Gene Expression Profiling Prognostication of Posterior Uveal Melanoma

Journal Pre-proof GEP prognostication of posterior uveal melanoma: Does size matter? Elaine M. Binkley, James F. Bena, Jacquelyn M. Davanzo, Connie Hinz, H. Culver Boldt, Arun D. Singh PII:

S2468-6530(20)30002-6

DOI:

https://doi.org/10.1016/j.oret.2019.12.020

Reference:

ORET 689

To appear in:

Ophthalmology Retina

Received Date: 15 November 2019 Revised Date:

19 December 2019

Accepted Date: 30 December 2019

Please cite this article as: Binkley E.M., Bena J.F., Davanzo J.M., Hinz C., Boldt H.C. & Singh A.D., GEP prognostication of posterior uveal melanoma: Does size matter?, Ophthalmology Retina (2020), doi: https://doi.org/10.1016/j.oret.2019.12.020. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © YEAR Published by Elsevier Inc. on behalf of American Academy of Ophthalmology

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Title: GEP prognostication of posterior uveal melanoma: Does size matter?

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Authors: Elaine M. Binkley,1,2 James F. Bena,2 Jacquelyn M. Davanzo,2 Connie Hinz,1 H. Culver Boldt,1

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Arun D. Singh2

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Meeting presentation: This work was presented at the American Academy of Ophthalmology meeting in San Francisco CA, October 2019

Corresponding Author: Arun D. Singh, MD Cole Eye Institute, Cleveland Clinic 9500 Euclid Avenue, Cleveland, Ohio 44195, USA E-mail: [email protected] Phone: (216) 445-9479 Financial Support: None

Conflict of interest: No conflicting relationship exists for any author

Running head: GEP and tumor size

Address for reprints: Arun D. Singh, MD Cole Eye Institute, Cleveland Clinic 9500 Euclid Avenue, Cleveland, Ohio 44195, USA E-mail: [email protected] Phone: (216) 445-9479

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1

Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, IA, 52242, USA

2

Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195, USA

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Abstract Purpose: To investigate the influence of tumor size by American Joint Committee on Cancer (AJCC) stage, Collaborative Ocular Melanoma Study (COMS) size, tumor largest basal diameter (LBD), and tumor thickness on prognostication by gene expression profiling (GEP) class. Design: Two center, retrospective Subjects: 215 consecutive patients diagnosed with posterior uveal melanoma over a five-year period who were evaluated with prognostic fine needle aspiration biopsy (FNAB) at the time of primary treatment. Methods: Patient demographics, tumor clinical size, AJCC stage, COMS size, GEP class, presence of metastasis, and mortality data were collected. Metastasis-free-survival (MFS) was defined as time to metastasis or death from any cause. Comparisons were made using Pearson chi-square tests or Fisher exact tests for categorical factors, and t-tests or Kruskal-Wallis tests for continuous measures. Cox proportional hazards models were fit to identify whether size measurements increased the prognostic discrimination index (C-statistic). Main Outcome Measures: Metastasis-free-survival Results: The average follow-up interval was 22.0 months [12.0, 37.0]. Eighty-nine tumors were class 1A, 48 class 1B, and 78 class 2. Twenty-one patients developed metastatic disease detected by surveillance and confirmed by liver biopsy. Three-year MFS was 96% for class 1 and 63% for class 2. Five-year MFS was 96% for class 1 and 49% for class 2. All size measures significantly improved prognostic discrimination index by GEP class as shown by increase in the C-statistic with addition of size variables (C-statistic 0.750 for GEP alone, 0.830 GEP with AJCC (p=0.016), 0.822 GEP with COMS (p<0.001), 0.842 GEP with LBD (p<0.001), and 0.847 GEP with tumor thickness (p<0.001)). Class 2 patients with metastasis had larger tumors compared to non-metastatic class 2 tumors (AJCC class p=0.004; COMS class p=0.024; with metastasis mean thickness 6.5 mm [3.8, 9.5], without metastasis 3.9 mm [3.1, 6.0] (p=0.008), with metastasis mean LBD 14.9±2.8 mm, without metastasis, 12.3±2.7 mm p<0.001). All class 1 tumors with metastasis were large requiring enucleation. Conclusions: Incorporation of tumor size enhances the prognostic discrimination index of the GEP test in patients with posterior uveal melanoma. All size tumor parameters are equivalent in their ability to enhance GEP prognostication.

3 71

Introduction:

72

Metastasis free survival seems to be the most appropriate outcome measure for assessing prognostic

73

tests. However, many published studies have not indicated the methods used to establish the diagnosis

74

of metastasis (Table 1). 1-11 As described in COMS report no. 15, the variability in methods by which

75

metastasis and mortality have been identified such as patient or family contact, death certificates, liver

76

function studies, hepatic imaging, and biopsy/autopsy, have resulted in a wide range of reported

77

incidence of metastasis. 12 An accurate description of the methods by which metastatic disease is

78

diagnosed and confirmed is imperative for predicting prognosis.

79 80

Being able to identify those tumors that are at high risk for metastasis and provide patients with

81

accurate prognostic information has been a challenge. Histopathologic features have been used to

82

design prognostic models since the 1970’s. 13-16 The collaborative ocular melanoma study (COMS)

83

established tumor size as a key clinical prognostic indicator, and the American Joint Committee on

84

Cancer (AJCC) staging criteria which incorporates tumor size, location, and extra-ocular extension has

85

been validated as an effective prognostic tool in a worldwide, multicenter study.13,17

86 87

More recently, it has been shown that the AJCC classification can be further enhanced by incorporation

88

of chromosomal status 3 and 8 status irrespective of the technique used to ascertain the chromosomal

89

status (FISH, SNP, MLPA).18 In fact, the commercially available prognostic multiplex ligation-

90

dependent probe amplification based test (MLPA, Impact Genetics, Toronto, Canada)

91

incorporates, size, location, extent, and histopathology to ascertain survival estimates.19,20 The other

92

commercial test based upon gene expression profiling (GEP, DecisionDx-UM; Castle Biosciences,

93

Inc., Phoenix, Arizona, USA ), in a multi-center study was shown to have superior prognostic accuracy

94

relative to (AJCC-TNM) staging and chromosome 3 status.9 However, subsequent work has shown that

4 95

tumor size plays an important role in prognostication that should be considered when interpreting GEP

96

results. Walter et al. showed that tumors with a class 2 molecular profile had a better prognosis if the

97

largest basal diameter (LBD) was less than 12 millimeters at the time of treatment and that large class 1

98

tumors had poor prognosis similar to small class 2 tumors.3,5 Demirci et al. similarly found worse

99

prognosis in patients with class 2 tumors and largest basal diameter greater than 12 millimeters.8

100

Combining GEP prognostic information with clinical variables such as tumor size may therefore enhance

101

prognostication, however, the precise relationships between clinical and molecular variables and how to

102

best incorporate these data into practical prognostic models requires further study.

103 104

We therefore explore the relationship between tumor size and GEP, and investigate the impact of

105

incorporating tumor size into GEP prognostication estimates using pooled data derived over a five-year

106

time period at two institutions.

107 108

Methods:

109

IRB approval was obtained from both the University of Iowa (IRB # 201708718)

110

and the Cleveland Clinic (IRB #16-1150). This research adhered to the tenets of the Declaration of

111

Helsinki. Records of consecutive patients treated for uveal melanoma who were evaluated with FNAB

112

and prognostic GEP testing were reviewed for a five-year period (12/2012-12/2017, the University of

113

Iowa began offering GEP testing in 12/2012, the Cleveland Clinic in 11/2013). Patients were excluded if

114

biopsy results were unavailable due to technical failure (n=10, six of these patients have no metastatic

115

disease at last follow up, two have developed metastatic disease, and two have no follow-up data

116

available). Primary iris melanomas were excluded.

117

5 118

Patient age at diagnosis, tumor largest basal diameter and thickness, tumor location, treatment

119

modality (episcleral plaque brachytherapy, enucleation, transpupillary thermotherapy), date of

120

treatment, biopsy technique, GEP class, GEP discriminate score, date of last follow up, presence of

121

metastasis, and status at last follow up (alive or deceased) was recorded for each patient. The date of

122

last follow up was determined by review of the electronic medical record and was defined as the date of

123

last follow up with either the ocular oncology or medical oncology service. For patients who transferred

124

care outside of either institution the date of last follow up with local providers was recorded where

125

available. Each tumor was also classified using the 8th edition of the AJCC staging manual for uveal

126

melanoma and by the COMS small, medium, and large criteria.21,22

127 128

For patients who developed metastatic disease, the modality by which metastasis was detected and

129

confirmed was recorded. A scoring system modified from the Collaborative Ocular Melanoma Study

130

(COMS) was used to grade the metastatic status at death. Each deceased patient was assigned a score as

131

follows: 1=dead with melanoma metastasis confirmed, 2=no evidence of metastasis 3=insufficient

132

evidence to establish the presence of metastasis.12 These data were obtained by review of the electronic

133

medical record and review of outside documents including imaging results and pathologic reports where

134

available. For patients who were deceased, cause and date of death was determined by review of the

135

electronic medical record, obituaries, notification by family or friends where applicable, and report by

136

the cancer registry at either the University of Iowa or Cleveland Clinic. The means by which these data

137

were obtained (electronic medical record, cancer registry, family or friend report, obituary) was

138

recorded for each patient. Mortality data were verified with the records of the oncology registry at the

139

University of Iowa and the tumor registry at the Cleveland Clinic Taussig cancer institute respectively.

140

6 141

Metastasis-free-survival was defined as time to metastasis diagnosis or death from any cause as events.

142

Last follow-up date was used for censoring those without these events. Categorical factors were

143

described using frequencies and percentages, while continuous measures were described with means

144

and standard deviations or medians and interquartile ranges. Comparisons were made using Pearson

145

chi-square tests or Fisher exact tests for categorical factors, and t-tests or Kruskal-Wallis tests for

146

continuous measures. Cox proportional hazards models were fit for metastasis-free survival to assess

147

whether size measurements increased the prognostic ability of the GEP grade. Prognostic ability was

148

defined using the discrimination index (C-statistic) developed by Uno et al23, which measures the ability

149

of the model to correctly predict higher risk of metastasis or death based on the occurrence and earlier

150

timing of these events in groups with different follow-up times. Analysis was performed using SAS

151

software (version 9.4; Cary, NC).

152 153

Results:

154

A. General demographic data

155

There were 92 patients from the University of Iowa and 123 patients from the Cleveland Clinic. Average

156

age at diagnosis was 60.1 (±12.0 years). There were 114 right eyes and 101 left eyes. The average tumor

157

largest basal diameter (LBD) was 12.6±3.0 millimeters and thickness 4.1 [2.9, 7.0] millimeters. One-

158

hundred and seventy-two tumors involved the choroid only and 43 involved the ciliary body±choroid. By

159

AJCC staging 49 tumors were stage I, 81 IIA, 56 IIB, 22 IIIA, and 7 IIIB. By COMS classification 25 tumors

160

were small, 156 tumors were medium, and 34 tumors were large. Six eyes had extra-ocular extension

161

and 209 did not. Thirty eyes were treated with enucleation, 184 with I-125 episcleral plaque

162

brachytherapy, and one with transpupillary thermotherapy. Fine-needle aspiration biopsy approach was

163

transvitreal in 47 cases and trans-scleral in 168 cases (table 2).

164

7 165

B. Metastasis

166

There were 89 class 1A tumors, 48 class 1B tumors, and 78 class 2 tumors. Over a mean follow-up

167

interval of 22.0 [12.0, 37.0] months, twenty-one patients developed metastatic disease. Metastasis was

168

diagnosed by surveillance imaging (MRI, CT, or right upper quadrant ultrasound) rather than symptoms

169

in all cases and was confirmed by biopsy in all cases. Twenty patients were deceased. Of those patients,

170

13 patients had metastatic disease, 3 had no metastatic disease, and 4 had insufficient evidence to

171

determine the presence of metastatic disease. Patient status was determined by review of medical

172

records in 200 cases, cancer registry in 3 cases, family report in 3 cases, and obituary in 9 cases (table 3).

173

The three-year metastasis-free survival was 96% for class 1 patients and 63% for class 2 patients. The

174

five-year metastasis-free survival was 96% for class 1 patients and 49% for class 2 patients (figure 1).

175 176

The number of patients with class 1 tumors who developed metastatic disease was too small for

177

statistical analysis to compare to those without metastatic disease. For the two patients with class 1A

178

tumors who developed metastatic disease, both were COMS large tumors. One was AJCC class IIIA and

179

the other IIIB. The average largest basal diameter was 17.0 mm (14.5-19.5) and average height 11.8 mm

180

(10.5-13.0). The ciliary body was involved in both cases, neither patient had extra-ocular extension. Both

181

were treated with enucleation.

182 183

Patients with GEP class 2 tumors with and without metastasis where compared based upon tumor size

184

measured by AJCC stage, COMS size, tumor LBD, and tumor thickness. The eye involved, tumor location

185

(choroid or ciliary body+/- choroid), treatment type, biopsy type, follow up interval, and discriminant

186

score were also compared between these groups. Those patients with metastasis had larger tumors by

187

all size classifications. No significant differences in other characteristics including follow-up length or

188

discriminant score were observed (table 4).

8 189 190

C. GEP and tumor size: Distribution

191

GEP class was compared to tumor size measured by AJCC stage, COMS size, tumor LBD, and tumor

192

thickness. There appeared to be a trend towards patients with GEP class 2 tumors being larger across all

193

classification schemes. However, none of these differences were statistically significant (figure 2).

194

Conversely, larger tumors were also more likely to be class 2 as measured by AJCC stage (p=0.004),

195

COMS size (p=0.024), tumor LBD (p=<0.001), and tumor thickness (0.008).

196 197

D. GEP and tumor size: prognostic discrimination index (C-statistic)

198

Incorporation of all size measures (AJCC stage, COMS size, tumor LBD, tumor thickness) significantly

199

improved the ability of the model to correctly identify higher risk cases (prognostic discrimination index)

200

relative to the model with the GEP test alone (figure 3, table 5). The C-statistic value significantly

201

increased with the addition of each size variable (C-statistic 0.750 for GEP alone, C-statistic of 0.830 for

202

GEP combined with AJCC (p=0.016), 0.822 for GEP combined with COMS (p<0.001), 0.842 for GEP

203

combined with LBD (p<0.001), and 0.847 for GEP combined with tumor thickness (p<0.001)). However,

204

the prognostic discrimination among with models with each of the size measures did not significantly

205

differ from one another (figure 3, table 5).

206 207

Discussion:

208

We observed that incorporation of all tumor size measures (AJCC stage, COMS size, tumor LBD, and

209

tumor thickness) significantly improved the prognostic discrimination of GEP testing in patients with

210

posterior uveal melanoma as shown by higher C-statistic value with the addition of each size variable.

211

No size classification scheme was superior to another in its ability to enhance prognostic discrimination.

212

This is a key finding, and supports previous work by Correa, Demirci, and Walter, who reported that

9 213

tumor size measured by largest basal diameter and AJCC class influences GEP interpretation.3,5,8

214

Moreover, the present study suggests that the particular strategy used for grading tumor size (AJCC

215

stage, COMS size, tumor LBD, tumor thickness) may be less important than ensuring that some size

216

measure is incorporated into the prognostic model.3,5,8 Although we are unable to identify a LBD cutoff

217

as previously reported, to be of prognostic significance, it is possible that this parameter would also

218

reach significance as more patients are followed over longer time.3,5,8

219 220

We identified two patients with class 1A tumors who developed metastatic disease. Both cases had

221

large tumors that were managed by enucleation suggesting adverse prognostic effect of increasing

222

tumor size even in tumors others classified to have low risk of metastasis by GEP. It is possible that

223

additional testing with Preferentially Expressed Antigen in Melanoma (PRAME) testing would have

224

improved prediction in these cases.24

225 226

A strength of our study is the presence of two-center data, with standardized methods for collecting

227

metastasis and mortality data. Much of the previous work analyzing GEP prognostication relative to

228

tumor size has not stated the methods by which these data were collected (Table 1), and it is possible

229

that some patients with metastatic disease or death could have been left out of the analysis.

230

Establishing methods to effectively monitor patients for the development of metastatic disease is

231

imperative to collecting data for prognostic studies and takes on added significance in more rural

232

settings where patients may be co-managed with local medical oncologists or primary care physicians

233

for metastatic surveillance. Further, all patients in our study with metastatic disease had their

234

metastases detected by surveillance imaging rather than by clinical symptoms. Previous variability in

235

detecting and recording of metastasis may explain the better 5-year-metastasis free survival for patients

236

with class 2 tumors in our cohort than predicted by GEP (49% observed compared to 28% predicted). It

10 237

will be important to continue to follow these patients over time as more patients may develop

238

metastasis in the future.

239 240

In summary, we observed that AJCC stage, COMS size, tumor LBD, and tumor thickness, all equally

241

enhance prognostic discrimination by GEP class. Further, patients with class 1 and class 2 tumors who

242

developed metastasis had larger tumors when compared with non-metastasizing tumors. While GEP

243

testing is an important prognostic tool for patients with posterior uveal melanoma, tumor size is an

244

important variable that seems to enhance prognostic discrimination. Future work should be directed at

245

studying the tumor and host characteristics of those individuals whose metastatic outcomes deviate

246

from the predicted risk.

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Figure legends Figure 1: Kaplan-Meier survival curve for class 1 and class 2 patients. The three-year metastasis-free survival was 96% for class 1 patients and 63% for class 2 patients. The five-year metastasis-free survival was 96% for class 1 patients and 49% for class 2 patients. Figure 2: Distribution of patients in each GEP class by AJCC, COMS, and LBD size classifications. There appeared to be a trend towards patients with GEP class 2 tumors being larger across all classification schemes. However, none of these differences were statistically significant. Figure 3: Receiver Operating Characteristic (ROC) curve showing the ability to detect metastasis-free survival at 36 month based on GEP alone versus GEP with various tumor size measures. Incorporation of all size measures (AJCC stage, COMS size, tumor LBD, tumor thickness) improved the ability of the model to correctly identify higher risk cases (prognostic discrimination index) relative to the model with the GEP test alone.

Table 1. GEP prognostication of posterior uveal melanoma: Published studies Author, Year

Study Design

N

Follow up interval

Variables

Results

How metastasis/death determined Date of death attributable to metastatic disease recorded

Chappell et al. 2012

Masked, retrospective, single institution case series

197

Median 21.7 months

GEP class, clinical characteristics, development of metastasis, tumor regression rate

Class 2 tumors had higher initial mean thickness, more likely to metastasize

Onken et al. 2012

Prospective, multicenter

459

Median 17.4 months

GEP class, TNM class, presence of metastasis, monosomy 3 status

GEP had improved prognostic accuracy compared to monosomy 3 and TNM class

Last known patient survival status, presence or absence of metastasis, date metastasis detected, date of death/last follow up recorded

Correa et al. 2016

Prospective, single institution, interventional case series with a prognostic model

299

Median 32.2 months, mean 33 months

GEP class, clinical characteristics, metastasis

Both GEP class and LBD of the tumor are independent prognostic factors

Not stated

Plasseraud K et al. 2016

Prospective, multi-center

70

Median 27.3 months

GEP class, metastatic surveillance, metastasis

Not stated

Mruthyunjaya P et al. 2017.

Multicenter, retrospective

353

Median 2.1 years

GEP class, change in tumor height following brachytherapy

Tumor LBD and ciliary body involvement were not significant prognostic markers, tumor thickness was prognostic GEP class 2 associated with older patient age, larger tumor basal diameter and thickness, percent tumor height reduction greater

Not applicable

Walter et al. 2016

Retrospective, observational, two centers

339

Mean 30.8 months, median 24.5 months

GEP class, clinical size characteristics, metastasis

Nguyen et al. 2018

Retrospective, single center

83

Not applicable

Clinical findings, GEP class, AJCC class

Berry et al. 2018

Retrospective, multicenter

379

Not applicable

GEP, clinical features

Berry et al. 2018

Retrospective, multicenter

360

Not applicable

GEP, AJCC class

Cai et al. 2018

Retrospective, single center cohort

240

Median 29 months, mean 42 months

GEP class, PRAME expression, AJCC staging

for class 1 at 3 and 6 months, but no difference 9 months Class 2 tumors with LBD less than 12 mm had better prognosis Clinical high-risk features were not associated with tumor class, LBD and AJCC stage were associated with GEP class Increasing size measured by LBD and tumor thickness associated with class 2 tumors AJCC stage 1 tumors with larger thickness and LBD were more likely to be class 2, larger AJCC tumor group and stage had higher odds of worse prognosis by GEP class GEP, PRAME, LBD, and TNM stage had strongest association with metastasis. LBD was the only TNM variable that

Not stated

Not applicable

Not applicable

Not applicable

Not stated

Demirci et al. 2018

Retrospective, two center cohort

293

Median 23 months, mean 26 months

GEP class, AJCC stage, LBD, metastasis free survival

Table 1: Previous studies examining gene expression profile (GEP) prognostic class and tumor size parameters. LBD=Largest basal diameter PRAME=Preferentially expressed antigen in melanoma

contributed independent prognostic information Class 2 tumors with LBD ≥12 mm and class 2 and 1B tumors with AJCC stage III had worse prognosis. GEP and LBD independently predict metastasis

Medical record review where available. If not available, status updated by patient or primary care physician contact and cause of death identified by contacting family

Table 2. GEP prognostication of posterior uveal melanoma: General demographic data Iowa (N=92) Factor Age at diagnosis Eye . OD . OS Tumor Size: LBD Tumor Size: Height Location . Choroid Only . Ciliary body +/- Choroid COMS . Small . Medium . Large AJCC . I . IIA . IIB . IIIA . IIIB Treatment . Enucleation . Plaque . TTT Biopsy Type . TSV . TSC

Total (N=215) 60.1±12.0 114(53.0) 101(47.0) 12.6±3.0 4.1[2.9,7.0]

n

Statistics

n

Statistics

p-value

92 92

60.2±12.0

123 123

60.0±12.1

0.89a 0.30c

92 92 92

172(80.0) 43(20.0)

45(48.9) 47(51.1) 12.5±2.2 3.5[2.8,4.7]

123 123 123

72(78.3) 20(21.7) 92

25(11.6) 156(72.6) 34(15.8)

18(14.6) 77(62.6) 28(22.8)

16(17.4) 48(52.2) 19(20.7) 9(9.8) 0(0.0)

30(14.0) 184(85.6) 1(0.47)

0.32b 33(26.8) 33(26.8) 37(30.1) 13(10.6) 7(5.7) <0.001c

123 0(0.0) 92(100.0) 0(0.0)

92

30(24.4) 92(74.8) 1(0.81) <0.001c

123 0(0.0) 92(100.0)

Statistics presented as Mean ± SD, Median [P25, P75] or N (column %). p-values: a=ANOVA, b=Kruskal-Wallis test, c=Pearson's chi-square test, d=Fisher's Exact test. TSV=transvitreal, TSC=transscleral, TTT=transpupillary thermotherapy

<0.001b 0.58c

0.18b

123

92

0.74a

100(81.3) 23(18.7)

7(7.6) 79(85.9) 6(6.5)

49(22.8) 81(37.7) 56(26.0) 22(10.2) 7(3.3)

69(56.1) 54(43.9) 12.7±3.5 5.0[3.2,9.0]

123

92

47(21.9) 168(78.1)

CCF (N=123)

47(38.2) 76(61.8)

Table 3. GEP prognostication of posterior uveal melanoma: Metastasis data University of Iowa (N=92) Factor Follow up interval (months) GEP Results . 1A . 1B . 2 Alive/Dead Metastasis Metastatic status at death . Metastasis . No Metastasis . Insufficient Evidence Source of patient status data . Medical Records . Registry . Family . Obituary

Total (N=215) 22.0[12.0,37.0] 89(41.4) 48(22.3) 78(36.3) 20(9.3) 21(9.8)

n

Statistics

n

Statistics

p-value

92 92

25.5[16.0,44.0]

123 123

18.0[11.0,29.0]

<0.001b 0.43b

92 92 8

13(65.0) 3(15.0) 4(20.0)

32(34.8) 28(30.4) 32(34.8) 8(8.7) 7(7.6)

123 123 12

4(50.0) 2(25.0) 2(25.0) 92

200(93.0) 3(1.4) 3(1.4) 9(4.2)

Cleveland Clinic (N=123)

57(46.3) 20(16.3) 46(37.4) 12(9.8) 14(11.4) 9(75.0) 1(8.3) 2(16.7)

0.26d

123 85(92.4) 1(1.1) 3(3.3) 3(3.3)

Statistics presented as Mean ± SD, Median [P25, P75] or N (column %). p-values: a=ANOVA, b=Kruskal-Wallis test, c=Pearson's chi-square test, d=Fisher's Exact test.

0.79c 0.36c 0.53d

115(93.5) 2(1.6) 0(0.0) 6(4.9)

Table 4. GEP prognostication of posterior uveal melanoma: Class 2 tumors with and without metastasis No (N=59) Factor

n

COMS . Small . Medium . Large AJCC . I . IIA . IIB . IIIA . IIIB Tumor Size: LBD Tumor Size: Height Eye . OS . OD Location . Choroid Only . Ciliary Body +/- Choroid Treatment . Enucleation . Plaque Biopsy type . TSV . TSC Follow up interval (months) GEP Discriminant Score

59

Statistics presented as Mean ± SD, Median [P25, P75], or N (column %). p-values: a=ANOVA, b=KruskalWallis test, c=Pearson's chisquare test, d=Fisher's Exact test.

Statistics

Yes (N=19) n

59

0(0.0) 13(68.4) 6(31.6)

19 19 19

27(45.8) 32(54.2) 59

1(5.3) 5(26.3) 7(36.8) 5(26.3) 1(5.3) 14.9±2.8 6.5[3.8,9.5]

0.10c

19

59

12(63.2) 7(36.8) 0.22c

19 6(10.2) 53(89.8)

59

4(21.1) 15(78.9) 0.16c

19 11(18.6) 48(81.4) 22.0[11.0,44.0] 0.84±0.36

<0.001a 0.008b 0.60c

10(52.6) 9(47.4)

48(81.4) 11(18.6)

59 59

0.004b

19 12(20.3) 27(45.8) 15(25.4) 3(5.1) 2(3.4) 12.3±2.7 3.9[3.1,6.0]

p-value 0.024b

19 5(8.5) 47(79.7) 7(11.9)

59 59 59

Statistics

19 19

1(5.3) 18(94.7) 25.0[18.0,36.0] 0.92±0.36

0.66b 0.40a

Table 5: GEP prognostication of posterior uveal melanoma: Prognostic discrimination index Model GEP Only

GEP + AJCC

Variable GEP Results (2-level)

Hazard Ratio (95% CI)

137

4 (3%)

95.9 (92.0,99.9)

1.00 (REF)

78

24 (31%)

48.8 (29.0,68.6)

9.00 (3.11,26.05)

GEP Results (2-level)

.

Wald p-value . . < 0.001 .

1

137

4 (3%)

95.9 (92.0,99.9)

1.00 (REF)

2

78

24 (31%)

48.8 (29.0,68.6)

10.00 (3.36,29.76)

.

. < 0.001 .

81

9 (11%)

73.0 (49.4,96.6)

1.42 (0.30,6.63)

0.66

.

IIB

56

7 (13%)

73.6 (55.6,91.6)

2.00 (0.41,9.70)

0.39

.

IIIA

22

8 (36%)

43.8 (5.5,82.1)

5.76 (1.19,27.92)

0.030

.

IIIB

7

2 (29%)

0.0 (0.0,0.0)

28.75 (3.72,222.32)

0.001

.

. 95.9 (92.0,99.9)

1.00 (REF)

2

78

24 (31%)

48.8 (29.0,68.6)

8.86 (3.06,25.64)

.

. < 0.001 .

Small

25

0 (0%)

0.0 (0.0,0.0)

1.00 (REF)

Medium

156

19 (12%)

77.0 (63.7,90.3)

NA

0.99

Large

34

9 (26%)

44.5 (14.1,74.9)

NA

0.99

GEP Results (2-level) 2 Tumor Size: LBD GEP Results (2-level) 1 2 Tumor Size: Height

.

.

.

137

4 (3%)

95.9 (92.0,99.9)

1.00 (REF)

78

24 (31%)

48.8 (29.0,68.6)

8.52 (2.94,24.67)

< 0.001

215

28 (13%)

72.5 (59.6,85.4)

1.29 (1.13,1.49)

< 0.001

137

4 (3%)

95.9 (92.0,99.9)

1.00 (REF)

78

24 (31%)

48.8 (29.0,68.6)

9.30 (3.21,26.94)

< 0.001

215

28 (13%)

72.5 (59.6,85.4)

1.26 (1.12,1.41)

< 0.001

.

.

. .

0.822

<0.001

0.842

<0.001

0.847

<0.001

< 0.001

IIA

4 (3%)

0.016

.

1.00 (REF)

.

0.830

.

0.0 (0.0,0.0)

137

P-value vs. GEP Only

. < 0.001

2 (4%)

1

C-Statistic 0.750

.

49

GEP Results (2-level)

.

Overall p-value < 0.001

I

1

GEP + Height

5-Year Metastasis % (95% CI)

2

COMS

GEP + LBD

Events

1

AJCC

GEP + COMS

N .

.

< 0.001 . . 0.006 . . . < 0.001 . . < 0.001 < 0.001 . . < 0.001

Précis: Tumor size data improves prognostication in patients with posterior uveal melanoma undergoing gene expression profile testing at the time of treatment.