterations determined by microsatellite analysis in uveal melanoma: a long-term follow-up study

Background:

In uveal melanoma (UM), the most frequent primary intraocular tumour in adults, loss of one entire chromosome 3 (monosomy 3 (M3)) is observed in ∼50% of tumours and is significantly associated with metastatic disease. The strong association of metastatic disease with M3 offers the opportunity for molecular prognostic testing of UM patients.

Methods:

To re-evaluate M3 as prognostic marker in our clinical and laboratory setting and to determine the metastatic potential of rare tumours with partial M3, we performed a comprehensive study on 374 UM patients treated by enucleation in our clinic within 10 consecutive years, starting in 1998. Genotyping of all tumours was performed by microsatellite analysis.

Results:

Median follow-up time was 5.2 years. The disease-specific mortality rates (death by UM metastases) for tumours with disomy 3 (D3) and M3 were 13.2% and 75.1%, respectively. The disease-specific survival was worse when M3 was observed together with chromosome 8 alterations (P=0.020). Death of UM metastases was also observed in 12 patients (9%) with D3 tumours. The metastasising D3 tumours showed a larger basal tumour diameter (P=0.007), and were more frequently of mixed or epitheloid cell type (P<0.0001) than D3 tumours that did not metastasise. Mortality rate of tumours showing partial M3 (8.3%) was as low as that for tumours with D3.

Conclusion:

This shows that large tumours with disomy 3 have an increased risk to develop metastases. On the basis of these results, our clinic offers routine prognostic testing of UM patients by chromosome 3 typing.     

Expression and prognostic significance of iNOS in uveal melanoma

Uveal melanoma (UM) is the most common primary intraocular tumor in adults. Disease metastasis occurs in half of the patients and is uniformly fatal despite systemic therapy. Inducible nitric oxide synthase (iNOS) is associated with disease progression in various malignancies including cutaneous melanoma. In this retrospective cohort, we examined the prognostic value of iNOS in UM by performing immunohistochemistry on paraffin‐embedded sections of primary tumors (90 patients) and matched primary and metastatic hepatic tumors (19 patients) with complete histopathological and clinical data. We show that iNOS is expressed in UM (57% of the patients) and high iNOS levels significantly (p = 0.04; hazard ratio (HR) = 2.3) predict disease‐specific survival (DSS) as assessed by Kaplan‐Meier analysis and univariate Cox's proportional hazards regression model. Furthermore, high iNOS expression in the UM primary tissue was significantly associated with metastatic disease and vice versa. Expression of iNOS in hepatic metastases significantly (p = 0.02) predicted a shortened survival as assessed by Kaplan‐Meier analysis. However, iNOS did not appear to be a significant (p = 0.16; HR = 1.9) factor in the multivariate Cox's regression analysis performed together with the clinical parameters tumor diameter, tumor cell type, and tumor location in which only tumor diameter predicted DSS. In conclusion, iNOS predicts DSS in UM and may play a role in disease progression but it is not an independent prognostic factor.     

A randomized phase II trial of gemcitabine plus treosulfan versus treosulfan alone in patients with metastatic uveal melanoma.

BACKGROUND: Several trials demonstrated efficacy of the gemcitabine/treosulfan (GeT) combination in metastatic uveal melamoma. This randomized phase II trial compared the GeT combination versus treosulfan alone (T) in this rare disease. PATIENTS AND METHODS: Chemotherapy-naive patients with proven metastatic uveal melanoma were randomly assigned to receive 1000 mg/m(2) of gemcitabine plus 3500 mg/m(2) of treosulfan (GeT) or 3500 mg/m(2) of T. Chemotherapy was administered on days 1 and 8 in both arms, cycles were repeated on day 29. Primary end point was rate of responses and disease stabilizations. RESULTS: Forty-eight patients were randomized. Seven confirmed stable diseases (SDs) and one partial remission (PR) were observed in 24 patients treated with the GeT regimen, whereas no PR and only three SDs were observed in the T arm (P = 0.08). Median progression-free survival (PFS) was 3 months (95% CI 1.1-4.9) and 2 months (95% CI 1.7-2.3) in the GeT and T arm (P = 0.008, log-rank). Six and 12 months PFS was 34.8% and 17.9% and 16.7% and 0% always favoring the GeT arm. CONCLUSIONS: This first randomized trial in metastatic uveal melanoma showed a superior PFS and a trend for a higher response/stabilization rate of the GeT combination over T.     

Relationship of uveal and cutaneous malignant melanoma in persons with multiple primary tumors

Uveal and cutaneous melanomas differ in tumor biology, immunophenotypes and the demographic correlates of their occurrence. As a means to examine the possibility of some shared etiologic factors, we wished to learn if the 2 cancers occurred in the same individual more often than would be expected by chance. Data from the Surveillance, Epidemiology and End Results (SEER) program from 1973-1998 were utilized for this purpose. The number of persons who went on to develop a second melanoma was compared to that expected based on the incidence of each type of melanoma in the general population, after adjusting for age, sex, calendar year and residence. Given an initial cutaneous melanoma, there was a 10-fold increased risk of developing a second cutaneous melanoma (95% confidence interval [CI] = 9.4-10.6). Persons with uveal melanoma went on to develop cutaneous melanoma 4.6 times (95% CI = 2.9-6.8) more often than the population at large. In contrast, persons with cutaneous melanoma were not subsequently diagnosed with uveal melanoma at an appreciably elevated rate (standardized incidence ratio [SIR] = 1.4; 95% CI = 0.5-3.0). While these data offer some support for the hypothesis that uveal and cutaneous melanomas have 1 or more etiologies in common, the lack of symmetry in the pattern of second uveal and second cutaneous melanomas remains unexplained.     

Oncogenic GNAQ mutations are not correlated with disease-free survival in uveal melanoma

Background:

Recently, oncogenic G protein alpha subunit q (GNAQ) mutations have been described in about 50% of uveal melanomas and in the blue nevi of the skin.

Methods:

GNAQ exon 5 was amplified from 75 ciliary body and choroidal melanoma DNAs and sequenced directly. GNAQ mutation status was correlated with disease-free survival (DFS), as well as other clinical and histopathological factors, and with chromosomal variations detected by FISH and CGH.

Results:

Of the 75 tumour DNA samples analysed, 40 (53.3%) harboured oncogenic mutations in GNAQ codon 209. Univariate and multivariate analysis showed that GNAQ mutation status was not significantly correlated with DFS.

Conclusion:

The GNAQ mutation status is not suitable to predict DFS. However, the high frequency of GNAQ mutations may render it a promising target for therapeutic intervention.     

Association of specific chromosome alterations with tumour phenotype in posterior uveal melanoma

Posterior uveal melanomas have recurrent alterations of chromosomes 1, 3, 6 and 8. In particular, changes of chromosomes 3 and 8 occur in association, appear to characterize those tumours with a ciliary body component, and have been shown to be of prognostic significance. The relevance of other chromosome alterations is less certain. We have performed cytogenetic analysis on 42 previously untreated primary posterior uveal melanomas. Of interest was the observation that as tumour size increased the involvement of specific chromosome changes, and the amount of chromosome abnormalities likewise increased. Loss, or partial deletions, of the short arm of chromosome 1 were found to associate with larger ciliary body melanomas; typically, loss of the short arm resulted from unbalanced translocations, the partners of which varied. Trisomy of chromosome 21 occurred more often in ciliary body melanomas, whilst rearrangements of chromosomes 6 and 11 were primarily related to choroidal melanomas. Our results imply that alterations of chromosome 1 are important in the progression of some uveal melanomas, and that other chromosome abnormalities, besides those of chromosomes 3 and 8, are associated with ocular tumours of particular locations.     

Episodic Src activation in uveal melanoma revealed by kinase activity profiling

Background:

The RAS/RAF/MEK/ERK pathway is involved in the balance between melanocyte proliferation and differentiation. The same pathway is constitutively activated in cutaneous and uveal melanoma (UM) and related to tumour growth and survival. Whereas mutant BRAF and NRAS are responsible for the activation of the RAS/RAF/MEK/ERK pathway in most cutaneous melanoma, mutations in these genes are usually absent in UM.

Methods:

We set out to explore the RAS/RAF/MEK/ERK pathway and used mitogen-activated protein kinase profiling and tyrosine kinase arrays.

Results:

We identified Src as a kinase that is associated with ERK1/2 activation in UM. However, low Src levels and reduced ERK1/2 activation in metastatic cell lines suggest that proliferation in metastases can become independent of Src and RAS/RAF/MEK/ERK signalling. Inhibition of Src led to the growth reduction of primary UM cultures and cell lines, whereas metastatic cell line growth was only slightly reduced.

Conclusion:

We identified Src as an important kinase and a potential target for treatment in primary UM. Metastasis cell lines seemed largely resistant to Src inhibition and indicate that in metastases treatment, a different approach may be required.     

SUMOylation regulates Rb hyperphosphorylation and inactivation in uveal melanoma

Small ubiquitin‐like modifier (SUMO)ylation is one of the posttranslational modifications and is implicated in many tumor types. Modulation of SUMOylation can affect tumor progression, but the underlying mechanisms remain unclear. Here, we show that, for the first time, in uveal melanoma (UM), the most common intraocular malignancy in adults, global SUMOylation is upregulated and participates in tumor growth. Inhibition of SUMOylation in UM is sufficient to reduce tumor growth both in vitro and in vivo. Furthermore, we found that retinoblastoma protein (Rb) is a target protein and a critical downstream effector of the upregulated SUMOylation activity in UM. Increased SUMOylation of the Rb protein leads to its hyperphosphorylation and inactivation in UM cells, promoting UM cell proliferation. In summary, our results provide novel insight into the mechanism underlying SUMOylation‐regulated tumor growth in UM.     

OSuvm: An interactive online consensus survival tool for uveal melanoma prognosis analysis

Uveal melanoma (UM) is a rare, aggressive, but the most frequent primary intraocular malignancy in adults, and up to 50% of patients develop a tendency of liver metastases. Great efforts have been made to develop biomarkers that facilitate diagnosis, prediction of the risk, and response to treatment of UM. However, a biologically informative and highly accurate gold standard system for prognostic evaluation of UM remains to be established. To facilitate assessment of the prognosis of UM patients, we established a user-friendly Online consensus Survival tool for uveal melanoma, named OSuvm, by which users can easily estimate the prognostic values of genes of interest by the Kaplan-Meier survival plot with hazard ratio and log-rank test. OSuvm comprises four independent cohorts including 229 patients with both gene expression profiles and relevant clinical follow-up information, and it has shown great performance in evaluating the prognostic roles of previously reported biomarkers. Using OSuvm enables researchers and clinicians to rapidly and conveniently explore the prognostic value of genes of interest and develop new potential molecular biomarkers for UM. OSuvm can be accessed at http://bioinfo.henu.edu.cn/UVM/UVMList.jsp .     

Nuclear HER3 is associated with favorable overall survival in uveal melanoma

HER3 is a member of the epidermal growth factor receptor (EGFR) family and is expressed in several types of cancer. Both the cytoplasmic and nuclear appearances of the receptor have been reported. Here, we investigate the expression and subcellular distribution of HER3 in uveal melanoma (UM) cells and tissues and its potential impact on clinical outcome of patients. Paraffin-embedded samples from 128 consecutive UM patients, enucleated without alternative treatment on UM diagnosis, were evaluated for HER3 using immunohistochemistry. Immunoreactivity was scored for frequency, intensity of positive cells, and subcellular distribution. The results were correlated with the established clinicopathological parameters using univariate and multivariate statistical analyses. HER3 expression was shown in 70% of the cases (89/128). This contrasts with the other EGFR family receptors (EGFR, HER2 and HER4) that are infrequently expressed in UM. Surprisingly, HER3 was found to be localized solely in the cell nuclei in 56 cases. The remaining 33 HER3 positive cases showed diffuse distribution (cytoplasmic ± nuclear). Nuclear HER3 was independently correlated with a more favorable overall survival (p = 0.043 and hazard ratio = 0.618) compared to cases with diffuse and/or no HER3. Nuclear localization of HER3 was also confirmed in fresh UM material and in UM cell lines. In conclusion, HER3 is frequently localized solely in the cell nuclei in UM and as such it predicts a more favorable overall survival.