Sarah L. Lake

Genetic Heterogeneity in Uveal Melanoma Assessed by Multiplex Ligation-Dependent Probe Amplification

PURPOSE To determine intratumor genetic heterogeneity in uveal melanoma (UM) by multiplex ligation-dependent probe amplification (MLPA) in formalin-fixed, paraffin-embedded (FFPE) tumor tissues. METHODS DNA was extracted from whole tumor sections and from two to nine different areas microdissected from 32 FFPE UMs. Thirty-one loci on chromosomes 1, 3, 6, and 8 were tested with MLPA for copy number changes. The tumor was considered heterogeneous at a locus if (1) the difference in dosage quotients (DQs) of any two areas was 0.2 or more, and (2) the DQs of the areas belonged to different ranges. RESULTS Comparison of MLPA data obtained from microdissected areas of the UMs showed heterogeneity in 1 to 26 examined loci in 24 (75%) tumors, with only 25% of the tumors being homogeneous. Intratumor heterogeneity of 3p12.2, 6p21.2, and 8q11.23 was most common, occurring in >30% of the UMs. Gains of chromosome 3 were observed in four UMs, with three of these tumors showing the highest degree of heterogeneity. Copy number variation was associated with differences in tumor cell type, but not with differences in tumor pigmentation or reactive inflammation. UMs with genetic heterogeneity across multiple sample sites showed equivocal MLPA results when the whole tumor section was examined. These results suggest that different clones dilute MLPA results. CONCLUSIONS Heterogeneity of chromosomal abnormalities of chromosomes 1, 3, 6, and 8 is present in most UMs. This heterogeneity causes equivocal MLPA results. One random tumor sample may not be representative of the whole tumor and, therefore, may be insufficient for prognostic testing.

Multiplex ligation-dependent probe amplification of conjunctival melanoma reveals common BRAF V600E gene mutation and gene copy number changes.

PURPOSE To determine the occurrence of BRAF V600E gene mutations and copy number changes of all autosome arms and genes known to be frequently altered in tumorigenesis in primary and metastatic conjunctival melanomas (CoMs). METHODS DNA (200 ng) was analyzed by three multiplex ligation-dependent probe amplification assays (P027 uveal melanoma, P036 human telomere, and P206 spitzoid melanoma). RESULTS Eight of 16 primary tumor samples and 4 of 6 metastatic samples showed BRAF V600E gene mutations. CDKN1A and RUNX2 (both 6p21.2) were amplified in 11 and 16 of 21 primary CoMs, respectively. In metastatic CoMs, MLH1 (3p22.1) and TIMP2 (17q25.3) were frequently amplified, and MGMT (20q26.3) and ECHS1 (10q26.3) were frequently deleted. The BDH (3q), FLJ20265 (4p), OPRL1 (20q), and PAO (10q) genes, representing the telomeres of their respective chromosome arms in the P036 assay, were frequently amplified in metastatic CoMs. No statistically significant associations were identified between BRAF mutation or CDKN1A or RUNX2 amplification and sex, age, histologic cell type, or patient survival. CONCLUSIONS No copy number changes were associated exclusively with metastatic CoMs. However, further investigation of the role of CDKN1A and RUNX2 in CoMs development and that of MLH1, TIMP2, MGMT, and ECHS1 in metastatic CoMs is warranted. Validation of the observed gene and chromosome arm copy number changes in a larger cohort of primary and metastatic CoMs is necessary to identify the patients at highest risk for CoMs metastasis.

Single nucleotide polymorphism array analysis of uveal melanomas reveals that amplification of CNKSR3 is correlated with improved patient survival.

Metastatic death from uveal melanoma occurs almost exclusively with tumors showing monosomy of chromosome 3. However, approximately 5% of patients with a disomy 3 uveal melanoma develop metastases, and a further 5% of monosomy 3 uveal melanoma patients exhibit disease-free survival for >5 years. In the present study, whole-genome microarrays were used to interrogate four clinically well-defined subgroups of uveal melanoma: i) disomy 3 uveal melanoma with long-term survival; ii) metastasizing monosomy 3 uveal melanoma; iii) metastasizing disomy 3 uveal melanoma; and iv) monosomy 3 uveal melanoma with long-term survival. Cox regression and Kaplan-Meier survival analysis identified that amplification of the CNKSR3 gene (log-rank, P = 0.022) with an associated increase in its protein expression (log-rank, P = 0.011) correlated with longer patient survival. Although little is known about CNKSR3, the correlation of protein expression with increased survival suggests a biological function in uveal melanoma, possibly working to limit metastatic progression of monosomy 3 uveal melanoma cells.

Combined p53 and MDM2 biomarker analysis shows a unique pattern of expression associated with poor prognosis in patients with renal cell carcinoma undergoing radical nephrectomy.

Whats known on the subject? and What does the study add?

Proteomics of Uveal Melanomas Suggests HSP-27 as a Possible Surrogate Marker of Chromosome 3 Loss

PURPOSE To compare the proteomic profiles of primary uveal melanomas, with and without loss of chromosome 3. METHODS Frozen specimens from three uveal melanomas with disomy 3 and from four tumors with monosomy 3, according to fluorescence in situ hybridization (FISH) analysis, were subjected to high-resolution, two-dimensional (2-D) gel electrophoresis. The protein expression profiles of the two uveal melanoma cytogenetic groups were compared: Proteins that differed significantly were excised and analyzed by tandem mass spectrometry. Differentially expressed proteins were further analyzed with Western blot analysis. An independent cohort of 41 formalin-fixed, paraffin-embedded (FFPE) uveal melanomas, whose chromosome 3 status had been determined by multiplex ligation-dependent probe amplification (MLPA), was examined for the appropriate antigens by immunohistochemistry. RESULTS Four protein spots were 1.5-fold (Students t-test, P < 0.05) differentially expressed in the two uveal melanoma types: two spots were overexpressed in the disomy 3 group compared with the monosomy 3 group, whereas two spots were underexpressed. Identification of the four spots yielded nine proteins. Western blot analysis confirmed the results for heat shock protein (HSP)-27, vimentin, and pyruvate dehydrogenase beta (PDHB), with a statistical significance for the first two proteins. HSP-27 was significantly downregulated, whereas vimentin was upregulated in the monosomy 3 tumors (Students t-test, P = 0.003 and P = 0.005, respectively). Immunohistochemistry confirmed low-to-negative HSP-27 protein expression in monosomy 3 uveal melanomas (Students t-test; P = 0.011). CONCLUSIONS Low-to-negative HSP-27 protein expression in uveal melanoma correlates strongly with monosomy 3. Further validation is necessary to determine whether immunohistochemical assessment of HSP-27 expression correlates with metastatic mortality.

Determination of genomic DNA sequences for beta-tubulin isotype 1 from multiple species of cyathostomin and detection of resistance alleles in third-stage larvae from horses with naturally acquired infections

BackgroundGenetic resistance against benzimidazole (BZ) anthelmintics is widespread in cyathostomins, the commonest group of intestinal parasitic nematodes of horses. Studies of BZ-resistant nematodes of sheep, particularly Haemonchus contortus, have indicated that an anthelmintic resistance-conferring T/A polymorphism, encoding an F (phenylalanine) to Y (tyrosine) substitution, in beta-tubulin isotype 1 is present at two loci, codons 167 and 200 (F167Y, F200Y). Recent studies using complementary (c) DNA derived from BZ-susceptible and -resistant cyathostomins identified statistical differences in the frequency of the BZ-resistant A allele at these loci. However, the lack of high-throughput genomic DNA-based detection of polymorphisms limits the study of eggs or larvae from field isolates. In the present study, we report genomic DNA sequences for beta-tubulin isotype 1 from multiple cyathostomin species, thus facilitating the development of pyrosequencing assays to genetically characterize third-stage larvae (L3s) of cyathostomins from mixed-species field isolates.ResultsSequence analysis of the beta-tubulin isotype 1 gene in a common species, Cylicocyclus nassatus, indicates a revised genomic structure to published data, revealing that codons 167 and 200 are located on separate exons. A consensus sequence was generated from 91 and 76 individual cyathostomins for the regions spanning codons 167 and 200, respectively. A multi-species genomic DNA-based assay was established to directly pyrosequence individual L3 from field samples of unknown species and BZ sensitivity in a 96-well plate. In this format, the assay to detect F167Y gave a 50-90% success rate. The optimisation of the assay at codon 200 is currently underway. Subsequently, the genotype at F167Y was determined for 241 L3s, collected prior to and after BZ treatment. These results demonstrated a reduction in the heterozygous genotype, TTC/TAC, and an increase in the homozygous resistant genotype TAC/TAC in post-treatment samples. However, the differences in allele frequencies determined before and after BZ treatment were not statistically significant.ConclusionExtensive genomic DNA sequence, spanning codons 167 and 200 of the beta-tubulin isotype 1 gene, was generated from multiple cyathostomin species. The data facilitated the development of a pyrosequencing assay, capable of detecting the genotype of individual cyathostomin L3s derived from mixed-species field samples. Differences in codon 167 allele frequencies were observed in L3s isolated pre- and post-BZ treatment.

Mutations in G Protein Encoding Genes and Chromosomal Alterations in Primary Leptomeningeal Melanocytic Neoplasms

Limited data is available on the genetic features of primary leptomeningeal melanocytic neoplasms (LMNs). Similarities with uveal melanoma were recently suggested as both entities harbor oncogenic mutations in GNAQ and GNA11. Whether primary LMNs share additional genetic alterations with uveal melanoma including copy number variations is unknown. Twenty primary LMNs ranging from benign and intermediate-grade melanocytomas to melanomas were tested by direct sequencing for hotspot mutations in the genes GNA11, GNAQ, BRAF, NRAS and HRAS. Furthermore, the lesions were tested for copy number variations of chromosomes frequently present in uveal melanoma (1p, 3, 6 and 8q) by multiplex ligation-dependent probe amplification (MLPA). Genome-wide analyses of copy number alterations of two leptomeningeal melanocytic neoplasms were performed using the OncoScan SNP-array. GNAQQ209 mutations were present in eleven LMNs, while two of 20 cases carried a GNA11Q209 mutation. No BRAF, HRAS or NRAS hotspot mutations were detected. Monosomy 3 and gain of 8q were present in one leptomeningeal melanoma, and one intermediate-grade melanocytoma harbored a gain of chromosome 6. With MLPA, the melanocytomas did not show any further gross chromosomal variations. Our data shows that primary LMNs, like uveal melanoma, harbor oncogenic mutations in GNAQ and GNA11 but lack mutations in BRAF, NRAS and HRAS. This finding may help in the differential diagnosis between a primary LMN and a metastasis from a cutaneous melanoma to the central nervous system. Copy number variations in some aggressive LMNs resemble those present in uveal melanoma but their prognostic significance is unclear.

GDF-15: a novel serum marker for metastases in uveal melanoma patients

BackgroundAbout 50% of patients with uveal melanoma (UM) develop metastases during the course of their disease. We analyzed serum levels of Growth Differentiation Factor-15 (GDF-15), with the aim of identifying patients with early metastases.MethodsGDF-15 concentration was measured using an enzyme-linked immunosorbent assay (ELISA) in serum samples from 188 UM patients (170 patients without metastases; 18 patients with clinically detectable metastases) and 18 healthy control individuals. Data were analyzed with respect to differences between patients with and without clinically detectable UM metastases. GDF-15 serum levels were further analyzed with regard to significant patient and tumor characteristics as revealed by histology and multiplex ligation-dependent probe amplification (MLPA) to determine chromosome 3 copy number. GDF-15 expression in UM was investigated by immunohistochemistry.ResultsPatients with clinically detectable metastases had significantly higher GDF-15 serum levels compared to those without clinically detectable metastases as well as to healthy individuals (ANOVA; p < 0.001). GDF-15 concentrations in UM patients with overt clinically detectable metastases were significantly higher than those in UM patients with a second malignancy in remission but without clinically detected UM metastases (ANOVA; p < 0.001). No association between serum concentration of GDF-15 and clinical, pathological, and genetic features was observed. GDF-15 protein was only expressed in a minority of UM cells in most tumors.ConclusionsOur data suggest that GDF-15 can be used as a serum marker for the diagnosis of metastases in UM patients. Further data collection and analysis are necessary to evaluate a possible prognostic role of GDF-15 in predicting early metastases.

Multiplex ligation-dependent probe amplification analysis of uveal melanoma with extraocular extension demonstrates heterogeneity of gross chromosomal abnormalities.

PURPOSE To determine whether biopsy of extraocular extension of uveal melanoma (UM) is representative of the intraocular tumor with respect to copy number of chromosomes 1p, 3, 6, and 8. METHODS Multiplex ligation-dependent probe amplification (MLPA) using the P027 assay was performed on formalin-fixed, paraffin-embedded sections from 10 UMs. The intraocular and extraocular parts of the tumor were microdissected and analyzed separately. RESULTS Of the 10 UMs analyzed, seven showed heterogeneity for at least one chromosome arm; the most frequently heterogeneous chromosome arm was 6p. No heterogeneity of 8p was observed between the intraocular and extraocular areas of the tumor. One tumor showed monosomy 3 in the intraocular area of the tumor but loss of the 3q arm only for the extraocular area. CONCLUSIONS Biopsy of an extraocular tumor extension may not be representative of the underlying UM with respect to chromosome 1p, 3, 6, and 8q abnormalities detectable by MLPA. These results suggest that for UM with extraocular extension, both the intraocular and the extraocular parts of the tumor should be sampled for accurate genetic prognostic testing.

Insights into genetic alterations of liver metastases from uveal melanoma

The liver is the organ usually affected by metastatic uveal melanoma (MUM). Current treatments are almost always ineffective and mortality remains high. In this study, copy number variations (CNVs) were identified in 12 metastatic and five matched primary UMs (PUMs). Our data revealed a wide spectrum of genetic alterations in MUM. Most common were amplifications of chromosome (chr.) 8q; alterations on chr. 3 included monosomy, isodisomy, and large regions of homozygosity (ROH). Genomic profiles of PUM‐MUM pairs varied in their degree of similarity and complexity. However, within the pairs, 135 genes were consistently altered. Protein expression of C‐MYC and BAP1 was examined by immunohistochemistry (IHC); a positive association between IHC and CNVs was seen for C‐MYC. This comprehensive catalogue of CNVs associated with MUM should facilitate the identification of key alterations that drive tumor growth. This would have the potential to select urgently needed novel, targeted, therapeutic regimens.