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Originally published as JCO Early Release 10.1200/JCO.2005.02.2418 on December 12 2005 © 2006 American Society of Clinical Oncology. High Frequency of Submicroscopic Hemizygous Deletion Is a Major Mechanism of Loss of Expression of PTEN in Uveal MelanomaFrom the Department of Ophthalmology, the Ohio State University, Columbus; Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute and Department of Genetics, Case Western Reserve University School of Medicine, Cleveland, OH; and Cancer Research UK Human Cancer Genetics Research Group, University of Cambridge, Cambridge, United Kingdom Address reprint requests to Charis En, MD, PhD, Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, 9500 Euclid Ave, NE-50, Cleveland, OH 44195; e-mail: engc{at}ccf.org or spsmce{at}netscape.net
PURPOSE: Although cytogenetic aberrations at 10q have been reported in up to 27% of uveal melanomas, the role of PTEN in the pathogenesis of uveal melanoma is largely unknown. Our aim was to determine the frequency and clinical significance of PTEN alterations in uveal melanomas. PATIENTS AND METHODS: We examined PTEN expression using immunohistochemistry in 75 sporadic uveal melanomas, with an average follow-up of 89 months. Molecular cytogenetic alterations were studied using comparative genomic hybridization (CGH). Genotyping was carried out using an intragenic PTEN marker and two flanking markers. Mutational analysis of PTEN was also carried out. RESULTS: Of the 75 tumors, 12 (16%) showed no PTEN immunostaining, 32 (42.7%) showed weak to moderate staining and the remaining 31 (38.2%) showed staining similar to the normal internal controls. Using CGH, only two (15.4%) of 13 samples showed any loss of 10q. However, in the 38 tumors with informative genotyping, we found that 29 (76.3%) had loss of heterozygosity (LOH) of at least one PTEN marker, and 15 (39.5%) showed LOH of at least two markers. Mutations in the coding region of PTEN were identified in four (11.4%) of 35 tumors. Further, loss of cytoplasmic PTEN expression by immunohistochemistry was associated with shortened disease-free survival (P = .029). CONCLUSION: This is the first demonstration that PTEN is a tumor suppressor involved in uveal melanoma pathogenesis and may be associated with clinical outcome. Our data also suggest that submicroscopic deletion, but not large deletions, is the major mechanism of loss of PTEN expression in uveal melanomas.
PTEN (MMAC1/TEP1), a tumor suppressor gene on 10q23.3, is variably mutated and/or deleted in a variety of human cancers.1-3 Somatic mutation and/or deletion of PTEN occurs to a greater or lesser extent in a wide variety of human cancers that show loss of heterozygosity (LOH) in this region.4 Loss of genetic material on the long arm of chromosome 10 has been detected in 30% to 50% of both early- and advanced-stage sporadic cutaneous melanomas, and has been associated with poor clinical outcome.5-8 Despite inconsistent results, it would appear that homozygous deletions and intragenic mutations of PTEN occurred more frequently in the metastatic cutaneous melanomas and cell lines. Epigenetic inactivation has been proposed as an important alternative mechanism for PTEN inactivation in cutaneous melanomas.9 Moreover, nuclear-cytoplasmic partitioning of PTEN might also play a role in melanoma progression.10 Uveal melanoma is the most common adult primary intraocular malignancy in the United States.11 Gross loss of chromosome 10, by cytogenetic analysis, has been reported in approximately 27% of uveal melanomas.12 However, little is known about the role of PTEN in the pathogenesis of uveal melanomas. Only one study has reported no cytogenetic or molecular cytogenetic alterations at 10q23, the chromosomal location of PTEN, in nine uveal melanoma cell lines. Also, that study did not detect any mutations in PTEN in those cell lines. Currently, there are no published data on the frequency of deletions of 10q involving the PTEN region, nor on PTEN mutations in primary noncultured uveal melanomas. On the basis of genome-wide expression profiling, we identified a relative decrease in PTEN expression in aggressive primary uveal melanomas compared to nonaggressive tumors.13 This observation led us to investigate further the role of PTEN in the pathogenesis of uveal melanomas.
Tumor Samples and Tissue Microarray Preparation Specimens were obtained from the archives of the Departments of Ophthalmology and Pathology, The Ohio State University (Columbus, OH). A total of 75 tumors, collected from 1979 to 2004, were included. Of those 75, 36 were from men and 39 from women. The average age of the 75 patients was 59.6 years (range, 27 to 83 years). Sample collection and clinical information acquisition were in accordance with an institutional review board–approved protocol (2003C0057). Follow-up data were obtained from patient charts, and data were available for 71 patients, and of these, 25 had developed metastases. For patients with metastases, the average duration from the time of diagnosis to death as a result of metastasis was 50 months (range, 8 to 138 months). The average follow-up for patients who did not develop metastasis was 89 months (range, 16 to 150 months). One to four 1.5-mm representative cores from each tumor were included in three tissue microarrays (TMAs) according to previously published protocols.14 Sections from 27 tumors, 22 of those included in the TMAs, were also studied.
RNA and DNA Extraction
Quantitative Reverse Transcription Polymerase Chain Reaction Assay
Immunohistochemistry
Immunohistochemical Analyses
LOH Analysis
Comparative Genomic Hybridization
PTEN Mutation Analysis
Statistical Analysis
Validation of PTEN Gene Expression Profiling Using QRT-PCR Eleven uveal melanomas with available expression profiling data and sufficient high-quality RNA were studied. Three cutaneous melanomas, two with CGH-detectable loss of 10q, and choroidal tissue from a nontumor eye were included as controls. Confirming our array data, QRT-PCR revealed decreased PTEN expression in the primary uveal melanomas which subsequently metastasized (M002, M004, M018 and M007, Fig 1), and in the positive controls, the metastatic cutaneous melanomas with CGH-detectable loss of 10q including PTEN, compared with both the normal controls and the rest of the tumors (Fig 1).
PTEN IHC in Uveal Melanoma Seventy-five sporadic uveal melanoma samples were studied using three separate TMAs (70 samples) and/or tissue sections [27 samples] (Fig 2A, Table 1). Expression of PTEN in the normal retina and/or in vascular endothelial cells served as an internal positive control for this study (Fig 2B- 2E). Of these 75 tumors, 12 (16%) showed  15/300 cytoplasmic final score and were considered as null for PTEN immunoreactivity and graded negative (Fig 2B). Nuclear staining was also absent in the tumors scored as negative. Thirty-two tumors (42.7%) scored from > 15 to 150 of 300 for cytoplasmic staining and were considered weakly to moderately stained in comparison with the healthy tissue (and internal controls) and were graded + (Fig 2C-2D). The remaining 31 tumors (38.2%) scored  150/300 and were graded ++ (Fig 2E).
Nuclear PTEN was scored negative for tumors with < 5% cells with nuclear staining, + for tumors with < 50% staining and ++ for tumors with 50% staining. The quality and intensity of PTEN immunostaining in the nucleus and cytoplasm were relatively uniform throughout each specimen in 49 of 63 of the tumors with normal (++) or weak (+) PTEN immunostaining. In the remaining 14 tumors, five with normal and eight with weak PTEN staining, only cytoplasmic staining was detected and no nuclear staining was seen, suggesting nucleocytoplasmic dissociation of PTEN immunostaining in these.
CGH Analysis
LOH Analysis Of 46 samples examined, eight were excluded because they either were not informative at all markers or were likely to fail multiple PCR attempts. LOH at 10q23 was scored when one or more of the panel of three polymorphic loci showed a ratio < 0.67 or > 1.5 when comparing the two alleles from either the tumor or healthy tissue to each other, as is standard. Of the 38 samples that were informative for at least one marker, 29 (72.5%) had LOH at one marker and 15 (39.5%) for at least two markers (Table 2). We then compared the LOH data of the samples with CGH data (Tables 1 and 2). In sample M02, which showed CGH evidence of 10q23.3 loss, LOH was found at PTEN flanking markers D10S1765 and D10S541 as well as in the telomeric marker D10S10230. In contrast, retention of heterozygosity (ROH) was detected at all three polymorphic markers in tumor M017, which also showed 10q23 loss by CGH. Immunostaining of a full section of M017 identified a mixed pattern of PTEN immunostaining with approximately 25% of the tumor showing loss of PTEN and 75% showing immunostaining with similar intensity as the internal control (data not shown). In five of seven samples with no detectable molecular cytogenetic alteration by CGH, LOH was found at one or more of the three studied polymorphic markers nearby or within PTEN (Fig 3, Table 2).
PTEN Mutation Analysis DGGE screening for PTEN mutations occurred in 35 of 38 samples with informative genotyping data. In 30 of those 35 samples, mutational screening of exons 1 through 8 were successful, whereas in the remaining five samples, several of the exons did not amplify by PCR on multiple attempts, likely due to dwindling template. Eight sequence variants were detected, four in the coding region suggesting potential pathogenic mutations. All of the sequence variants were missense mutations (Table 2). Three of the exonic mutations were in exon 5, and the remaining one in exon 8. In one, UM128, DGGE repeatedly identified a sequence variant in exon 8 in the tumor but not in the healthy tissue. However sequencing did not detect sequence variation in the exon or in the immediate nearby intronic sequence.
Comparison of PTEN Genetic Alterations to PTEN Protein Expression by IHC There were four samples with somatic PTEN missense mutations in the coding region and all had weak PTEN protein expression (Table 2, M062, M103, M122, M202). Two of these tumors showed LOH of at least one PTEN marker and this is sufficient to explain the weak protein expression.
Survival Analysis
There is currently strong evidence supporting the role of PTEN in cutaneous melanoma tumorigenesis.18 Although physical loss of chromosome 10 has been reported in up to 27% of uveal melanomas,12 little is known about the role of PTEN in primary uveal melanoma tumorigenesis. Before now, only a single study was available on the frequency of PTEN mutations (none were found) in uveal melanomas, and in that study, no primary tumors were included.19 In the present study, we found a high (58.7%) frequency of decreased or complete loss of PTEN expression by immunostaining in a series of 75 primary uveal melanomas. We further investigated the mechanism responsible for the decreased PTEN expression using a combination of molecular cytogenetic and molecular genetic techniques. Using CGH, which has a 10- to 20-Mb resolution,16 we identified a deletion in only 15% of primary uveal melanoma, which is similar to the published low frequency of loss of chromosome 10 detected by conventional cytogenetic techniques.19 In contrast, LOH analysis revealed 76.3% of samples having lost of at least one of the three examined markers defining PTEN, and in approximately 40%, at least two markers showed LOH, which is within the frequency range of samples with decreased or no PTEN expression by IHC. To identify whether submicroscopic deletion (< 10 to 20Mb in size) in 10q is localized to the PTEN region or involves other regions of 10q in uveal melanomas, we included two additional markers, D10S1239 and D10S1230, which are 13.7 Mb and 33 Mb telomeric to the chromosomal location of PTEN, respectively. Overall, our results suggest that submicroscopic deletion of the PTEN region explains the underexpression and it also suggests that small deletions telomeric of 10q24 also occur in addition to and independently of PTEN loss. Taking together the IHC, CGH, mutational analysis and LOH data, our observations indicate that small submicroscopic deletion is the major mechanism of PTEN inactivation in uveal melanomas. Because four of 17 tumors scored + had ROH and no PTEN mutation in the coding region, an epigenetic mechanism of PTEN silencing likely does occur as well, but at a lesser frequency. Whether somatic PTEN alterations in uveal melanomas are associated with tumor initiation and/or progression remains unclear. In most tumors with mixed morphology, including areas of highly anaplastic epithelioid cells, PTEN loss was similar in both epithelioid and spindle cell regions. Surprisingly, in two tumors, the spindle cell regions, a histologic marker of a less aggressive tumor, showed total absence of PTEN while the anaplastic epithelioid cell regions showed a decrease but not total absence of PTEN expression (Fig 5). Considering the monoclonal origin of all tumors, in mixed tumors, the highly anaplastic epithelioid cells have been hypothesized to have evolved from the less aggressive-looking spindle cells. If this hypothesis were true, and that PTEN loss is associated with tumor progression, then loss of PTEN expression should be more profound in the epithelioid regions of mixed tumors. Our converse observation may suggest, although not conclusively, that PTEN loss occurs relatively early in the development of uveal melanomas; that the two components of mixed tumors evolve from separate stem cell clones; or that PTEN expressional loss occurs in the less aggressive-looking spindle cells and these cells evolve into the more anaplastic epithelioid cells which may have acquired genetic/epigenetic alterations downstream of PTEN, thus making PTEN expression itself superfluous.
Based on our survival analysis, we have shown that loss of PTEN expression by IHC was associated with a less favorable survival profile for patients presenting with primary uveal melanoma (Fig 4). Patients with total loss of PTEN had a median survival of about 60 months compared to more than 120 months for patients with normal or nearly normal PTEN expression. The prognostic significance of loss of PTEN expression has been reported in a wide variety of cancers including those of prostate,20,21 endometrium22,23 and breast,24 pediatric malignant astrocytomas,25 and hepatocellular carcinoma.26 Although PTEN expressional loss in uveal melanomas was associated with more aggressive tumors, favoring metastasis, we did observe that two of the 12 patients, whose tumors had total loss of PTEN expression (M069 and M092) had a rather long disease-free survival of 113 and 120 months, respectively. This observation suggests that, in addition to PTEN loss, other genetic alterations (ie, "additional hits") are necessary for the progression of uveal melanomas. Alternatively, equally plausibly, these rare tumors that are PTEN null yet are not associated with poor prognosis, may have other alterations, perhaps downstream of PTEN, which counteract the PTEN null status. Similarly, uveal melanomas that express PTEN (+ or ++) are associated with prolonged disease-free survival but there clearly are a subset of tumors that have (+ or ++) PTEN expression that are associated with decreased disease-free survival as well (Fig 4). There are two possible explanations. First, a few of these uveal melanomas could have somatic biallelic intragenic mutations, which may not lead to PTEN null status by IHC, but the downstream effect is equivalent. We have shown that biallelic intragenic mutations do not occur (Table 2). Second, loss of chromosome 3 and/or gain of 8q have been well-documented to be associated with more aggressive tumors,27 could confer such additional genetic hits. Our CGH data, although limited, suggest that such interactions may be important. However, further studies to elucidate the interaction between PTEN loss and other genetic alteration in uveal melanomas would be warranted. Metastatic uveal melanomas are highly aggressive tumors that usually kill the patients within less than a year of diagnosis.28 Among different therapeutic modalities, only local treatment using chemoembolization with cisplatin-based regimens produced clinically meaningful response rates.28 The outlook for systemic chemotherapy for uveal melanomas including combination of systemic chemotherapy and interferon therapy is currently pessimistic.29 Our observations have implications for prognostication and potentially therapy. Establishing PTEN status in a primary uveal melanoma may be useful because those that are null may be a signal to a clinician to adjucate closer follow-up and/or consideration of adjuvant therapy. Our findings may suggest that mammalian target of rapamycin (mTOR) inhibitors such as rapamycin or its analogs can be utilized for treatment of metastatic uveal melanomas. Recent studies have demonstrated that mTOR inhibition has remarkable activity against a wide range of human cancers in vitro and in human tumor xenograft models, especially when PTEN is dysfunctional.30 The clinical challenge for the application of this class of anticancer drugs is the ability to prospectively identify which tumors will be sensitive to mTOR inhibition. For uveal melanomas, it will remain to be identified whether total versus partial PTEN loss has any effect on tumor response to mTOR therapy. In conclusion, our observations suggest that PTEN is a tumor suppressor involved in the pathogenesis of uveal melanomas. Our data suggest that submicroscopic deletions but not large deletions or intragenic mutations, are the major mechanism of loss of PTEN expression in uveal melanomas. Finally, our results suggest that total loss of PTEN expression in primary uveal melanomas is associated with more aggressive tumors, portending future metastases. Thus, PTEN status in primary uveal melanomas may help indicate whether closer follow-up is indicated, and in the future, guide targeted therapy.
The authors indicated no potential conflicts of interest.
Supported by a clinical cancer genetics fellowship funded by the Department of Internal Medicine, The Ohio State University (M.A.-R.) and a Doris Duke Distinguished Clinical Scientist Award (C.E.), and partially funded by the Patti Blow Research fund in Ophthalmology. Presented in part at the 96th Annual Meeting of the American Association for Cancer Research, Anaheim, CA, April 16-20, 2005. Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
