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
    Cancer Research UK Human Cancer Genetics Research Group, University of Cambridge, Cambridge, United Kingdom

    ABSTRACT

    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.

    INTRODUCTION

    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.

    PATIENTS AND METHODS

    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

    RNA was extracted from fresh frozen tumor tissues using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Genomic DNA was extracted from tumor and matched healthy tissue with a QIAamp DNA Mini Kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Normal genomic DNA was extracted from the sclera and retina away from the tumor by macrodissection of normal and tumor areas of paraffin-embedded archival tissue using standard protocols.

    Quantitative Reverse Transcription Polymerase Chain Reaction Assay

    TaqMan (Applied Biosystems, Foster City, CA) 5' nuclease quantitative reverse transcription polymerase chain reaction (QRT-PCR) assays were carried out using the iCycler real-time PCR system (Bio-Rad, Hercules, CA). The RNA was DNAse I treated to eliminate the PTEN pseduogene. The reactions were carried out in triplicates for each sample in 25 μL. The PTEN primers used were forward 5'-CAGCCATCATCAAAGAGATCG (exon 1) and reverse 5'-TTGTTCCTGTATACGCCTTCAA (exon 2). In addition to PTEN, two endogenous controls, GPI and GAPDH, were tested in separate reactions. The PCR reaction settings were 95°C for 3 minutes, then 40 to 50 cycles of 95°C for 15 seconds, and 60°C for 1 minute. The relative expression levels were assessed by both standard curve and comparative CT methods.14

    Immunohistochemistry

    Immunohistochemistry (IHC) was carried out at the histology core facility, Department of Pathology, The Ohio State University. Monoclonal antibody 6H2.1 against the terminal 100 amino acids of human PTEN (Cascade Bioscience, Winchester, MA) was used at a dilution of 1:100 for either 60 minutes at room temperature or overnight at 4 C°. For detection of the immunostaining, we used either DAKO LSAB + Kit or Vectastain Elite (both Vector Laboratories, Burlingame, CA). Both detection systems produced a dark red staining to minimize the interference from melanin pigment.

    Immunohistochemical Analyses

    Immunostaining was evaluated independently by two investigators (M.A., who is a pathologist, and Y.Y.) without knowledge of the clinical and pathologic parameters. Samples with more than 20% difference between these two investigators were jointly reviewed and rescored. Selected samples were rereviewed by an experienced ophthalmic pathologist (E.C.). The immunostaining signals were scored on a scale of 0 to 300, with the intensity of the staining scored from 0 to 3, and the percentage of the cells stained was recorded from 1% to 100%. The percentage of tumor cells showing nuclear stain was scored from 1% to 100%. The final 0-to-300 score was derived from multiplication of the two scores. We used a combination of internal and external controls for scoring of our immunohistochemical samples. Staining of the retina and/or endothelium of healthy blood vessels was used as an internal positive control for PTEN, as previously described.9 Also, immunostaining for vimentin was used as external controls to ensure homogeneous staining of all tumors. For tumors with insufficient internal control elements represented in the TMA cores, full sections of the tumors were studied.

    LOH Analysis

    Forty-six tumors with sufficient tumor and corresponding normal DNA were analyzed for LOH at three polymorphic markers flanking and within the PTEN gene. The marker order, from centromere to telomere is D10S1765-IVS4 + 109ins/delTCTTA-D10S541, as described previously.9 To get an indication of the telomeric extent of LOH, two markers, D10S1239 and D10S1230, which are 13.7 Mb and 33 Mb telomeric to the PTEN gene, were also included. Polymerase chain reaction (PCR) conditions for these markers are described elsewhere.15 The PCR products were analyzed using an ABI 377 sequencer and the GeneScan and Genotyper softwares (Applied Biosystems). The genotyping was carried out at least twice for all the samples.

    Comparative Genomic Hybridization

    Molecular cytogenetic changes were studied using comparative genomic hybridization (CGH) in 13 tumors with high-quality DNA extracted from fresh-frozen tissue, as described previously.14,16 In nine of those tumors, M02, M04, M06, M011, M017, M021, M4033, M4035, and M4036, genotyping were also possible.

    PTEN Mutation Analysis

    Genomic DNA from 35 of 38 samples with LOH data were scanned for somatic mutations by denaturing gradient gel electrophoresis (DGGE) as described previously.17 Exon 9 was excluded because of high frequency of PCR failure and because exon 9 mutations are extremely rare. Samples showing DGGE variation were reamplified with the same set of primers, column purified, and subjected to semi-automated sequence analysis as previously published.17

    Statistical Analysis

    GB-STAT version 10 software (Dynamic Microsystems Inc, Silver Spring, MD) was used for analysis. For survival analysis, we used the Kaplan-Meier cumulative survival analysis curves, and log-rank and generalized Wilcoxon tests for equality of survival for detection of significance.

    RESULTS

    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

    Molecular cytogenetic changes close to the 10q23.3 chromosomal region, the physical location of PTEN, were detected in two of the 13 tumors studied, M02 and M017 (Tables 1 and 2). One of these tumors, M02, had a large deletion spanning 10q23-10qter. The other sample, M017, showed a small deletion in the 10q23 region.

    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

    Both immunohistochemical and informative LOH data were available for a total of 38 samples (Table 2). Among these 38, seven were PTEN null by IHC and six had LOH of at least one PTEN marker, whereas one, M128, had ROH at the two flanking markers; unfortunately, the intragenic marker was not informative (homozygous in the germline). Notably, two tumors that were PTEN null by IHC and PTEN-marker LOH showed ROH at the marker distant to the 3' end of PTEN. Of the 22 samples that scored + for PTEN expression, 17 had LOH at least one PTEN marker, whereas the remaining five had ROH. Again, at least four tumors with LOH in the PTEN region showed ROH at one and/or the other of the two markers 14 and 33 Mb telomeric of the 3' end of PTEN (Table 4). Finally, the remaining nine graded ++ all had ROH at at least one marker. However, one sample with ++ PTEN expression (M89) showed LOH in two flanking markers, although the intragenic marker was not informative. Re-reviewing the immunostaining score for sample M89 indicated a relatively mild decrease in the level of PTEN expression compared with the internal control.

    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

    Patients with complete cytoplasmic PTEN loss (PTEN negative) in their tumors had a significantly shorter disease-free survival compared to patients with normal (++) PTEN immunostaining (log-rank P = .029 for equality of survival; Fig 4A). Decreased cytoplasmic PTEN expression (+) was also associated with a shorter survival but the trend was not statistically significant. Absent nuclear PTEN expression was also associated with a decrease in survival when compared with high nuclear PTEN expression (log-rank P = .039 for equality of survival; Fig 4B).

    DISCUSSION

    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.

    Authors' Disclosures of Potential Conflicts of Interest

    The authors indicated no potential conflicts of interest.

    Author Contributions

    Conception and design: Mohamed H. Abdel-Rahman, Charis Eng

    Financial support: Frederick H. Davidorf, Charis Eng

    Provision of study materials or patients: Mohamed H. Abdel-Rahman, Ying Yang, Elson L. Craig, Frederick H. Davidorf, Charis Eng

    Collection and assembly of data: Mohamed H. Abdel-Rahman, Ying Yang

    Data analysis and interpretation: Mohamed H. Abdel-Rahman, Charis Eng

    Manuscript writing: Mohamed H. Abdel-Rahman, Frederick H. Davidorf, Charis Eng

    Final approval of manuscript: Mohamed H. Abdel-Rahman, Ying Yang, Xiao-Ping Zhou, Elson L. Craig, Frederick H. Davidorf, Charis Eng

    GLOSSARY

    CGH (comparative genomic hybridization):

    A molecular cytogenetic method of screening tumor samples for genetic changes showing characteristic patterns for copy number changes (mutations cannot be detected by CGH) at chromosomal and subchromosomal levels. Alterations in patterns are classified as DNA gains and losses. The method consists of isolating DNA from tumors and healthy tissues (reference) and labeling each with a different "color" or fluor. The two samples are then mixed and hybridized to normal metaphase chromosomes. In the case of array or matrix CGH, the hybridization mixing is done on a slide with thousands of DNA probes. The detection system is varied, but basically determines the color ratio along the chromosomes to determine DNA regions that might be gained or lost in tumor samples.

    DGGE (denaturing gradient gel electrophoresis):

    DGGE is a rapid mutation-scanning technology that is based on the melting characteristics of double stranded DNA. Based on the fact that identical DNA molecules, which differ by only one nucleotide within a low melting domain, will have different melting temperatures, DGGE is typically performed on polymerase chain reaction products. In the procedure, double-stranded DNA is electrophoresed through an acrylamide gel containing a gradient of denaturant, which increases in the direction of electrophoresis. When appropriate denaturing conditions exist, the DNA molecules melt (melting domain). When the characteristic melting temperature is achieved, the aplicon melts and a single band is observed. Mismatched DNA (mutations) will retard the movement of the DNA through the gel, with sequence differences of one base having the ability to significantly alter the stability of the melting domains, and more than one band will be observed.

    Epigenetic:

    The transfer of information from one cell to its descendants without the information's being encoded in the nucleotide sequence of the DNA. The methylation of the promoter to inactivate a gene is an example of an epigenetic change. Epigenetic inheritance is typically transmitted in dividing cells. Although rare, it is occasionally seen in traits being transmitted from one generation to another. Epigenetic variants can arise spontaneously and just as spontaneously revert.

    Genotyping:

    The process used for obtaining the genotype of a given gene. Typically, polymerase chain reaction–based methods are used. However, in the case of single nucleotide polymorphism genotyping, microarray platforms are used routinely. Genotyping data serves several purposes, including a means to determine genetic diversity, to identify important genetic traits and in forensic and population studies. It is used increasingly in determining paternity of offspring. From a somatic point of view (within a tumor), genotyping is used to determine loss of heterozygosity.

    LOH (loss of heterozygosity):

    A situation where one chromosome has a normal allele of a gene and one chromosome has a mutant or deleted allele.

    Molecular cytogenetics:

    Cytogenetic studies that probe the molecular make up of chromosomes. Several techniques have been developed that have advanced the field of molecular cytogenetics, including fluorescence in situ hybridization and array-based comparative genomic hybridization.

    PTEN (phosphatase and tensin homolog):

    PTEN is a tumor suppressor gene, with a gamut of regulatory activities. The gene product is a multifunctional molecule. The predominant activity identified for PTEN is its lipid phosphatase activity, which converts inositol trisphosphates into inositol bisphosphates, thus inhibiting survival and proliferative pathways that are activated by inositol trisphosphates.

    QRT-PCR:

    Quantitative polymerase chain reaction (qPCR), also known as real-time PCR, consists of detecting PCR products as they accumulate. It can be applied to gene expression quantification by reverse transcription of RNA into cDNA, thus receiving the name of quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). In spite of its name—quantitative—results are usually normalized to an endogenous reference. Current devices allow the simultaneous assessment of many RNA sequences.

    TMA (tissue microarray):

    Used to analyze the expression of genes of interest simultaneously in multiple tissue samples, tissue microarrays consist of hundreds of individual tissue samples placed on slides ranging from 2 to 3 mm in diameter. Using conventional histochemical and molecular detection techniques, tissue microarrays are powerful tools to evaluate the expression of genes of interest in tissue samples. In cancer research, tissue microarrays are used to analyze the frequency of a molecular alteration in different tumor type, to evaluate prognostic markers, and to test potential diagnostic markers.

    NOTES

    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.

    REFERENCES

    Li DM, Sun H: TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta. Cancer Res 57:2124-2129, 1997

    Li J, Yen C, Liaw D, et al: PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275:1943-1947, 1997

    Steck PA, Pershouse MA, Jasser SA, et al: Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat Genet 15:356-362, 1997

    Eng C: PTEN: One gene, many syndromes. Hum Mutat 22:183-198, 2003

    Healy E, Belgaid C, Takata M, et al: Prognostic significance of allelic losses in primary melanoma. Oncogene 16:2213-2218, 1998

    Herbst RA, Weiss J, Ehnis A, et al: Loss of heterozygosity for 10q22-10qter in malignant melanoma progression. Cancer Res 54:3111-3114, 1994

    Tsao H, Zhang X, Benoit E, et al: Identification of PTEN/MMAC1 alterations in uncultured melanomas and melanoma cell lines. Oncogene 16:3397-3402, 1998

    Rodolfo M, Daniotti M, Vallacchi V: Genetic progression of metastatic melanoma. Cancer Lett 214:133-147, 2004

    Zhou XP, Gimm O, Hampel H, et al: Epigenetic PTEN silencing in malignant melanomas without PTEN mutation. Am J Pathol 157:1123-1128, 2000

    Whiteman DC, Zhou XP, Cummings MC, et al: Nuclear PTEN expression and clinicopathologic features in a population-based series of primary cutaneous melanoma. Int J Cancer 99:63-67, 2002

    Singh AD, Topham A: Incidence of uveal melanoma in the United States: 1973-1997. Ophthalmology 110:956-961, 2003

    Hoglund M, Gisselsson D, Hansen GB, et al: Dissecting karyotypic patterns in malignant melanomas: Temporal clustering of losses and gains in melanoma karyotypic evolution. Int J Cancer 108:57-65, 2004

    Abdel-Rahman MH, Shen L, Jones D et al: Molecular genetic profiling of uveal melanoma reveals clinically significant molecular subclasses. Presented at the American Society of Human Genetics Annual Meeting, Toronto, Canada, October 26-30, 2004

    Abdel-Rahman MH, Craig EL, Davidorf FH, et al: Expression of vascular endothelial growth factor in uveal melanoma is independent of 6p21-region copy number. Clin Cancer Res 11:73-78, 2005

    Marsh DJ, Dahia PL, Coulon V, et al: Allelic imbalance, including deletion of PTEN/MMACI, at the Cowden disease locus on 10q22-23, in hamartomas from patients with Cowden syndrome and germline PTEN mutation. Genes Chromosomes Cancer 21:61-69, 1998

    Kallioniemi OP, Kallioniemi A, Piper J, et al: Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors. Genes Chromosomes Cancer 10:231-243, 1994

    Mutter GL, Lin MC, Fitzgerald JT, et al: Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers. J Natl Cancer Inst 92:924-930, 2000

    Wu H, Goel V, Haluska FG: PTEN signaling pathways in melanoma. Oncogene 22:3113-3122, 2003

    Naus NC, Zuidervaart W, Rayman N, et al: Mutation analysis of the PTEN gene in uveal melanoma cell lines. Int J Cancer 87:151-153, 2000

    McMenamin ME, Soung P, Perera S, et al: Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced stage. Cancer Res 59:4291-4296, 1999

    Koksal IT, Dirice E, Yasar D, et al: The assessment of PTEN tumor suppressor gene in combination with Gleason scoring and serum PSA to evaluate progression of prostate carcinoma. Urol Oncol 22:307-312, 2004

    Terakawa N, Kanamori Y, Yoshida S: Loss of PTEN expression followed by Akt phosphorylation is a poor prognostic factor for patients with endometrial cancer. Endocr Relat Cancer 10:203-208, 2003

    Kanamori Y, Kigawa J, Itamochi H, et al: PTEN expression is associated with prognosis for patients with advanced endometrial carcinoma undergoing postoperative chemotherapy. Int J Cancer 100:686-689, 2002

    Depowski PL, Rosenthal SI, Ross JS: Loss of expression of the PTEN gene protein product is associated with poor outcome in breast cancer. Mod Pathol 14:672-676, 2001

    Raffel C, Frederick L, O'Fallon JR, et al: Analysis of oncogene and tumor suppressor gene alterations in pediatric malignant astrocytomas reveals reduced survival for patients with PTEN mutations. Clin Cancer Res 5:4085-4090, 1999

    Hu TH, Huang CC, Lin PR, et al: Expression and prognostic role of tumor suppressor gene PTEN/MMAC1/TEP1 in hepatocellular carcinoma. Cancer 97:1929-1940, 2003

    White VA, Chambers JD, Courtright PD, et al: Correlation of cytogenetic abnormalities with the outcome of patients with uveal melanoma. Cancer 83:354-359, 1998

    Bedikian AY, Legha SS, Mavligit G, et al: Treatment of uveal melanoma metastatic to the liver: A review of the M.D. Anderson Cancer Center experience and prognostic factors. Cancer 76:1665-1670, 1995

    Kivela T, Suciu S, Hansson J, et al: Bleomycin, vincristine, lomustine and dacarbazine (BOLD) in combination with recombinant interferon alpha-2b for metastatic uveal melanoma. Eur J Cancer 39:1115-1120, 2003

    Dutcher JP: Mammalian target of rapamycin (mTOR) inhibitors. Curr Oncol Rep 6:111-115, 2004

查询更多Uveal相关信息在本站>>

《临床肿瘤学医学期刊》2006年1月第24卷第1期