Targeting tumour-associated macrophages with CCR2 inhibition in combination with FOLFIRINOX in patients with borderline resectable and locally advanced pancreatic cancer: a single-centre, open-label, dose-finding, non-randomised, phase 1b trial.

BACKGROUND: In pancreatic ductal adenocarcinoma, the CCL2-CCR2 chemokine axis is used to recruit tumour-associated macrophages for construction of an immunosuppressive tumour microenvironment. This pathway has prognostic implications in pancreatic cancer, and blockade of CCR2 restores anti-tumour immunity in preclinical models. We aimed to establish the safety, tolerability, and recommended phase 2 oral dose of the CCR2 inhibitor PF-04136309 in combination with FOLFIRINOX chemotherapy (oxaliplatin and irinotecan plus leucovorin and fluorouracil). METHODS: We did this open-label, dose-finding, non-randomised, phase 1b study at one centre in the USA. We enrolled treatment-naive patients aged 18 years or older with borderline resectable or locally advanced biopsy-proven pancreatic ductal adenocarcinoma, an Eastern Cooperative Oncology Group performance status of 1 or less, measurable disease as defined by Response Evaluation Criteria in Solid Tumors version 1.1, and normal end-organ function. Patients were allocated to receive either FOLFIRINOX alone (oxaliplatin 85 mg/m(2), irinotecan 180 mg/m(2), leucovorin 400 mg/m(2), and bolus fluorouracil 400 mg/m(2), followed by 2400 mg/m(2) 46-h continuous infusion), administered every 2 weeks for a total of six treatment cycles, or in combination with oral PF-04136309, administered at a starting dose of 500 mg twice daily in a standard 3 + 3 dose de-escalation design. Both FOLFIRINOX and PF-04136309 were simultaneously initiated with a total treatment duration of 12 weeks. The primary endpoints were the safety, tolerability, and recommended phase 2 dose of PF-04136309 plus FOLFIRINOX, with an expansion phase planned at the recommended dose. We analysed the primary outcome by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01413022. RESULTS: Between April 19, 2012, and Nov 12, 2014, we treated 47 patients with FOLFIRINOX alone (n=8) or with FOLFIRINOX plus PF-04136309 (n=39). One patient had a dose-limiting toxic effect in the dose de-escalation group receiving FOLFIRINOX plus PF-04136309 at 500 mg twice daily (n=6); this dose was established as the recommended phase 2 dose. We pooled patients in the expansion-phase group (n=33) with those in the dose de-escalation group that received PF-04136309 at the recommended phase 2 dose for assessment of treatment-related toxicity. Six (75%) of the eight patients receiving FOLFIRINOX alone were assessed for treatment toxicity, after exclusion of two (25%) patients due to insurance coverage issues. The median duration of follow-up for treatment toxicity was 72·0 days (IQR 49·5-89·0) in the FOLFIRINOX alone group and 77·0 days (70·0-90·5) in the FOLFIRINOX plus PF-04136309 group. No treatment-related deaths occurred. Two (5%) patients in the FOLFIRINOX plus PF-04136309 group stopped treatment earlier than planned due to treatment-related toxic effects. Grade 3 or higher adverse events reported in at least 10% of the patients receiving PF-04136309 included neutropenia (n=27), febrile neutropenia (n=7), lymphopenia (n=4), diarrhoea (n=6), and hypokalaemia (n=7). Grade 3 or higher adverse events reported in at least 10% of patients receiving FOLFIRINOX alone were neutropenia (n=6), febrile neutropenia (n=1), anaemia (n=2), lymphopenia (n=1), diarrhoea (n=2), hypoalbuminaemia (n=1), and hypokalaemia (n=3). Therapy was terminated because of treatment-related toxicity in one (17%) of the six patients receiving FOLFIRINOX alone. 16 (49%) of 33 patients receiving FOLFIRINOX plus PF-04136309 who had undergone repeat imaging achieved an objective tumour response, with local tumour control achieved in 32 (97%) patients. In the FOLFIRINOX alone group, none of the five patients with repeat imaging achieved an objective response, although four (80%) of those patients achieved stable disease. INTERPRETATION: CCR2-targeted therapy with PF-04136309 in combination with FOLFIRINOX is safe and tolerable. FUNDING: Washington University-Pfizer Biomedical Collaborative.

Tumor-induced STAT3 activation in monocytic myeloid-derived suppressor cells enhances stemness and mesenchymal properties in human pancreatic cancer.

Pancreatic cancer (PC) mobilizes myeloid cells from the bone marrow to the tumor where they promote tumor growth and proliferation. Cancer stem cells (CSCs) are a population of tumor cells that are responsible for tumor initiation. Aldehyde dehydrogenase-1 activity in PC identifies CSCs, and its activity has been correlated with poor overall prognosis in human PC. Myeloid cells have been shown to impact tumor stemness, but the impact of immunosuppressive tumor-infiltrating granulocytic and monocytic myeloid-derived suppressor cells (Mo-MDSC) on ALDH1(Bright) CSCs and epithelial to mesenchymal transition is not well understood. In this study, we demonstrate that Mo-MDSC (CD11b(+)/Gr1(+)/Ly6G(-)/Ly6C(hi)) significantly increase the frequency of ALDH1(Bright) CSCs in a mouse model of PC. Additionally, there was significant upregulation of genes associated with epithelial to mesenchymal transition. We also found that human PC converts CD14(+) peripheral blood monocytes into Mo-MDSC (CD14(+)/HLA-DR(low/-)) in vitro, and this transformation is dependent on the activation of the STAT3 pathway. In turn, these Mo-MDSC increase the frequency of ALDH1(Bright) CSCs and promote mesenchymal features of tumor cells. Finally, blockade of STAT3 activation reversed the increase in ALDH1(Bright) CSCs. These data suggest that the PC tumor microenvironment transforms monocytes to Mo-MDSC by STAT3 activation, and these cells increase the frequency of ALDH1(Bright) CSCs. Therefore, targeting STAT3 activation may be an effective therapeutic strategy in targeting CSCs in PC.

BAP1 deficiency causes loss of melanocytic cell identity in uveal melanoma.

BACKGROUND: Uveal melanoma is a highly aggressive cancer with a strong propensity for metastasis, yet little is known about the biological mechanisms underlying this metastatic potential. We recently showed that most metastasizing uveal melanomas, which exhibit a class 2 gene expression profile, contain inactivating mutations in the tumor suppressor BAP1. The aim of this study was to investigate the role of BAP1 in uveal melanoma progression. METHODS: Uveal melanoma cells were studied following RNAi-mediated depletion of BAP1 using proliferation, BrdU incorporation, flow cytometry, migration, invasion, differentiation and clonogenic assays, as well as in vivo tumorigenicity experiments in NOD-SCID-Gamma mice. RESULTS: Depletion of BAP1 in uveal melanoma cells resulted in a loss of differentiation and gain of stem-like properties, including expression of stem cell markers, increased capacity for self-replication, and enhanced ability to grow in stem cell conditions. BAP1 depletion did not result in increased proliferation, migration, invasion or tumorigenicity. CONCLUSIONS: BAP1 appears to function in the uveal melanocyte lineage primarily as a regulator of differentiation, with cells deficient for BAP1 exhibiting stem-like qualities. It will be important to elucidate how this effect of BAP1 loss promotes metastasis and how to reverse this effect therapeutically.

Inflammatory monocyte mobilization decreases patient survival in pancreatic cancer: a role for targeting the CCL2/CCR2 axis.

PURPOSE: To determine the role of the CCL2/CCR2 axis and inflammatory monocytes (CCR2(+)/CD14(+)) as immunotherapeutic targets in the treatment of pancreatic cancer. EXPERIMENTAL DESIGN: Survival analysis was conducted to determine if the prevalence of preoperative blood monocytes correlates with survival in patients with pancreatic cancer following tumor resection. Inflammatory monocyte prevalence in the blood and bone marrow of patients with pancreatic cancer and controls was compared. The immunosuppressive properties of inflammatory monocytes and macrophages in the blood and tumors, respectively, of patients with pancreatic cancer were assessed. CCL2 expression by human pancreatic cancer tumors was compared with normal pancreas. A novel CCR2 inhibitor (PF-04136309) was tested in an orthotopic model of murine pancreatic cancer. RESULTS: Monocyte prevalence in the peripheral blood correlates inversely with survival, and low monocyte prevalence is an independent predictor of increased survival in patients with pancreatic cancer with resected tumors. Inflammatory monocytes are increased in the blood and decreased in the bone marrow of patients with pancreatic cancer compared with controls. An increased ratio of inflammatory monocytes in the blood versus the bone marrow is a novel predictor of decreased patient survival following tumor resection. Human pancreatic cancer produces CCL2, and immunosuppressive CCR2(+) macrophages infiltrate these tumors. Patients with tumors that exhibit high CCL2 expression/low CD8 T-cell infiltrate have significantly decreased survival. In mice, CCR2 blockade depletes inflammatory monocytes and macrophages from the primary tumor and premetastatic liver resulting in enhanced antitumor immunity, decreased tumor growth, and reduced metastasis. CONCLUSIONS: Inflammatory monocyte recruitment is critical to pancreatic cancer progression, and targeting CCR2 may be an effective immunotherapeutic strategy in this disease.

Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma.

Uveal melanoma is the most common primary cancer of the eye and often results in fatal metastasis. Here, we describe mutations occurring exclusively at codon 625 of the SF3B1 gene, encoding splicing factor 3B subunit 1, in low-grade uveal melanomas with good prognosis. Thus, uveal melanoma is among a small group of cancers associated with SF3B1 mutations, and these mutations denote a distinct molecular subset of uveal melanomas.

Collaborative Ocular Oncology Group report number 1: prospective validation of a multi-gene prognostic assay in uveal melanoma.

PURPOSE: This study evaluates the prognostic performance of a 15 gene expression profiling (GEP) assay that assigns primary posterior uveal melanomas to prognostic subgroups: class 1 (low metastatic risk) and class 2 (high metastatic risk). DESIGN: Prospective, multicenter study. PARTICIPANTS: A total of 459 patients with posterior uveal melanoma were enrolled from 12 independent centers. TESTING: Tumors were classified by GEP as class 1 or class 2. The first 260 samples were also analyzed for chromosome 3 status using a single nucleotide polymorphism assay. Net reclassification improvement analysis was performed to compare the prognostic accuracy of GEP with the 7th edition clinical Tumor-Node-Metastasis (TNM) classification and chromosome 3 status. MAIN OUTCOME MEASURES: Patients were managed for their primary tumor and monitored for metastasis. RESULTS: The GEP assay successfully classified 446 of 459 cases (97.2%). The GEP was class 1 in 276 cases (61.9%) and class 2 in 170 cases (38.1%). Median follow-up was 17.4 months (mean, 18.0 months). Metastasis was detected in 3 class 1 cases (1.1%) and 44 class 2 cases (25.9%) (log-rank test, P<10(-14)). Although there was an association between GEP class 2 and monosomy 3 (Fisher exact test, P<0.0001), 54 of 260 tumors (20.8%) were discordant for GEP and chromosome 3 status, among which GEP demonstrated superior prognostic accuracy (log-rank test, P = 0.0001). By using multivariate Cox modeling, GEP class had a stronger independent association with metastasis than any other prognostic factor (P<0.0001). Chromosome 3 status did not contribute additional prognostic information that was independent of GEP (P = 0.2). At 3 years follow-up, the net reclassification improvement of GEP over TNM classification was 0.43 (P = 0.001) and 0.38 (P = 0.004) over chromosome 3 status. CONCLUSIONS: The GEP assay had a high technical success rate and was the most accurate prognostic marker among all of the factors analyzed. The GEP provided a highly significant improvement in prognostic accuracy over clinical TNM classification and chromosome 3 status. Chromosome 3 status did not provide prognostic information that was independent of GEP.

Frequent mutation of BAP1 in metastasizing uveal melanomas.

Metastasis is a defining feature of malignant tumors and is the most common cause of cancer-related death, yet the genetics of metastasis are poorly understood. We used exome capture coupled with massively parallel sequencing to search for metastasis-related mutations in highly metastatic uveal melanomas of the eye. Inactivating somatic mutations were identified in the gene encoding BRCA1-associated protein 1 (BAP1) on chromosome 3p21.1 in 26 of 31 (84%) metastasizing tumors, including 15 mutations causing premature protein termination and 5 affecting its ubiquitin carboxyl-terminal hydrolase domain. One tumor harbored a frameshift mutation that was germline in origin, thus representing a susceptibility allele. These findings implicate loss of BAP1 in uveal melanoma metastasis and suggest that the BAP1 pathway may be a valuable therapeutic target.

Association between gene expression profile, proliferation and metastasis in uveal melanoma.

PURPOSE: Uveal melanomas cluster into two molecular groups based on their gene expression profile. Tumors with the class 1 signature rarely metastasize, whereas those with the class 2 signature have a very high rate of metastasis. However, the biological basis for this metastatic propensity of class 2 tumors remains unclear. Towards such an explanation, this study was conducted to determine whether class 2 tumors have a higher proliferative rate than class 1 tumors. MATERIALS AND METHODS: The study included 28 primary uveal melanomas with extensive clinical, pathologic, and genetic annotation, including age, gender, ciliary body involvement, tumor basal diameter, thickness, cell type, gene expression profile, status of chromosomes 3 and 8p, aneuploidy, and clinical outcome. Immunopositivity for Ki-67 was determined by counting all positive nuclei in representative whole tumor sections. RESULTS: Ki-67 positivity was significantly associated with class 2 gene expression profile, loss of chromosome 3 and increased aneuploidy (P = 0.04, P = 0.004, and P = 0.03, respectively). Ki-67 positivity showed a borderline significant association with epithelioid cell type (P = 0.07). Receiver operating characteristic (ROC) analysis of Ki-67 positivity, using the class 2 signature as an endpoint, identified a Ki-67 score of approximately 20 cells per high power field as the optimal cut-off point between low and high risk for metastasis (log rank test, P = 0.01). CONCLUSIONS: On average, class 2 uveal melanomas have a higher proliferative rate than class 1 tumors. Further work is needed to determine whether loss of chromosome 3, increased aneuploidy, or other factors may be responsible for the increased proliferation.

An accurate, clinically feasible multi-gene expression assay for predicting metastasis in uveal melanoma.

Uveal (ocular) melanoma is an aggressive cancer that often forms undetectable micrometastases before diagnosis of the primary tumor. These micrometastases later multiply to generate metastatic tumors that are resistant to therapy and are uniformly fatal. We have previously identified a gene expression profile derived from the primary tumor that is extremely accurate for identifying patients at high risk of metastatic disease. Development of a practical clinically feasible platform for analyzing this expression profile would benefit high-risk patients through intensified metastatic surveillance, earlier intervention for metastasis, and stratification for entry into clinical trials of adjuvant therapy. Here, we migrate the expression profile from a hybridization-based microarray platform to a robust, clinically practical, PCR-based 15-gene assay comprising 12 discriminating genes and three endogenous control genes. We analyze the technical performance of the assay in a prospective study of 609 tumor samples, including 421 samples sent from distant locations. We show that the assay can be performed accurately on fine needle aspirate biopsy samples, even when the quantity of RNA is below detectable limits. Preliminary outcome data from the prospective study affirm the prognostic accuracy of the assay. This prognostic assay provides an important addition to the armamentarium for managing patients with uveal melanoma, and it provides a proof of principle for the development of similar assays for other cancers.

Oncogenic mutations in GNAQ occur early in uveal melanoma.

PURPOSE: Early/initiating oncogenic mutations have been identified for many cancers, but such mutations remain unidentified in uveal melanoma (UM). An extensive search for such mutations was undertaken, focusing on the RAF/MEK/ERK pathway, which is often the target of initiating mutations in other types of cancer. METHODS: DNA samples from primary UMs were analyzed for mutations in 24 potential oncogenes that affect the RAF/MEK/ERK pathway. For GNAQ, a stimulatory alpha(q) G-protein subunit which was recently found to be mutated in UMs, resequencing was expanded to include 67 primary UMs and 22 peripheral blood samples. GNAQ status was analyzed for association with clinical, pathologic, chromosomal, immunohistochemical, and transcriptional features. RESULTS: Activating mutations at codon 209 were identified in GNAQ in 33 (49%) of 67 primary UMs, including 2 (22%) of 9 iris melanomas and 31 (54%) of 58 posterior UMs. No mutations were found in the other 23 potential oncogenes. GNAQ mutations were not found in normal blood DNA samples. Consistent with GNAQ mutation being an early or initiating event, this mutation was not associated with any clinical, pathologic, or molecular features associated with late tumor progression. CONCLUSIONS: GNAQ mutations occur in about half of UMs, representing the most common known oncogenic mutation in this cancer. The presence of this mutation in tumors at all stages of malignant progression suggests that it is an early event in UM. Mutations in this G-protein-coupled receptor provide new insights into UM pathogenesis and could lead to new therapeutic possibilities.

A metastasis modifier locus on human chromosome 8p in uveal melanoma identified by integrative genomic analysis.

PURPOSE: To identify genes that modify metastatic risk in uveal melanoma, a type of cancer that is valuable for studying metastasis because of its remarkably consistent metastatic pattern and well-characterized gene expression signature associated with metastasis. EXPERIMENTAL DESIGN: We analyzed 53 primary uveal melanomas by gene expression profiling, array-based comparative genomic hybridization, array-based global DNA methylation profiling, and single nucleotide polymorphism-based detection of loss of heterozygosity to identify modifiers of metastatic risk. A candidate gene, leucine zipper tumor suppressor-1 (LZTS1), was examined for its effect on proliferation, migration, and motility in cultured uveal melanoma cells. RESULTS: In metastasizing primary uveal melanomas, deletion of chromosome 8p12-22 and DNA hypermethylation of the corresponding region of the retained hemizygous 8p allele were associated with more rapid metastasis. Among the 11 genes located within the deleted region, LZTS1 was most strongly linked to rapid metastasis. LZTS1 was silenced in rapidly metastasizing and metastatic uveal melanomas but not in slowly metastasizing and nonmetastasizing uveal melanomas. Forced expression of LZTS1 in metastasizing uveal melanoma cells inhibited their motility and invasion, whereas depletion of LZTS1 increased their motility. CONCLUSIONS: We have described a metastatic modifier locus on chromosome 8p and identified LZTS1 as a potential metastasis suppressor within this region. This study shows the utility of integrative genomic methods for identifying modifiers of metastatic risk in human cancers and may suggest new therapeutic targets in metastasizing tumor cells.

Micro-RNAs associated with metastasis in uveal melanoma identified by multiplexed microarray profiling.

Uveal (ocular) melanoma is a highly aggressive cancer that leads to metastatic death in up to half of patients despite successful local therapy. Biomarkers of metastatic risk are critically needed to institute new adjuvant treatment strategies in high-risk patients. Previously, we showed that two prognostically significant molecular subtypes of uveal melanoma could be identified based on gene expression profiling of the primary tumor. In this study, we investigated the value of micro-RNA (miRNA) expression patterns in predicting metastatic risk. A genome-wide, microarray-based approach was used to screen for differentially expressed miRNAs using the Agilent miRNA microarray (Agilent Technologies, Foster City, California, USA) platform containing probes for 470 human miRNAs. Unsupervised analysis was performed using principal component analysis, and supervised analysis was performed using significance analysis of microarrays. Tumors readily clustered based on miRNA expression into two groups that corresponded to the gene expression-based subtypes: class 1 (low metastatic risk) and class 2 (high metastatic risk). The most significant discriminators were let-7b and miR-199a, and the expression of these miRNAs was validated by quantitative PCR. A classifier that included the top six miRNA discriminators accurately distinguished class 1 from class 2 tumors with 100% sensitivity and specificity. miRNA expression may represent a highly accurate biomarker for metastatic risk in uveal melanoma. In addition, these results may provide new insights into the role of miRNAs in tumor progression and the metastatic phenotype.

Prognostic biomarkers in uveal melanoma: evidence for a stem cell-like phenotype associated with metastasis.

Uveal melanomas frequently metastasize and cause patient death. Many clinical, histopathologic, molecular, and genetic factors have been linked to metastasis. We hypothesized that understanding the relationships between, and relative prognostic significance of these factors would provide new insights into the pathogenesis of metastasis. To this end, we collected clinical, pathologic, and molecular data for 65 uveal melanomas, including patient age, sex, tumor size, location, cell type, vasculogenic mimicry looping matrix patterns, gene expression profiles, and immunohistochemistry for cytokeratin-18, vascular endothelial cadherin, E-cadherin, beta-catenin, and hypoxia-inducible factor 1alpha. In addition, we used Gene Set Enrichment Analysis to identify statistically significant overlap in genes that were differentially expressed in metastasizing tumors and those expressed in other well-characterized biological systems. Our results show that the class 2 gene expression signature was the most accurate predictor of metastasis (P=0.0001) and that the biomarkers most strongly associated with the class 2 signature included epithelioid cell type, beta-catenin, E-cadherin, and hypoxia-inducible factor 1alpha (P< or =0.001 for each). Thus, the class 2 gene expression signature continues to be the most accurate predictor of uveal melanoma metastasis and can, therefore, serve as a benchmark for evaluating other biomarkers. Importantly, Gene Set Enrichment Analysis showed a significant association between genes expressed in class 2 tumors and those expressed in primitive ectodermal and neural stem cells. Taken together with the constellation of biomarkers associated with the class 2 signature, this suggests the presence of cancer cells with a primitive neural/ectodermal stem cell-like phenotype that may be responsible for metastasis in these highly aggressive tumors.

Loss of heterozygosity of chromosome 3 detected with single nucleotide polymorphisms is superior to monosomy 3 for predicting metastasis in uveal melanoma.

PURPOSE: Loss of chromosome 3 is strongly associated with metastasis in uveal melanoma and has been proposed as the basis for clinical prognostic testing. It is not known whether techniques that identify loss of heterozygosity for chromosome 3 predict metastasis more accurately than those that detect only numerical loss of chromosome 3 (monosomy 3). EXPERIMENTAL DESIGN: Fifty-three uveal melanomas were analyzed by 28 single nucleotide polymorphisms (SNP) across chromosome 3. SNP was compared with fluorescence in situ hybridization (FISH) and array-based comparative genomic hybridization (aCGH) for metastasis prediction by sensitivity, specificity, and Kaplan-Meier survival analysis, using our validated gene expression-based classifier as a reference standard. RESULTS: By Kaplan-Meier analysis, only the gene expression-based classifier (P=0.001) and SNP-based detection of loss of heterozygosity for chromosome 3 (P=0.04) were significantly associated with metastasis. Sensitivity and specificity were 95.2% and 80.8%, respectively, for SNP, 77.8% and 64.7%, respectively, for FISH, and 85.0% and 72.0%, respectively, for aCGH. Isodisomy 3 was identified by SNP but undetected by aCGH and FISH in three tumors. CONCLUSIONS: Prognostic tests based on SNP platforms, which detect both chromosomal homologues and their subregions, may be superior to techniques that only detect changes in chromosome number. These observations could have important implications for efforts to detect genetic alterations in cancer genomes with CGH-based approaches.

Transcriptomic versus chromosomal prognostic markers and clinical outcome in uveal melanoma.

PURPOSE: To compare a gene expression-based classifier versus the standard genetic prognostic marker, monosomy 3, for predicting metastasis in uveal melanoma. EXPERIMENTAL DESIGN: Gene expression profiling, fluorescence in situ hybridization (FISH), and array comparative genomic hybridization (aCGH) were done on 67 primary uveal melanomas. Clinical and pathologic prognostic factors were also assessed. Variables were analyzed by Cox proportional hazards, Kaplan-Meier analysis, sensitivity, specificity, positive and negative predictive value, and positive and negative likelihood ratios. RESULTS: The gene expression-based molecular classifier assigned 27 tumors to class 1 (low risk) and 25 tumors to class 2 (high risk). By Cox univariate proportional hazards, class 2 signature (P = 0.0001), advanced patient age (P = 0.01), and scleral invasion (P = 0.007) were the only variables significantly associated with metastasis. Only the class 2 signature was needed to optimize predictive accuracy in a Cox multivariate model. A less significant association with metastasis was observed for monosomy 3 detected by aCGH (P = 0.076) and FISH (P = 0.127). The sensitivity and specificity for the molecular classifier (84.6% and 92.9%, respectively) were superior to monosomy 3 detected by aCGH (58.3% and 85.7%, respectively) and FISH (50.0% and 72.7%, respectively). Positive and negative predictive values (91.7% and 86.7%, respectively) and positive and negative likelihood ratios (11.9 and 0.2, respectively) for the molecular classifier were also superior to those for monosomy 3. CONCLUSIONS: Molecular classification based on gene expression profiling of the primary tumor was superior to monosomy 3 and clinicopathologic prognostic factors for predicting metastasis in uveal melanoma.

Prognostic testing in uveal melanoma by transcriptomic profiling of fine needle biopsy specimens.

Many uveal melanoma patients die of metastasis despite ocular treatment. Transcriptomic profiling of enucleated tumors can identify patients at high metastatic risk. Because most uveal melanomas do not require enucleation, a biopsy would be required for this analysis. Here, we establish the feasibility of transcriptomic analysis of uveal melanomas from fine needle aspirates. Transcriptomic profiles were analyzed from postenucleation "mock" needle biopsies and matching tumors from eight enucleated eyes and from fine needle aspirates in 17 uveal melanomas before radiotherapy. Predictive accuracy was assessed using a weighted voting classifier optimized for probe set selection using a minimal redundancy/maximum relevance algorithm. Transcriptomic profiles from mock biopsies were highly similar to those from their matching tumor samples (P < 0.0001). Transcriptomic profiles from fine needle aspirates clustered into two classes with discriminating probe sets that overlapped significantly with those for our published classification (P < 0.00001). No loss of predictive accuracy was identified among eight needle aspirates obtained from a distant location. Thus, it is feasible to obtain RNA of adequate quality and quantity to perform transcriptomic analysis on uveal melanoma samples obtained by fine needle biopsy. This method can be applied to specimens obtained from distant geographic locations and can stratify uveal melanoma patients based on metastatic risk.

Functional gene expression analysis uncovers phenotypic switch in aggressive uveal melanomas.

Microarray gene expression profiling is a powerful tool for generating molecular cancer classifications. However, elucidating biological insights from these large data sets has been challenging. Previously, we identified a gene expression-based classification of primary uveal melanomas that accurately predicts metastatic death. Class 1 tumors have a low risk and class 2 tumors a high risk for metastatic death. Here, we used genes that discriminate these tumor classes to identify biological correlates of the aggressive class 2 signature. A search for Gene Ontology categories enriched in our class-discriminating gene list revealed a global down-regulation of neural crest and melanocyte-specific genes and an up-regulation of epithelial genes in class 2 tumors. Correspondingly, class 2 tumors exhibited epithelial features, such as polygonal cell morphology, up-regulation of the epithelial adhesion molecule E-cadherin, colocalization of E-cadherin and beta-catenin to the plasma membrane, and formation of cell-cell adhesions and acinar structures. One of our top class-discriminating genes was the helix-loop-helix inhibitor ID2, which was strongly down-regulated in class 2 tumors. The class 2 phenotype could be recapitulated by eliminating Id2 in cultured class 1 human uveal melanoma cells and in a mouse ocular melanoma model. Id2 seemed to suppress the epithelial-like class 2 phenotype by inhibiting an activator of the E-cadherin promoter. Consequently, Id2 loss triggered up-regulation of E-cadherin, which in turn promoted anchorage-independent cell growth, a likely antecedent to metastasis. These findings reveal new roles for Id2 and E-cadherin in uveal melanoma progression, and they identify potential targets for therapeutic intervention.

Association between microarray gene expression signature and extravascular matrix patterns in primary uveal melanomas.

PURPOSE: To determine whether there is an association between gene expression profile and looping extravascular matrix patterns in primary uveal melanomas. DESIGN: Laboratory investigation. METHODS: Formalin-fixed, paraffin-embedded sections from 22 primary uveal melanomas that previously were analyzed by microarray gene expression profiling were stained for periodic acid-Schiff and scored in a masked fashion for the presence of looping extravascular matrix patterns. RESULTS: A strong association was observed between looping patterns and the unfavorable class 2 molecular prognostic signature (P < .001). CONCLUSIONS: Looping extravascular matrix patterns occur almost exclusively in class 2 uveal melanomas. This finding may provide new insights into the pathogenesis and management of uveal melanoma.

Functional analysis of the p53 pathway in response to ionizing radiation in uveal melanoma.

PURPOSE: Uveal melanomas are notoriously radioresistant and thus necessitate treatment with extremely high radiation doses that often cause ocular complications. The p53 tumor suppressor pathway is a major mediator of the cellular response to radiation-induced DNA damage, suggesting that this pathway may be defective in uveal melanoma. The current study was conducted to analyze the functional integrity of the p53 pathway in primary uveal melanoma cells. METHODS: The p53 gene was sequenced in three primary uveal melanoma cells lines. Cultured primary uveal melanoma cells (MM28, MM50, Mel202, Mel270, and Mel290), MCF7 breast carcinoma cells, normal uveal melanocytes (UM47), and normal human diploid fibroblasts (NHDFs) were irradiated at 250 kVp and 12 mA at a dose rate of 1.08 Gy/min for a total dose of up to 20 Gy. Cell viability was analyzed with trypan blue exclusion. Western blot analysis was used to analyze the expression of p53, p53-phospho-Ser15, p21, Bax, PUMA, and Bcl-x(L). RESULTS: No p53 gene mutations were found in MM28, MM50, or Mel270 cells. Upstream signaling to p53 was intact, with normal induction of p53 and phosphorylation of p53-Ser15, in all five cell lines. Radiation-induced downstream activation of p21 was defective in MM28 and MM50 cells, and activation of Bax was defective in MM50 and Mel290 cells. MM28, MM50, and Mel202 cells failed to deamidate Bcl-x(L) in response to radiation-induced DNA damage. Overall, four of the five uveal melanoma cell lines exhibited at least one downstream defect in the p53 pathway. CONCLUSIONS: Expression of p53 and upstream signaling to p53 in response to radiation-induced DNA damage appear to be intact in most uveal melanomas. In contrast, functional defects in the p53 pathway downstream of p53 activation appear to be common. Further elucidation of p53 pathway abnormalities in uveal melanoma may allow therapeutic interventions to increase the radiosensitivity of the tumors.