Background Accurate survival prognostication for patients with uveal melanoma (UM) enables effective patient counselling and permits personalised systemic surveillance for the early detection of metastases and, in high-risk patients, enrolment in any trials of systemic adjuvant therapy. The aim of this work is to determine the success of prognostic UM tumour biopsy using an improved surgical approach and optimised sample handling workflow.
Methods Patients with UM treated by primary radiotherapy between 2011 and 2013 and who underwent a prognostic biopsy with cytology, multiplex ligation-dependent probe amplification and/or microsatellite analysis were included. The main outcomes and measures were success of cytology and genetic studies, and surgical complications.
Results The cohort comprised 232 patients with UM having a median age of 59 years (range, 25–82) at treatment. The median largest basal diameter was 11.4 mm (range, 4.1–20.8) and tumour height was 3.4 mm (range, 0.7–10.3). Ciliary body involvement was noted in 42 cases. Treatment consisted of Ru-106 brachytherapy in 151 cases (65%) and proton beam radiotherapy in 81 cases (35%). With improvements in surgical techniques and laboratory methods over time, cytology success increased from 92% (131/142) to 99% (89/90) and the numbers of samples with sufficient DNA for genetic testing increased from 79% (104/131) to 93% (83/89). Overall, chromosome 3 loss was noted in 64/187 (34%) cases. Surgical complications, including transient localised bleeding, vitreous haemorrhage and retinal perforation, decreased over time. Eight patients required additional surgery.
Conclusions Improved surgical techniques and laboratory methods yielded successful cytology and genetic information in the majority of cases.
Precis Analysis of data from 232 patients with uveal melanoma undergoing prognostic tumour biopsy demonstrated that improved surgical techniques and laboratory methods yielded successful cytology and genetic information in 99% and 89% of cases, respectively.
- uveal melanoma
- choroidal melanoma
- molecular analysis
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Uveal melanomas (UM) have been analysed genetically for survival prognostication since the late 1990s.1–5 Initially, tissue was processed from large UM samples, harvested after enucleation or local resection. Refinement of surgical and laboratory techniques enabled genetic analysis of tumour biopsies in patients undergoing eye-conserving treatment, such as plaque brachytherapy or proton beam radiotherapy (PBR).2–8 With experience, complications of intraocular biopsy are rare.4 8–10 Genetic typing of UM is successful in 85%–99% of cases.1 4 8 10 Biopsy failure is not only wasteful of resources but also distressing for all those involved and obviously for patients.11 12
The aims of this study were to determine (1) whether changes in the surgical and laboratory techniques over time improved results, (2) the incidence and type of complications in these patients and (3) the success rates of cytology and genetic typing of choroidal melanomas biopsied before or after radiotherapy at our centre between 2011 and 2013.
Materials and methods
Patients were included if treated for a choroidal melanoma with Ruthenium-106 (Ru-106) brachytherapy or PBR, and who underwent prognostic biopsy at the Liverpool Ocular Oncology Centre between January 2011 and June 2013. They were excluded if the tumour arose in the iris or if the biopsy was performed solely for diagnosis. The study closure end date of June 2013 allowed for a follow-up period of at least 3.5 years at analysis and also for the author MA to complete this study as part of her PhD thesis.
Tumours were diagnosed clinically and measured with B-scan ultrasound, as per standard practice. Tumours were biopsied trans-sclerally or transretinally depending on their size, location and treatment. Those treated with a Ru-106 plaque were biopsied either (a) trans-sclerally if they extended pre-equatorially and if the tumour thickness exceeded 2 mm or (b) transretinally if the tumour was considered inaccessible with this approach because it was small and/or posterior. With few exceptions, tumours treated with PBR were biopsied transretinally as soon as possible after completion of the radiotherapy, that is, on the last day of the radiotherapy or, if this was not possible, within 2 weeks. Transretinal biopsies were performed under local anaesthesia with a 25 G vitreous cutter, as previously described.9 Trans-scleral fine-needle aspiration biopsies (FNAB) were performed with a 25 G needle attached to a 20 mL syringe by flexible plastic tubing. From July 2012, trans-scleral FNAB of anterior tumours was replaced by incisional biopsy with Essen forceps.13 14 The deep sclerotomy was performed under a lamellar scleral flap, which was closed with histoacryl glue. The specimen was collected with the Essen forceps through a full thickness scleral cut down and the sample obtained by flushing into a sterile universal tube.
Any biopsy-related complications were recorded prospectively in a systematic fashion at the time of surgery and at the first postoperative examination. The electronic database was reviewed to document longer-term effects.
Before July 2012, we analysed several cytospin preparations with the May-Grunewald-Giemsa (MGG) stain and immunocytochemistry, using the remaining tissue for genetic analysis. From July 2012, only a single cytospin was prepared for the MGG stain, thereby retaining a larger sample for genetic studies. However, no genetic analysis was performed without prior morphological examination using MGG and confirmation of the presence of UM cells in the sample by an experienced cellular pathologist (SEC). Chromosomal analysis was performed by multiplex ligation-dependent probe amplification (MLPA; MRC-Holland) or, if the DNA yield was <100 ng, by microsatellite analysis (MSA). Methods for DNA extraction, quantification, quality control and MLPA or MSA have been described.6 In the case of a large epithelioid ciliary body melanoma with disomy 3 MLPA results occurring, we would perform additional MSA where possible to exclude the possibility of an isodisomy 3, as MLPA cannot detect this anomaly.
SPSS (IBM) V.20 (SPSS Science) was used for all statistical analyses, with statistical significance set at p<0.05.
Patients were grouped according to whether the biopsy was performed before or after July 2012, when we started using Essen forceps and when we stopped performing immunocytochemistry. The seventh edition of the American Joint Committee on Cancer tumour node metastasis (TNM) classification scheme was used for tumour size.15
This audit adhered to the tenets of the Declaration of Helsinki and was approved by the Royal Liverpool University Hospital (Ref 4031-11/129).
The cohort comprised 232 patients with choroidal melanoma (123 men, 109 women) having a median age of 59 years (range, 25–82) at treatment. The tumours had a median largest basal diameter and thickness of 11.4 mm (range, 4.1–20.8) and 3.4 mm (range, 0.7–10.3), respectively (figure 1), and ciliary body involvement was noted in 42 cases. Treatment consisted of Ru-106 brachytherapy in 151 cases (65%) and PBR in 81 cases (35%). Biopsy was performed before July 2012 in 142 patients (ie, ‘group 1’) and after this date in 90 patients (ie, ‘group 2’). There were no significant clinical differences between these two groups (table 1).
Intraoperative complications were recorded for 49/218 patients and are detailed in table 2, subdivided according to the surgical technique used. Follow-up of complications was possible in 200 patients, which had a median of 9.8 months (range, 4.2–50.0). Bleeding was the main surgical complication, which spontaneously resolved in all but six patients (3%) who underwent vitrectomy with no further problems. One patient, with a large tumour with exudative detachment at the time of treatment, underwent vitrectomy for non-clearing vitreous haemorrhage and persistent retinal detachment, but then chose enucleation over additional surgery in a non-seeing eye. One other case in this series underwent enucleation for relapse of peri-papillary melanoma. Overall, as can be seen in table 2, there was a decrease in the number of surgical complications over time.
Histopathology and genetic testing
Cytological assessment was possible for 131/142 (92%) cases in group 1 and 89/90 (99%) cases in group 2. Details of cytomorphology are shown in table 1.
Of the 131 confirmed UM cases in group 1, 104 (79%) had sufficient good-quality DNA for genetic testing; 17 transretinal samples and 10 trans-scleral FNAB yielded insufficient DNA. Six further cases were excluded from genetic testing, as the DNA concentration was below that required for MLPA and matched bloods were not available to perform MSA. Thus, 98/131 cases underwent genetic testing; 66 were analysed by MSA and 32 by MLPA. Fifty-one cases were reported as disomy 3 (D3) and 40 as monosomy 3 (M3); seven cases passed the quality assessment but could not be unequivocally classified as either D3 or M3.
Of the 89 confirmed UM cases in group 2, genetic analysis was possible in 83 (93%); two transretinal samples and four trans-scleral flap biopsies yielded insufficient DNA for genetic testing. Fifty-two cases were analysed by MSA and 31 by MLPA. Fifty-five cases were reported as D3 and 24 as M3. Four cases could not be clearly classified as either D3 or M3. Tumour thickness between those cases yielding sufficient DNA and those that did not was not significantly different (t-test, p=0.677).
In our retrospective audit, sufficient material for both cytological and genetic analysis was obtained initially in 73% (104/142) of all UM intraocular biopsies taken, but improved to 92% (83/90) of biopsies with modified surgical and laboratory-based technical approaches. This improvement was not dependent on tumour thickness: indeed, as shown in figure 1, most of the tumours in this cohort belong to the T1–T2 categories and have a reduced tumour thickness. Thin tumours are notoriously more challenging to biopsy.16 Overall, our success rate is slightly higher than figures previously reported for either cytological or genetic analyses alone, and may be due to differing surgical techniques and molecular tests performed.1 3 4 8
Another difference to previous studies1 5 10 17 18 is that we do not perform molecular genetic analyses on the specimen without cytomorphological examination, as we want to be sure that we are examining melanoma cells, and the presence of dense infiltrates of macrophages or leucocytes may affect the molecular genetic results, possibly providing false reassurances to the patients. Melanoma cells can be distinguished from other cell types on the basis of their cytoplasmic, nuclear and nucleolar features with high accuracy by experienced cytopathologists; only in rare cases of non-pigmented choroidal melanomas are confirmatory immunostains or guanine nucleotide-binding protein G(q) subunit alpha (GNAQ)/11 mutational tests required. Moreover, it has been long known that the presence of epithelioid melanoma cells is a poor prognostic factor,19 hence cytomorphological subtyping of the tumour cells (where possible) adds meaningful additional information to our predictive model. Other authors have recently highlighted the importance of cytomorphological examination of UM biopsies to ensure that the sample being tested is a melanoma.1 8 20–22
Our improved workflow and altered surgical technique particularly with the use of the Essen forceps through a lamellar scleral flap for the anteriorly placed choroidal melanomas (rather than the ‘blind’ FNAB approach) increased the numbers of samples undergoing genetic testing from 79% (104/131) to 93% (83/89). The Essen forceps have been especially designed for intraocular tumour biopsy. The standard use is through sutureless 23-gauge three-port vitrectomy, as first described by Akgul et al.13 The novelty introduced by author BED and used for all patients in group 2 undergoing transcleral biopsy is their usage through a fine scleral opening under a lamellar scleral flap, allowing direct access to the tumour with resultant increased tissue yield.14
This improved yield in turn increased the number of patients for whom genetic analysis was undertaken and an individualised prognostication curve generated using the ‘Liverpool UM Prognosticator Online’ (LUMPO), which also incorporates clinical, histological and genetic parameters.23 LUMPO was recently validated in a retrospective study using a cohort of 390 patients treated between 2002 and 2007 for choroidal melanoma at University College San Francisco.24 As for any study on prognostic biopsies in UM, one of the potential criticisms is that a single biopsy may be subject to sampling error for monosomy 3, as a result of intratumoral genetic heterogeneity.25 While this is an important consideration, previous data of ours suggest that a biopsy provides concordant results to a larger sample.6 In addition, we are aware that rare cases of isodisomy may be missed by MLPA and thus consideration of the clinical and histological characteristics of the tumour will also determine whether MLPA and/or MSA are performed.
A key concern when performing biopsy is the possibility of tumour seeding. We observed no local dissemination with extraocular recurrence in the cases analysed, although we recognise that the follow-up time in this cohort is short. The main complication associated with the surgical procedures performed was transient subretinal or peri-lesional bleeding requiring no further intervention, as observed by others.4 5 8 9
In conclusion, prognostic biopsy for UM when undertaken in experienced hands is a safe procedure that can yield useful information in >90% of cases. Using a variety of surgical techniques, depending on the size and location of the tumour, improves the chances of obtaining an adequate specimen. Cytomorphological examination of a cytospin from the specimen is essential to determine the actual composition of the sample and reduce the risk of a falsely reassuring prognosis. Using a combination of molecular prognostic techniques maximises the chance of providing a result from the biopsy specimen. A close collaboration between the clinical and molecular pathology team is essential to optimise the success of the overall process and provide the patient with prognostic information for meaningful discussions regarding further management.
Cancer Research UK funded the first author’s PhD, and this study was part of her PhD thesis, which was successfully awarded in 2015. The funding organisation had no role in the design or conduct of this research.
Contributors All of the authors have been involved in the following: substantial contributions to the conception or design of the work; the acquisition, analysis or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; final approval of the version to be published; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy of integrity of any part of the work are appropriately investigated and resolved.
Competing interests None declared.
Patient consent Obtained.
Ethics approval RLBUHT, Ref 4031-11/129.
Provenance and peer review Not commissioned; externally peer reviewed.
Correction notice This paper has been amended since it was published Online First. Owing to a scripting error, some of the publisher names in the references were replaced with 'BMJ Publishing Group'. This only affected the full text version, not the PDF. We have since corrected these errors and the correct publishers have been inserted into the references.