Imaging, Diagnosis, Prognosis Genotypic Profiling of 452 Choroidal Melanomas with Multiplex Ligation-Dependent Probe Amplification

Purpose:Metastasis from uvealmelanoma occurs almost exclusively with tumors showing chromosome 3 loss.We usedmultiplex ligation-dependent probe amplification (MLPA) to detect chromosome 1p, 3, 6p, 6q, 8p, and 8q abnormalities in uveal melanomas. The purpose of this study was to correlate our MLPA results with other risk factors and metastatic death. Experimental Design: Patients were included if they had a uveal melanoma involving choroid. Correlations between baseline risk factors were analyzed using the chi-square test (without Yates’s adjustment) and the Mann–Whitney test, with log-rank analysis for associations with metastatic death. Results: The patients (194 female; 258 male) had a median age of 59.4 years and a median follow-up of 1.89 years. MLPA abnormalities occurred in a wide variety of combinations. Ten-year disease-specific mortality was 0% in 133 tumors with no chromosome 3 loss, 55% in tumors with chromosome 3 loss but no chromosome 8q gain, and 71% in 168 tumors showing combined chromosome 3 loss and 8q gain. In tumors with both these abnormalities, epithelioid melanoma cytomorphology, closed loops, and high mitotic rate correlated with poor survival as did lack of chromosome 6p gain. Conclusions: These results support the use of MLPA for routine clinical prognostication, especially if the genetic data are considered together with clinical and histologic risk factors. We showed a wide variety of MLPA results, which suggests that chromosomal abnormalities in uveal melanoma accumulate in a variable sequence. Clin Cancer Res; 16(24); 6083–92. 2010 AACR. Approximately 90% of all uveal melanomas involve the choroid. Without timely treatment, these tumors result in a blind and painful eye. Despite successful ocular treatment, about 50% of patients die of metastatic disease, which usually involves the liver (1). Estimation of survival probability tends to be based on clinical features, particularly largest basal tumor diameter, tumor thickness, ciliary body involvement, and extraocular spread (2–4). These tumor characteristics form the basis of the 7th edition of the UICC/AJCC TNM staging system (5). Several pathologic predictors are recognized, the most widely used comprising: presence of epithelioid cells; high mitotic count; low HSP27 staining score; and the presence of PASþ closed connective tissue loops (6–9). In 1996, Prescher et al. showed a strong correlation between chromosome 3 loss and metastatic death (10). Since then, other chromosomal abnormalities have been shown to correlate with poor prognosis and these include: 8q gain; 8p loss; 1p loss; 6q loss and lack of 6p gain (11–13). White et al. suggested that chromosome 3 loss correlated with metastatic death only if combined with chromosome 8 gain (henceforth, summarized by us as "C38A" for "chromosome 3 and 8 abnormality"; ref. 11) Gene expression profiling has identified class 2 tumors with high risk of metastasis (14). In 1999, we started offering genetic tumor typing to all patients treated by local resection or enucleation, using fluorescence in situ hybridization (FISH; 15). This method lacked sensitivity, however, so that small chromosomal abnormalities were missed (16). To some extent, this problem was alleviated by neural networks we developed, which analyzed clinical, histologic, and genetic risk factors (17). In 2006, we replaced FISH with multiplex ligationdependent probe amplification (MLPA), which examines for genetic gains and losses by means of a multiplex polymerase chain reaction (PCR refs. 18, 19). We used a kit specifically designed for uveal melanoma (SALSA P027 (B1; MRC-Holland). This comprises 12 control probes and test probes directed at 7 loci on chromosome 1, 13 loci on chromosome 3, 6 loci on chromosome 6, and 5 loci on chromosome 8. We previously evaluated MLPA with 73 choroidal melanomas and found good correlations with survival (19). Important findings from that study were that chromosome 3 loss and chromosome 8q gain Authors' Affiliations: Liverpool Ocular Oncology Centre, St. Paul's Eye Clinic, Royal Liverpool University Hospital; and Department of Pathology, University of Liverpool, Liverpool, United Kingdom Corresponding Author: Bertil Damato, Ocular Oncology Service, Royal Liverpool University Hospital, Prescot St, Liverpool L7 8XP, United Kingdom. Phone: 44-(0)-151-7063973; Fax: 44-(0)-151-7065436; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-10-2076 2010 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 6083 were associated with increased risk of metastasis even when the MLPA values showed only borderline, or equivocal, abnormality. Such borderline MLPA abnormality was subsequently found to reflect intratumoral melanocytic clonal heterogeneity (20). Apart from increased sensitivity, MLPA had the additional benefit of requiring smaller samples, thereby making it possible to test tiny tumor specimens obtained by transscleral fine-needle aspiration biopsy or transretinal biopsy conducted with a 25-gauge vitreous cutter. Since 2007, therefore, we have also conductedMLPA on biopsies of uveal melanomas treated by radiotherapy or phototherapy. The aims of this study were to determine the prevalence of chromosomal abnormalities in choroidal melanomas, using MLPA, and to correlate these results with other survival predictors and metastatic death. Patients and Methods Patients Patients were included in the main analysis if treated for uveal melanoma and if MLPA data were available. They were excluded if: (1) the patient was not resident on mainland Britain; (2) both eyes had melanoma; (3) the tumor did not involve choroid or (4) the patient received primary treatment elsewhere or was not initially treated. We also included 68 patients treated between January 1998 and February 2000 and who were included in previous studies (19). This study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines. Consent for the use of tissues and data for research was obtained from all patients. Institutional Review Board/ Ethics committee approval was not required, this study being an audit of our routine clinical practice. Clinical methods Full ophthalmologic examination was conducted. Largest basal tumor diameter and tumor thickness were measured by B-scan echography (Eyecubed; Ellex). Systemic clinical examination was performed routinely with preoperative liver ultrasonography if the largest basal tumor diameter exceeded 16 mm. Postoperatively, patients were referred to an oncologist for systemic screening only if MLPA showed an increased risk of metastasis. The first choice of treatment was radiotherapy, delivered with a 106-ruthenium plaque or proton beam (21). Transscleral local resection was construction if the tumor was too bulky for radiotherapy. Transretinal endoresection was undertaken only if this offered the last chance of conserving useful vision. Enucleation was conducted if the patient was not motivated to undergo eye-conserving therapy or if such therapy was unlikely to succeed. Transpupillary thermotherapy was administered as a primary treatment only exceptionally, and was usually reserved for exudation after radiotherapy. Tumor sampling As soon as possible after local resection or enucleation, a fresh tumor sample was harvested by the pathologist (SEC) for genetic studies. The remainder was formalinfixed and embedded in paraffin for routine histopathologic and immunohistochemical analyses, as previously described (16). Tumors treated by ruthenium plaque brachytherapy were sampled by transscleral fine-needle aspiration biopsy, which was conducted just before ruthenium plaque insertion. With proton beam radiotherapy, transretinal biopsy was conducted as soon as possible after completion of the radiotherapy. Each tumor was biopsied twice, with 1 sample sent for histology and the other for the genetic analysis. Histopathologic examination The diagnosis of melanoma was confirmed using sections stained for hematoxylin and eosin (H&E) and, if necessary, immunohistochemically using Melan A (16). Spindle and epithelioid cell types were assessed using the modified Callender system (7). Extravascular matrix patterns were assessed using the periodic acid–Schiff (PAS) reagent, without counterstaining, to facilitate recognition of closed loops, as previously reported (16). The mitotic count was measured by counting the number of mitoses in 40 high-power fields (HPF) in the H&E sections (16). Assessments for closed connective loops and mitotic count were not possible with biopsy specimens. MLPA technique DNA extraction, DNA quality assessment and quantification, and MLPA were conducted as previously reported (18, 19). Briefly, the frozen uveal melanoma Translational Relevance Uveal melanomas are the most common primary intraocular malignancy, with more than 90% of these involving choroid. About 50% of patients with this cancer die of metastatic disease, which occurs almost exclusively in patients whose tumor shows chromosome 3 loss and chromosome 8q gain. Almost all patients whose tumor shows this lethal combination of chromosomal abnormalities die of uveal melanoma. For these reasons, genetic tumor typing is essential for prognostication; however, methods such as FISH are insensitive and more sophisticated tests are unaffordable for routine use in many centers. We therefore applied multiplex ligation-dependent probe amplification (MLPA) to uveal melanomas and have now been using this in routine clinical use for several years. This study shows that our MLPA results are highly predictive of survival, especially if the results are assessed in combination with histologic risk factors. MLPA of choroidal melanomas can therefore be applied in clinical practice. Damato et al. Clin Cancer Res; 16(24) December 15, 2010 Clinical Cancer Research 6084 samples and controls consisting of normal choroidal tissue were collected in microfuge tubes. Tissue lysis and protein digestion were conducted. Samples were incubated overnight at 56 C on Thermomixer comfort (Eppendorf). Fresh Proteinase K was added after 16 hours of incubation and samples were incubated for additional 4 hours. Genomic DNA was isolated using a high salt concentration and ethanol precipitation. DNA was dissolved in 20–100 mL of TE buffer, depending on pellet size. The DNA concentration and absorbance were measured with the NanoDrop Spectrophotometer (Thermo Scientific). Multiplex PCR was adapted from Dongen et al. (22). It was carried out on samples with a concentration exceeding 40 ng/mL to assess DNA quality using a Techne TC-412 thermal cycler (Techne). PCR products were visualized on a 2% agarose gel (150 mA for 30 minutes) stained with 1x SYBR Safe (Invitrogen), using the Bio Doc-It Imaging System (Ultra-Violet Products Ltd). The MLPA procedure and capillary electrophoresis were conducted as previously reported using the SALSA P027.B1 Uveal Melanoma Kit for 432 tumors. Briefly, 6 nontumor controls were used in each MLPA assay. MLPA reactions were conducted using a G-Storm GS1 thermal cycler (Gene Technologies Ltd) with fragment detection being conducted using the ABI-3130XL Genetic Analyzer and GeneMapper software (Applied Biosystems). Raw data were received as peak heights, as a measure of peak intensity, for each of the 43 probes (31 test probes and 12 control probes). MLPA was conducted in triplicate for each UM sample. The analysis was conducted using an adapted version of the Excel spreadsheet designed by the National Genetics Reference Laboratory (NGRL; http:/www.ngrl.org.uk/Manchester/mlpapubs.html), as described previously (19). We modified this method to exclude UM control loci that seemed abnormal (1920). The MLPA data were considered as reliable if 7 or more control probes were within the normal range. The dosage quotient (DQ) was categorized as suggested by the NGRL as: deletion (D), 0.65; borderline loss (B), 0.65–0.84; normal 1⁄4 diploid (N), 0.85–1.14; borderline amplification (Q), 1.15–1.35; and amplification (A) 1.35. Chromosomes were considered to be abnormal if any loci showed reproducible borderline or definite gain or loss, with such abnormality being described as partial or total according to whether any or all of the loci were abnormal. Statistical analyses Clinical, pathologic, and cytogenetic data were computerized into a customized database prospectively. Tumors were categorized as involving ciliary body if they extended anterior to ora serrata and were recorded as having epithelioid cells irrespective of the proportion of such cells in the tumor. Extraocular extension was recorded as being present irrespective of whether this was noted clinically or histopathologically. We notified the NHS Cancer Registry of all newly diagnosed patients with ocular melanoma. These were flagged at the Registry, which informed us automatically of the date and cause of any deaths. If we did not receive such information about any patient by the close of the study, we assumed that the patient was alive. Because of our reliance on the NHS Cancer Registry, patients from overseas were excluded from follow-up studies. Depending on the wording of the death certificate, death frommetastatic disease was coded as definitely or probably caused by uveal melanoma unless another primary malignancy was specified as the cause in the death certificate. Follow-up time was estimated from the time of treatment to the date of death or to the 18th March 2010, when the data were downloaded. Data analysis was done using a statistical program (SPSS; SPSS Inc.). Correlations between baseline risk factors were analyzed using the chi-square test (without Yates’s adjustment) and the Mann–Whitney test. The Kaplan–Meier method was used to compute metastatic mortality and groups were compared using the log-rank test. A P value of less than 0.05 was considered to be statistically significant. All statistical tests were 2sided.