Prognostication in UM

Transcription

1 Prognostication in UM

2 Prognostication in UM 1. Is there a preferred prognostic tool? 2. What is the role of prognostic biopsy?

3 Prognostic parameters in uveal melanoma Clinical Histomorphological Immunohistochemical Genetic (somatic alterations) Serological Combined

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10 Clinical prognostic parameters in UM Age Gender Tumour height Largest basal diameter Ciliary body involvement Extraocular melanoma growth (PET/CT) (Confocal indocyanine green angiography)

11 Age Seddon, J.M., et al., Arch. Ophthalmol., Retrospective, 267 patients, FU 17 years. Folberg R et al., 1993: Ophthalmology; retrospective, 234 choroidal melanomas, nine vascular patterns; age. The COMS randomized trial of iodine 125 brachytherapy for choroidal melanoma: V. Twelve-year mortality rates and prognostic factors: COMS report No. 28. Arch Ophthalmol. 2006; Retrospective, 1317 patients treated with plaque. Virgili, G et al Survival in patients with uveal melanoma in Europe. Arch. Ophthalmol.; Retrospective study; 5788 patients, 32 cancer registries, 16 European countries, FU 15 years. Shields, C.L. et al., Metastasis of uveal melanoma millimeter-by millimeter in 8033 consecutive eyes. Arch. Ophthalmol.; retrospective; 7256 eyes with choroidal melanoma. Gender Folberg R et al., 1993: Ophthalmology; retrospective, 234 choroidal melanomas, nine vascular patterns; males. Virgili, G et al Survival in patients with uveal melanoma in Europe. Arch. Ophthalmol.; Retrospective study; 5788 patients, 32 cancer registries, 16 European countries, FU 15 years. Damato and Coupland, patients, retrospective; differences in tumour location and size. Tumour height Shields, C.L. et al., Metastasis of uveal melanoma millimeter-by millimeter in 8033 consecutive eyes. Arch. Ophthalmol.; retrospective; 7256 eyes with choroidal melanoma, the mean tumor thickness was 5.5 mm and metastasis occurred in 8%, 15%, and 25% at 3, 5, and 10 years, respectively. Shields CL et al., 2013; 7731 patients; Retrospective, interventional case series; Compared with uveal melanoma classified as T1, the rate of metastasis and death was 2 times greater for T2, 4 times greater for T3, and 8 times greater for T4. Largest basal diameter Seddon, J.M., et al., Arch. Ophthalmol.; Retrospective, 267 patients, FU 17 years. Folberg R et al., 1993: Ophthalmology; retrospective, 234 choroidal melanomas, nine vascular patterns; tumour size The COMS randomized trial of iodine 125 brachytherapy for choroidal melanoma: V. Twelve-year mortality rates and prognostic factors: COMS report No. 28. Arch Ophthalmol. 2006; Retrospective, 1317 patients treated with plaque. Damato, B., Coupland, S.E., A reappraisal of the signicance of largest basal diameter of posterior uveal melanoma. Eye; retrospective; 1776 patients; FU 5 yrs; The LTD was greater in older patients (t-test, P<0.001). >LTD > greater metastatic death. Shields CL et al., 2013; 7731 patients; Retrospective, interventional case series; Compared with uveal melanoma classified as T1, the rate of metastasis and death was 2 times greater for T2, 4 times greater for T3, and 8 times greater for T4. Shields, C.L. et al., Metastasis of uveal melanoma millimeter-by millimeter in 8033 consecutive eyes. Arch. Ophthalmol.; retrospective; 7256 eyes with choroidal melanoma.

12 Ciliary body involvement Seddon, J.M., et al., A prognostic factor study of disease-free interval and survival following enucleation for uveal melanoma. Arch. Ophthalmol. 101, 1894e1899 Damato, B., Legacy of the collaborative ocular melanoma study. Arch. Ophthalmol. 125, 966e968 Damato, B., Coupland, S.E., A reappraisal of the signifcance of largest basal diameter of posterior uveal melanoma. Eye; retrospective; 1776 patients; FU 5 yrs; >LTD > greater association with ciliary body involvement. Shields, C.L. et al., Metastasis of uveal melanoma millimeter-by millimeter in 8033 consecutive eyes. Arch. Ophthalmol.; retrospective; 7256 eyes with choroidal melanoma. Extraocular melanoma growth Kujala et al., JCO, 2013; 7,369 patients; extraocular extension (EXE) was analyzed among 5,403 patients; EXE exceeding 5 mm in largest diameter carried a worse prognosis than smaller extensions (P <.001) Coupland et al., Ophthalmology, 2008; 847 patients; EXE in 124; Extraocular spread correlated with anterior tumor extension, large basal tumor diameter, epithelioid cellularity, closed loops, high mitotic rate, and monosomy 3; Extraocular spread correlated with increased mortality. (18-FDG PET/CT) Lee CS et al; BJO, 2011; retrospective review; 40 patients; 1 yr FU; inverse relationship btwn tumour uptake and time to death Reddy et al., BJO; 2005; 50 consecutive patients; The smallest tumour physiologically identifiable by PET/CT had basal dimensions of 3x5.9 and an apical height of 2.9 mm; PET/CT was most effective in detecting the physiological activity of AJCC-T3 and large choroidal melanomas. Calcagni ML, 2013, prospective study; 34 patients, comparison of standardized uptake value (SUV) and/or metabolic rate of glucose (MRglu); comparison with cell type; MRglu is useful for distinguishing the different cell types in uveal melanoma, as well as high-risk from low-risk lesions, while SUV is not; FU too short - role in prognosis? (Confocal indocyanine green angiography) Mueller et al. Ophthalmol. 2002; 98 patients; Observational case series, prospective, non-randomized; Time to growth of the tumor; 30%; parallel with cross-linking MCP (P < 0.001), arcs with branching MCP (P = 0.006), loops (P < 0.001), and networks (P < 0.001) associated with tumour growth.

13 Histopathological prognostic parameters in UM Cell type Mitotic count Mean diameter of ten largest nucleoli Presence of extravascular matrix patterns Microvascular density Extraocular melanoma growth

14 Cell type McLean, I.W., Zimmerman, L.E., Evans, R.M., Reappraisal of Callender s spindle a type of malignant melanoma of choroid and ciliary body. Am. J. Ophthalmol.; 105 melanomas re-evaluated; 3 subgroups. COMS Report 21. Arch Ophthalmol 2003; Kujala E, Mäkitie T, Kivelä T. Invest Ophthalmol Vis Sci. 2003;44: Mäkitie T, et al. IOVS Retrospective; 167 uveal melanomas; both MVD and microvascular patterns contribute independently to prognosis in uveal melanoma in addition to cell type and size of the tumour. Mitotic count Folberg R et al., 1993: Ophthalmology; retrospective, 234 choroidal melanomas, nine vascular patterns; mitotic count COMS Report 21. Arch Ophthalmol 2003; Coupland SE, Hiscott P, Smith P & Damato B Melanoma Res. Retrospective study; 864 choroidal melanomas; median FU = 4 years; increased mitotic counts correlated with other poor prognostic factors; correlated with higher mortality rates, even in absence of cytogenetic data. Onken MD, et al. Curr Eye Res. 2010: retrospective analysis, 28 uveal melanomas, 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). Mean diameter of ten largest nucleoli McLean IW, Keefe KS, Burnier MN. Ophthalmology. 1997; 496 posterior uveal melanomas; at 15 years, survival decreased from 67.5% to 33.8% when complete loops were present. Al-Jamal RT, Kivelä T. Curr Eye Res. 2006; retrospective; 167 uveal melanomas, Ki-67 score, MLN, and presence of extravascular loops and networks were independent predictors of melanoma-related mortality. Presence of extravascular matrix patterns (loops) Folberg R et al., 1993: Ophthalmology; retrospective, 234 choroidal melanomas, nine vascular patterns; closed loops & parallel cross linking. McLean IW, Keefe KS, Burnier MN. Ophthalmology. 1997; 496 posterior uveal melanomas; at 15 years, survival decreased from 67.5% to 33.8% when complete loops were present. Microvascular density (MVD) Foss AJE, et al. Cancer Res Retrospective, 123 cases, median, 117 months; together with tumour size, significant on multivariate analysis. Mäkitie T, et al. IOVS Retrospective; 167 uveal melanomas; both MVD and microvascular patterns contribute independently to prognosis in uveal melanoma in addition to cell type and size of the tumour Chen et al., IOVS, 2002; retrospective, 200 choroidal melanomas, Extraocular melanoma growth (see above)

15 Immunohistochemical prognostic parameters in UM Cellular proliferation markers Tumor-infiltrating lymphocytes Tumor-infiltrating macrophages (HLA Class I expression) (HSP-27 expression) (IGF1-R)

16 Cellular proliferation markers Seregard S et al. 1998; retrospective; PC-10 count Al-Jamal RT, Kivelä T. Curr Eye Res. 2006; retrospective; 167 uveal melanomas, Ki-67 score, MLN, and presence of extravascular loops and networks were independent predictors of melanoma-related mortality. Angi M et al. Acta Ophthalmol. 2011; retrospective, 132 choroidal melanomas; Ser-10 (PHH3) count Tumor-infiltrating lymphocytes Whelchel et al. IOVS, 1993; 34: de Waard-Siebinga et al. Graefe s Arch Clin Exp Ophthalmol. 1996, 234:34-42 Folberg R et al., 1993: Ophthalmology; retrospective, 234 choroidal melanomas. Mougiakakos et al, Cancer, 2010; 116: Jager et al., Prog. Retin Eye Res, 2011,30: Tumor-infiltrating macrophages COMS. Am J Ophthal.,1998, 125: Tobal et al., Melanoma Res. 1993; 3:63-5 de Waard-Siebinga et al. Graefe s Arch Clin Exp Ophthalmol. 1996, 234:34-42 Mäkitie et al., IOVS, 2001, 42: Maat et al. IOVS, 2008, 49: Jager et al., Prog. Retin Eye Res, 2011, 30: Herwig MC et al., Exp Eye Res Feb;107:52-8 (HLA Class I expression) Blomm et al., IOVS, 1997; retrospective, 30 UM; lack of expression of HLA-A and -B antigens correlated with a better patient survival Anastassiou G et al. Invest Ophthalmol Vis Sci (HSP-27 expression) Jmor F et al., Acta Ophthalmol. 2012; retrospective, 99 uveal melanomas; Low HSP-27 expression correlates with monosomy 3 in uveal melanoma and with increased predicted mortality (IGF1-R) All-Ericsson C et al., 2002; IOVS; retrospective, 36 uveal melanomas Al-Jamal RT, Kivelä T. Can J Ophthalmol Retrospective, 167 uveal melanomas, IGF-IR did not independently predict metastasis

17 Molecular testing for prognostication in UM Prescher G, Bornfeld N, Hirche H, Horsthemke B, Jockel KH, Becher R. Prognostic implications of monosomy 3 in uveal melanoma. Lancet. 1996; 347(9010):1222-5

18 Molecular prognostication techniques Karyotype FISH MLPA MSA acgh GEP asnp

19 Comparing various methods for UM subtyping Cytoge netics Marker Chr. 3 copy number Costs/ complexity FISH Chr. 3 copy number MSA Essen Chr. 3,8 LOH MLPA Liverpool Chr. 1,3,6,8 dosage Array Expression profile low low very high ( 1000 $) Equipment Microscope Sequencer Array scanner GEP Fluidics 15 gene expression? Real-time PCR Reliability Sensitivity Material Live cells Tissue/ cells DNA 1-20 ng DNA 150 ng RNA ng Convenience RNA Paraffin tissue No Yes Possible Possible but not preferred

20 Conventional typing only: i.e. karyotyping and CGH Authors Year Method Pts Ch 3 Chr 8 Outcome Comment Prescher et al 1996 Cytogenetics (Karyotyping) CGH 54 (16) (30) 8 (both) Yes No Median FU = 3.4 yrs Fresh samples 17 (57%) had loss of chromosome 3 No additional value of age, sex, EOM, tumour thickness No patient deaths in disomy 3 tumours Univariate analysis: Monosomy 3 strongest predictor of metastasis; followed by tumour location and LBD

21 Karytoyping and FISH Authors Year Method Pts Ch 3 Chr 8 Samples/Outcom e Sisley et al 1997 Karyotyping FISH Yes Yes Fresh samples Median of 31 months Comment Cytogenetics may be of value in prognostication in UM M3 in 21 (50%) of UM Reduced survival correlated with: CB involvement Patel et al FISH 33 Enucleation specimens 16 UM with M3 14 deaths by study end No relationship btwn age, Tu size or location, or morphology Possibility also exists for missing genetic changes because of the small number of cells studied Cut-off ca. 30% Sisley et al 2006 Multiplex- FISH 24 Yes Yes FU: 5-55 months (median 27) 1 metastatic death- M3 P8 Frequency of chr. 6 changes 60% Alterations of chromosomes 1, 6, 8, 11, 15 and 18 can be more fully classified using M-FISH

22 FISH only Authors Year Method Pts Ch 3 Chr 8 Outcome Comment Scholes et al Karotyping FISH 105 Yes No Local resection (n=25) or enucleation (n=74) Frozen samples Interrelationship btwn particular prognostic parameters apparent; idea of prognostic index Follow-up time: 3yrs 16 patients had died at study closure Significant assoc btwn M3 and: Increasing LBD Ciliary body involvement Epithelioid cellularity Damato et al 2007 FISH 356 Yes CEP BCL6 Yes Median follow-up time was 2.23 years 76 patients had died Significantly assoc btwn M3 and: Increasing LBD CB involvement Epithelioid cellularity Monosomy 3 was present in 23% of small UM (<10 mm in diameter) 15% false negative rate

23 FISH only Authors Year Method Pts Ch 3 Chr 8 Outcome Comment Scholes et al Karotyping FISH 105 Yes No Local resection (n=25) or enucleation (n=74) Frozen samples Interrelationship btwn particular prognostic parameters apparent; idea of prognostic index Follow-up time: 3yrs 16 patients had died at study closure Significant assoc btwn M3 and: Increasing LBD Ciliary body involvement Epithelioid cellularity Damato et al 2007 FISH 356 Yes CEP BCL6 Yes Median follow-up time was 2.23 years 76 patients had died Significantly assoc btwn M3 and: Increasing LBD CB involvement Epithelioid cellularity Monosomy 3 was present in 23% of small UM (<10 mm in diameter) 15% false negative rate

24 FISH and UM: Liverpool experience N=356 Cytogenetics A: 3c & 8q B: 3c & 8q+ C: 3c- & 8q D: 3c- & 8q+ Cytogenetics of uveal melanoma: a 7-year clinical experience. Damato B, Duke C, Coupland SE, et al.: Ophthalmology Oct; 114(10) :

25 FISH only in FNAB Authors Year Method Pts Ch 3 Chr 8 Samples/Outcom e Midena et al 2008 FISH 32 Yes No FNAB Median FU = 47 months Mean LBD = 12.5mm Comment M3: No correlation with tumour size or location Sufficient yield in 81% 17 (65%) with M3 5 (15%) metastatic deaths 1. Is there a preferred prognostic tool? o For enucleation/local resection specimens? o For intraocular biopsies?

26 FISH only enucleated eye using biopsy techniques Authors Year Method Pts Ch 3 Chr 8 Samples/Outcom e Naus et al 2008 FISH 40 Yes Yes FNAB Comment Chrom 6 analysis also Concordance between FISH biopsy and enucleated eye No follow-up data

27 MSA Authors Year Method Pts Ch 3 Chr 8 Samples/ Outcome Tschenscher et al MSA CGH 30 (20) Yes No Fresh No clinical outcome Comment Good concordance for chromosome 3 but not for 6 and 8. Thomas et al 2012 MSA 373 Yes No Enucleated (n=160) Endoresection (n=51) Biopsy (n=149) Median follow-up time =? Longest 10 yrs Variable proportion of M3 per specimen type Since examining chrom. 8 using MSA 57 patients had died M3 significantly assoc with: LBD

28 MSA only FNAB Authors Year Method Pts Ch 3 Chr 8 Outcome Comment Shields et al. (Trans Am Ophthalmol Soc.) (Arch Ophthal) 2007 MSA 140 Yes No Median follow-up time = 8 months M3 in 44 cases (31%) D3 in 76 cases (54%) Insufficient yield in 20 (14%) M3 found in both small & large UM M3 significantly assoc with: LBD Distance from the disc Transient vitreous haemorrhage No seeding or recurrence Deaths from metastasis not mentioned

29 MLPA Authors Year Method Pts Ch 3 Chr 8 Samples/Outcom e Damato et al 2009 MLPA FISH 73 9 Yes Yes Mean age: 60 yrs Median FU = 6.2 yrs Fresh samples Enucleation or resection Metastatic death in 28 pts Correlated with M3 and P8 Comment Chrom 1,3,6,8 Partial deletions detected with MLPA not seen with FISH Significance of partial deletions and borderline alterations of chrom. 3 Damato et al 2010 MLPA 452 yes yes Median follow-up time was 1.89 years 57 patients had died Significantly assoc with: LBD Monosomy 3 Polysomy 8q Combination of both Epithelioid cellularity High mitotic count Closed loops 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

30 MLPA vs Other techniques Authors Year Method Pts Ch 3 Chr 8 Samples/Outcom e Coupland et al 2012 ARVO abstract MLPA vs FISH MLPA vs acgh Yes Yes Comment yes Fresh and FFPE See extra slide MLPA vs MSA MLPA vs MLPA 24 Yes MSA* Low risk High risk Choroidal melanoma MLPA*

31 Results: Comparative quality assessment of MLPA MLPA vs FISH (Liverpool) n = 44 UM MLPA vs array CGH (Liverpool) n = 32 UM MLPA (Liv) vs MSA (Liverpool) n = 24 UM MLPA/MSA (Liv) vs MLPA/MSA (Essen) n = 12 UM FISH MLPA Disomy Partial del Monosomy Polysomy acgh MLPA Disomy Partial del. 9 8 Monosomy Polysomy MLPA MSA Disomy Monosomy MLPA/MSA-L MLPA/MSA -E Disomy Monosomy 3 6 6

32 MLPA versus FISH Authors Year Method Pts Ch 3 Chr 8 Outcome Comment Vaarwater et al 2012 MLPA vs FISH asnp 64 7 yes yes Median follow-up time: 1.89 yrs Fresh samples Loss of chromosome 3, loss or gain of 8p, and gain of 8q, found with MLPA, correlated with a significantly lower diseasefree survival (P<0.001) Sensitivity of MLPA to detect patients at risk for metastatic disease was higher than that of FISH (0.795 vs ) but the specificity was equal for both techniques (0.840).

33 SNP house designed Authors Year Method Pts Ch 3 Chr 8 Outcome Comment Onken et al SNP-custom designed (28 SNPs) 53 Yes, only No Fresh samples FU period: 25 months Lab-based SNP tool has not been externally validated FISH acgh Metastatic deaths in low risk: 10% with their SNP analysis, 25% with achg, Was a proof-ofprinciple study 35% with FISH

34 SNP array off the shelf Authors Year Method Pts Ch 1,3 Ewens et al July 2 nd SNP array, 5.0 or 6.0 Affymetrix, Santa Clara CA 254 biopsies 66 enuc Ch 6, 8 Outcome 320 Yes, Yes 60 pts developed mets Most within 48 months Significantly associated with poor metastatic outcome (univariate): complete or partial loss of chromosome 3 1p-loss 6q-loss 8p-loss 8q-gain Comment Limited discordancy between biopsy and enucleation. Loss of chromosome 3 is necessary, but not sufficient for UM metastasis and other independent changes in 1p and 8p contribute significantly. Significantly associated with poor outcome (multivariate): Loss of chromosome 3 Loss of chr. 1p Loss of 8p Predictive of metastasis: Gender (male) LTD TNM Stage

35 FISH versus asnp Authors Year Method Pts Ch 3 Chr 8 Specimens/Outco me Young T et al 2007 FISH vs asnp asnp (Illumina) 59 Yes CEP only Yes Biopsy cases only? Median follow-up time? patients with mets & died 49 (83%) of the cases yielded adequate DNA Comment Additional chromosomes (e.g. 1,4, 6, 9,11,16) examined using asnp Cut-off used for FISH not reported but 20% indicated from Fig. 1 38/59 (64%) cases with monosomy 3 revealed by FISH 43/59 (73%) cases with chr. 3 loss by asnp

36 FISH versus asnp Authors Year Method Pts Ch 3 Chr 8 Outcome Comment Singh et al 2012 FISH vs asnp ssnp (Illumina) 50 Yes CEP only no Enucleation cases only Median follow-up time was 35.5 months 30 cases with monosomy 3 revealed by FISH 31 cases with chr 3 loss by asnp No correlation with clinical or morphological features Emphasis placed on cut-off of percentage of cells with monosomy 3 for definition (8-20%) 17 (52%) patients with mets & died

37 GEP versus acgh Authors Year Method Pts Ch 3 Chr 8 Outcome Comment Petrausch 2008 GEP vs acgh 28 Yes Median FU = 28 months 3-7 yrs Enucleation or excision 12 developed mets Monosomy 3 strong risk factor for metastatic spread Two gene sets compared for GEP Both subdividing into two groups using clustering techniques

38 GEP versus SNP home made or acgh Authors Year Method Pts Ch 3 Chr 8 Outcome Comment Onken et al 2012 GEP vs SNP tool 459 (260 ) Yes n/a Median follow-up time was 17.4 years Metastasis was detected in 3 class 1 cases (1.1%) and 44 class 2 cases (25.9%) TNM staging An association between GEP class 2 and monosomy 3 54 of 260 tumors (20.8%) were discordant for GEP and chromosome 3 status Claims: 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. (Kivela response*) See later slide re TNM

39 GEP versus SNP assay 28 SNP assay

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41 GEP versus MLPA Authors Year Method Pts Ch 3 Chr 8 Samples/Outcome Comment Coupland et al 2013 ARVO abstract Manusc ript submitt ed BJO GEP vs MLPA 20 Yes Yes FFPE Enucleated eyes TNM staging Blind analysis LUMPO Class 1A, 1B, 2 MLPA Minimal, Low, High Median age 60 years FU months Good correlation of results Some minor discrepancies of Class 1A and 1B groups 5 patients developed mets: all identified as high-risk by both methods

42 MLPA versus GEP

43 MLPA versus GEP 5 metastatic deaths

44 TNM 7 th Edition Authors Year Metho d Kujala et al 2013 JCO Jul 1 Pts Ch 3 Chr 8 Patients/Outcome Comment 7 th TNM 20 n/a n/a 8,736 cases FU = 3.4 years 1,301 metastatic death T1 24 T2 33% T3 31% T4 12% Ten-year survival rates for stages I, IIA- B and IIIA-C were 88%, 80%, 67%, 45%, 27%, 10%, respectively (P<0.0001)

45 Prognostication TNM Staging System Largest tumour diameter Tumour height Cilary body involvement Extraocular spread

46 Prognostic factor combinations Authors Year Method Pts Patients/Outcome Comment Damato et al Ophthalm ology conditional hazard estimating neural network (CHENN) Taktak A et al., 2004 Artificial Neural Networks (ANN) Kaisermann et al. training set of 1780, test set of another 874 patients 2331, split randomly into training and test sets 2005 ANN 153 pts predicting 5-year mortality All-cause survival curves generated by the CHENN matched those produced with Kaplan-Meier analysis (Kolmogorov-Smirnov, P<0.05). In older patients, however, the estimated melanoma-related mortality was lower with the CHENN, which accounted for competing risks, unlike Kaplan-Meier analysis. AI system can match if not better the clinical expert's prediction logistic regression reached 86% forecasting accuracy, with a very low LR (0.8), whereas the human expert forecasting ability was <70% (LR, 1.85) Estimation of survival prognosis in patients with choroidal melanoma requires multivariate assessment of age, sex, clinical tumor stage, cytogenetic melanoma type, and histologic grade of malignancy No genetics included

47 Prognostication in uveal melanoma Liverpool Uveal Melanoma Predictor Online ( LUMPO ) 2654 UM patients: Training set = 1780 Test set = UM patients: Bootstrap re-sampling (200x) Bayesian regularization

48 Serological prognostic parameters in UM

49 Authors (year) Marker Study type Patients (n) Comment nmum MUM ctrl Schaller et al. (2002) MIA Case-control Prospective (8 months) at diag; 3 at f-up - Increased levels in the 3 pts who developed mets Missotten et al. (2003) S100B Prospective (60 months) at f-up 58 No significant difference btw pts and controls Reininger et al. (2005) MIA Case-control Prospective (18 months) at diag; 8 at f-up - Increased levels in the 8 pts who developed mets Kakdkol et al. (2006) OPN Case-control Increased levels in metastatic pts Reininger et al. (2007) OPN Case-control Increased levels in metastatic pts Missotten et al. (2007) MIA S100B Case-control Elevated in pts with mets: S100B 62%, MIA 52% (LDH 62% also) Barak V et al. (2007) Haritoglou et al. (2009) MIA, OPN S100B Case-control (8 before/after mets) 18 91% AUC for 3 markers combined MIA, OPN Case-control Increased levels in metastatic pts Barak V et al. (2011) MIA, OPN S100B, TPS Case-control (sequential samples over 4 years) Lead time for predicting mets: S100B mts, OPN mts Suesskind D et al. (2011) GDF-15 Case-control Increased levels in metastatic pts

50 21 pts with metastatic uveal melanoma (MUM) bi-pap real-time PCR assays for tumor-specific mutations in cfcdna: GNAQ626A>T GNAQ626A>C cfcdna was quantified using the LINE1 real-time PCR assay (LINE1 PCR) Activating mutations were detected in all but one (20 of 21) plasma samples from patients with MUM, whereas no positive detection was observed in plasma of 20 healthy volunteers.. Consecutive series of 28 pts with metastatic or extraocular uveal melanoma 7 pts treated with sorafenib (varying dosages) 1 patient had intrahepatic chemoembolization 1 month prior blood sample collection. Amplification of the GNAQ Q209 (298 bp), GNAQ R183 (212 bp), GNA11 Q209 (150 bp), and GNA11 R183 (249 bp) Regions performed using the universal tailed amplicon sequencing strategy (Roche, Mannheim, Germany). Mutant alleles of the GNA11 or GNAQ genes, which are highly specific for uveal melanoma, were identified in cfdna of 9 of 22 (41%) patients.

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52 Prognostication in UM 1. Is there a preferred prognostic tool? There is a paucity of studies that directly compare prognostic techniques in uveal melanoma. (Singh et al. 2012) Prognostication requires the incorporation of a number of parameters (clinical, histological and genetic): no single stand alone parameter or test has been convincingly proven (Damato, Eye, 2012). 2. What is the role of prognostic biopsy?

53 2. What is the role of prognostic biopsy? a) Patient counselling b ) Cook, S.A., et al., 2008: 298 pts, The main benefit perceived by patients was that they would have greater control and that screening for metastatic disease and early treatment might enhance chances of survival. McCannel T et al., J Genetic Counselling 2009: 99 UM patients; Psychological status did not vary significantly as a function of cytogenetic test result. Prognostic information was important to patients with choroidal melanoma, even in the absence of prophylactic measures which might improve prognosis. Patient demand (as above) c) Screening and possible metastectomy Mariani P, Servois V, Piperno-Neumann S. Therapeutic options in metastatic uveal melanoma. Dev Ophthalmol. 2011; 49: Frenkel S, Nir I, Hendler K, Lotem M, Eid A, Jurim O et al. Long-term survival of uveal melanoma patients after surgery for liver metastases. Br J Ophthalmol 2009; 93(8): Marshall E et al., Br J Ophthalmol. 2013; 188 patients; prospective single arm study; 6-montly MRI in high-risk UM patients, determined using AFT model (clinical, histological and genetic); detected mets in 83 (92%) of 90 pts systemic disease, with 49% of these having less than five hepatic lesions all measuring less than 2 cm in diameter. Of these 90 patients, 12 (14%) underwent hepatic resection, all surviving for at least a year afterwards. d) Clinical trials e.g. adjuvant Rx? Whitehead J, et al. IOVS, 2012: analysis of >4000 UM cases; estimated the number of patients that would need to be enrolled to have a 90% power to detect a 10% increase in survival from 50% to 60% in high-risk patients with monosomy 3 melanoma exceeding 15mm in basal tumour diameter. A standard randomised trial would require recruitment of 850 patients and a trial duration of >6 years. Harbour et al. Plos Current, 2013 (review).

54 2. What are the complications of intraocular biopsy? a) Insufficient yield b) Haemorrhage c) Seeding d) Infection e) Retinal detachment