Nicole C. Naus

Clinical significance of immunohistochemistry for detection of BAP1 mutations in uveal melanoma.

Uveal melanoma is a lethal cancer with a strong propensity to metastasize. Limited therapeutic options are available once the disease has disseminated. A strong predictor for metastasis is the loss of chromosome 3. Inactivating mutations in BAP1 encoding the BRCA1-associated protein 1 and located on chromosome 3p21.1, have been described in uveal melanoma and other types of cancer. In this study, we determined the prevalence of somatic BAP1 mutations and examined whether these mutations correlate with the functional expression of BAP1 in uveal melanoma tissue and with other clinical, histopathological and chromosomal parameters. We screened a cohort of 74 uveal melanomas for BAP1 mutations, using different deep sequencing methods. The frequency of BAP1 mutations in our study group was 47%. The expression of BAP1 protein was studied using immunohistochemistry. BAP1 staining was absent in 43% of the cases. BAP1 mutation status was strongly associated with BAP1 protein expression (P<0.001), loss of chromosome 3 (P<0.001), and other aggressive prognostic factors. Patients with a BAP1 mutation and absent BAP1 expression had an almost eightfold higher chance of developing metastases compared with those without these changes (P=0.002). We found a strong correlation between the immunohistochemical and sequencing data and therefore propose that, immunohistochemical screening for BAP1 should become routine in the histopathological work-up of uveal melanoma. Furthermore, our analysis indicates that loss of BAP1 may be particularly involved in the progression of uveal melanoma to an aggressive, metastatic phenotype.

Characterization of complex chromosomal abnormalities in uveal melanoma by fluorescence in situ hybridization, spectral karyotyping, and comparative genomic hybridization

Several nonrandom recurrent chromosomal changes are observed in uveal melanoma. Some of these abnormalities, e.g., loss of chromosome 3, gain of the q arm of chromosome 8, and chromosome 6 abnormalities, are of prognostic value. Cytogenetic analysis and/or fluorescence in situ hybridization (FISH) are used to detect these changes. In some cases, however, detailed cytogenetic analysis is not possible due to the presence of complex abnormalities. To define more accurately these cytogenetic changes, we have applied comparative genomic hybridization (CGH) and/or spectral karyotyping (SKY) to two uveal melanoma cell lines and five primary uveal melanomas, with partially defined and/or complex abnormalities. SKY provided additional information on 34/39 partially defined aberrant chromosomes and revealed a new abnormality, a der(17)t(7;17)(?;q?), that had not been recognized by conventional cytogenetics. Additionally, using SKY, abnormalities involving chromosome 6 or 8 were found to be twice as common as observed with cytogenetic analysis. CGH was especially useful in assigning the abnormalities identified by SKY to specific chromosomal regions and, in addition, resulted in the detection of a small deletion of chromosome region 3q13∼21. We conclude that SKY and CGH, as methods complementary to cytogenetic and FISH analysis, provide more complete information on the chromosomal abnormalities occurring in uveal melanoma.

Higher percentage of FISH-determined monosomy 3 and 8q amplification in uveal melanoma cells relate to poor patient prognosis

PURPOSE To investigate the relation between patient survival and incrementally increasing percentages of fluorescence in situ hybridization-determined complete loss of chromosome 3 (monosomy 3) and gain of chromosome 8q in primary uveal melanoma cells. METHODS Clinicopathological factors were related to disease-free survival. Fluorescence in situ hybridization was performed using probes on chromosomes 1, 3, 6, and 8. The percentages of UM cells with monosomy 3 or chromosome 8q gain were classified in groups with incrementally increasing percentages and related to disease-free survival. Correlations between clinical factors and cytogenetic aberrations were also analyzed. RESULTS Two-hundred twenty choroidal and ciliary body melanomas were analyzed. The following proved to be significant predictors of survival in univariate analysis: older patient age (P = 0.003); large tumor diameter (P < 0.001); mixed cell type (P = 0.001); presence of closed microvascular loops (P < 0.001); loss of chromosome 1p (P = 0.006); monosomy 3 (P < 0.001); gain of 6p (P < 0.001); and gain of chromosome 8q (P < 0.001). Multivariate Cox analysis displayed monosomy 3 (Hazard ratio [HR] 2.83, P = 0.002) and gain of chromosome 8q (HR 3.13, P = 0.002) as the most important independent prognostic factors of poor survival, followed by older patient age (HR 1.02, P = 0.017). Increasing percentages of monosomy 3 and gain of chromosome 8q in tumor cells showed a correlation with worse prognosis (Log-rank test 49.9 and 40.4, both P < 0.001) and increased number of additional copies of 8q correlated with shorter disease-free interval (Log-rank test 45.7, P < 0.001). CONCLUSIONS A high percentage monosomy 3 and chromosome 8q gain in primary UM cells showed a strong relation with poor disease-free survival compared with low percentage aberrations.

Gene expression profiling in uveal melanoma: two regions on 3p related to prognosis.

PURPOSE Although studies on uveal melanoma (UM) revealed prognostic significance of chromosomal aberrations, they resulted in classification errors in survival prediction. A robust prognostic classifier with strong predictive value and further insight in genes responsible for poor prognosis were obtained by performing a gene-expression profile in tumors of UM patients for which extensive clinical, histopathologic, cytogenetic, and follow-up data were available. Furthermore, the UM microarray expression data were compared with cytogenetic data. METHODS Gene-expression profiles of 46 UMs were obtained with microchip assays. Data were analyzed with cluster-analysis and predictive analysis of microarrays (PAM) software and validated with real-time PCR. The prognostic significance of UMs with specific molecular signatures was determined. Furthermore, LAP analysis resulted in the identification of differentially expressed chromosomal regions. RESULTS The primary UMs were classified in two distinct molecular classes with a strong prognostic value (P < 0.001; hazard ratio 7.7). Classifier gene sets for microarray class and disease-free survival were validated with real-time PCR, and the predictive value of the UM class marker set was validated with gene-expression profiles of tumors provided by other institutions, showing a sensitivity of 0.93 and specificity of 1.00 for class II tumors. A locally adaptive statistical procedure identified two regions on the short arm of chromosome 3 with decreased gene-expression in tumors with shorter disease-free survival. CONCLUSIONS Microarray classification outperforms known prognostic indicators for UM, such as clinical, histopathologic, and cytogenetic parameters. In addition, the identified regions with lower expressed genes on 3p could harbor genes that are responsible for the poor prognosis of patients with UM.

Chromosome 3 intratumor heterogeneity in uveal melanoma

PURPOSE To investigate the presence of focal or diffuse heterogeneity of monosomy 3 in uveal melanoma, by using fluorescence in situ hybridization (FISH). METHODS Direct interphase FISH in a series of 151 uveal melanomas revealed 82 tumors with loss of chromosome 3. Tumors with monosomy 3 were suspected to be heterogeneous if there were low percentages of monosomy 3, triploid clones, inconsistencies between FISH on centromere 3 and the long arm of chromosome 3, or discrepancies between fine-needle-aspiration biopsies (FNABs) and the main tumor. These tumors (n=16), all choroidal melanomas, were selected and analyzed for intratumor heterogeneity by using FISH on paraffin-embedded tissue sections. RESULTS Different sections of each tumor were evaluated with FISH: 6 tumors showed monosomy 3 in the same percentage throughout the tumor, and 10 showed multiple clones with different percentages of monosomy 3. However, these tumors did not show focal heterogeneity with respect to chromosome 3 status, and differences in monosomy 3 distribution between the base and apex of the tumor could not be identified. CONCLUSIONS Although a small number of uveal melanomas show heterogeneity for chromosome 3, it does not affect survival. In the presence of triploid clones, the loss of chromosome 3 is more difficult to interpret. In general, tumor biopsies in uveal melanoma provide an accurate prediction of the patients prognosis.

Multiplex ligation-dependent probe amplification equals fluorescence in-situ hybridization for the identification of patients at risk for metastatic disease in uveal melanoma.

In uveal melanoma, loss of chromosome 3 and gain of chromosome 8q are associated with a high risk of metastasis. In this study, we validated the use of multiplex ligation-dependent probe amplification (MLPA) in detecting patients at risk for metastatic disease in comparison with the predictive power of fluorescence in-situ hybridization (FISH). For 64 uveal melanoma samples, the MLPA results of chromosome 3 and 8 were compared with the results obtained by FISH. For seven samples, a single nucleotide polymorphism array was performed to clarify discrepancies. Clinical information together with the histopathology and chromosomal aberrations of chromosomes 1, 3, 6, and 8 were evaluated for correlation with the patients’ prognosis. Loss of chromosome 3, loss or gain of 8p, and gain of 8q, found with MLPA, correlated with a significantly lower disease-free survival (P<0.001). On the basis of the clinical outcome, 12 patients would have been classified incorrectly using MLPA results of chromosomes 3 and 8. FISH results led to the same incorrect classification. Four patients with abnormalities of chromosomes 3 and 8 in the tumor, detected with MLPA, are still alive without metastasis. Eight patients without concurrent aberrations of chromosomes 3 and 8 in the tumors died due to metastasis. The sensitivity of MLPA to detect patients at risk for metastatic disease is higher than with the results obtained with FISH (0.795 vs. 0.692). The specificity is equal for both techniques (0.840). MLPA is able to detect patients at risk for metastasis using the results for chromosomes 3 and 8. There is no significant difference in the predictive power of MLPA compared with FISH.

Prevalence and implications of TERT promoter mutation in uveal and conjunctival melanoma and in benign and premalignant conjunctival melanocytic lesions.

PURPOSE Hot-spot mutations in the promoter region of telomerase reverse transcriptase (TERT promoter mutations) occur frequently in cutaneous and conjunctival melanoma and are exceedingly rare in uveal melanoma. No information is available on the presence of these mutations in the conjunctival melanocytic precursor lesion primary acquired melanosis (PAM). We tested a cohort of uveal and conjunctival melanomas as well as conjunctival benign and premalignant melanocytic lesions for TERT promoter mutations in order to elucidate the role of these mutations in tumor progression. METHODS TERT promoter mutation analysis on fresh tumor DNA and DNA from formalin-fixed, paraffin-embedded specimens was performed by SNaPshot analysis in 102 uveal melanomas, 39 conjunctival melanomas, 26 PAM with atypia, 14 PAM without atypia, and 56 conjunctival nevi. RESULTS Mutations of the TERT promoter were not identified in conjunctival nevi or PAM without atypia, but were detected in 2/25 (8%) of PAM with atypia and 16/39 (41%) of conjunctival melanomas. A single TERT promoter mutation was detected in 102 uveal melanomas (1%). CONCLUSIONS We present the second documented case of TERT promoter mutation in uveal melanoma. In comparison with other types of melanoma, TERT promoter mutations occur at extremely low frequency in uveal melanoma. TERT promoter mutations are frequent in conjunctival melanoma and occur at lower frequency in PAM with atypia but were not detected in benign conjunctival melanocytic lesions. These findings favor a pathogenetic tumor progression role for TERT promoter mutations in conjunctival melanocytic lesions.

Lacrimal Gland Radiosensitivity in Uveal Melanoma Patients

PURPOSE To find a dose-volume effect for inhomogeneous irradiated lacrimal glands. METHODS AND MATERIALS Between 1999 and 2006, 72 patients (42 men and 30 women) were treated with fractionated stereotactic radiotherapy in a prospective, nonrandomized clinical trial (median follow-up, 32 months). A total dose of 50 Gy was given on 5 consecutive days. The mean of all Schirmer test results obtained > or =6 months after treatment was correlated with the radiation dose delivered to the lacrimal gland. Also, the appearance of dry eye syndrome (DES) was related to the lacrimal gland dose distribution. RESULTS Of the 72 patients, 17 developed a late Schirmer value <10 mm; 9 patients developed DES. A statistically significant relationship was found between the received median dose in the lacrimal gland vs. reduced tear production (p = 0.000) and vs. the appearance of DES (p = 0.003), respectively. A median dose of 7 Gy/fraction to the lacrimal gland caused a 50% risk of low Schirmer results. A median dose of 10 Gy resulted in a 50% probability of DES. CONCLUSION We found a clear dose-volume relationship for irradiated lacrimal glands with regard to reduced tear production and the appearance of DES.

Fractionated stereotactic radiotherapy for uveal melanoma, late clinical results

PURPOSE To determine local control, late toxicity and metastatic free survival (MFS) of patients treated with fractionated stereotactic radiation therapy (fSRT) for uveal melanoma (UM). METHODS AND MATERIALS Between 1999 and 2007, 102 UM patients were included in a prospective study of a single institution (median follow-up (FU) 32 months; median tumor thickness 6 mm); five fractions of 10 Gy were given. Primary endpoints were local tumor control and late toxicity (including visual outcome and eye preservation). Secondary endpoint was MFS. RESULTS Local tumor control was achieved in 96% of the patients. Fifteen enucleations were performed, 2-85 months after radiation. Four eyes were enucleated because of local tumor progression. Nine patients developed grade 3 or 4 neovascular glaucoma (NVG), 19 developed severe retinopathy, 13 developed opticoneuropathy grade 3 or 4, 10 developed cataract grade 3, and 10 patients suffered from keratitis sicca. Best corrected visual acuity (BCVA) decreased from a mean of 0.26 at diagnosis to 0.16, 3 months after radiation and it gradually declined to 0.03, 4 years after therapy. The 5-year actuarial MFS was 75% (95% CIs: 62-84%). CONCLUSIONS fSRT is an effective treatment modality for uveal melanoma with a good local control. With that, fSRT is a serious eye sparing treatment modality. However, our FU is relatively short. Also, the number of secondary enucleations is substantial, mainly caused by NVG.

Increased expression of p73Δex2 transcript in uveal melanoma with loss of chromosome 1p

The loss of chromosome 1p and chromosome 3 is associated with metastatic disease and decreased survival of uveal melanoma (UM) patients. The p53 homologues, p73 and p63, are located on chromosomes 1p and 3q, respectively. Both are able to activate p53 target genes, resulting in growth arrest, apoptosis and differentiation. N-terminally truncated isoforms of these genes may act as dominant negative inhibitors of wild-type p53 and transactivating activity. Although, p53 is frequently involved in several malignancies it does not play a major role in UM. Altered expression has been reported for both p63 and p73 in various malignancies. In this study, fluorescent in-situ hybridization was performed to identify gains or losses of p63 and p73 loci in UM. The expression of the different p63 and p73 isoforms was evaluated by reverse transcriptase PCR followed by Southern blot analysis. Furthermore, the expression pattern of the various ΔTAp73 transcripts was analysed in seven primary UMs and 11 UM-derived cell lines using isoform-specific real-time PCR. Our results indicated that the isoform p73Δex2/3 was abundantly expressed and a relative loss of the p73 locus was associated with the upregulation of p73Δex2 and TAp73 transcripts. N-terminal transactivation forms of both p73 and p63 were observed in primary and metastasis-derived cell lines, as well as in primary melanomas, but in only one of the cell lines a ΔNp63 mRNA transcript was observed. Our data suggest a potential function of p73 deletion transcripts in UM progression.