uveal melanoma ( um ) is a rare intraocular cancer affecting the choroid , ciliary body , or iris . at diagnosis , almost all patients will present without evidence of metastatic disease . despite high rates of primary tumor control , metastatic lesions , predominantly to the liver ,
occur in approximately 2050% of um patients . primary tumor gene expression profile ( gep ) testing has shown that patients can be accurately and reliably classified into low - risk class 1 and high - risk class 2 with significantly different metastatic potentials [ 25 ] .
these patient groups should be managed based upon their relative risk versus the overall population risk . for clinical care purposes
, a prognostic tool should be highly accurate in its risk prediction ( clinical validity ) , and its results should be implemented to inform patients ' subsequent management plans ( clinical utility ) .
clinical factors including age , extraocular extension , tumor size , and ciliary body involvement have been linked to higher risk for metastasis of um tumors [ 7 , 8 ] . however , none of these have offered the prognostic accuracy or reproducibility required for clinical implementation .
genetic analysis has led to the identification of metastasis - associated cytogenetic abnormalities on chromosomes 1 , 3 , 6 , and 8 [ 914 ] . in particular , the presence of monosomy 3 in primary tumor cells is a significant factor for predicting metastatic risk [ 10 , 15 , 16 ] , and fluorescence in situ hybridization ( fish ) , comparative genomic hybridization ( cgh ) , multiplex ligation - dependent probe amplification ( mlpa ) , and loss of heterozygosity ( loh ) have been developed to identify chromosome abnormalities [ 1720 ] .
however , to our knowledge , these molecular tests to detect gains and losses of chromosomes 1 , 3 , 6 , and 8 , including the commercially available mlpa platform [ 17 , 21 ] , have not been clinically validated in prospective , multicenter studies and published clinical utility is limited to high - risk patients .
the decisiondx - um gep test is an accurate prospectively validated molecular classifier , and its results are highly correlative to metastatic potential [ 2 , 5 ] , as reported by the collaborative ocular oncology group ( coog ) .
notably , because decisiondx - um has been extensively validated exclusively on pretreatment um specimens and the training set , to which the machine - learning algorithm compares each patient sample to generate a class 1 or 2 result , consists of only such samples , nonmelanoma samples are inappropriate for prognostication with the gep panel , consistent with the exclusion criteria of the coog study .
similarly , given the lack of validation on posttreatment tumors and the potential for radiobiological effects on tumor 's genomics , irradiated samples are also ineligible [ 23 , 24 ] .
decisiondx - um 's clinical utility has also been reported , indicating that the majority of ophthalmologists who order the test use the results to guide risk - appropriate treatment and management strategies for um patients .
herein are the first results from an interim analysis of the ongoing , prospective , multicenter clinical application of decisiondx - um gene expression assay results ( clear ) registry .
this study tracks decision impact ( surveillance regimens and treatment referral patterns ) , as well as clinical outcomes for decisiondx - um patients .
these results add additional prospective , multicenter evidence underscoring decisiondx - um as a highly accurate and clinically actionable assay for determining risk associated with primary um tumors .
after irb approval of the study ( number nct02376920 ; clinicaltrials.gov ) at participating centers ( mayo clinic , rochester mn ; university of virginia , charlottesville , va ; retinal consultants , sacramento , ca ; retina specialists of michigan , grand rapids , mi ) , patient consent was obtained .
data entry was performed at receipt of test results and semiannually ( censor date : june 2015 ) .
physician - obtained fnab or ffpe um tumor specimens were submitted to a centralized cap - accredited , clia - certified laboratory for gep .
frozen specimens , mostly fnab , were dispersed into rnase - free stabilization buffer immediately following biopsy , and all samples were shipped to the recipient lab on dry ice .
rna isolation was performed with the picopure rna isolation kit ( molecular devices , sunnyvale , ca ) .
all ffpe samples were prepared from enucleated globes , and tumor sections on microscope slides were shipped at room temperature to the recipient lab .
tumor tissue was macrodissected from slides using a sterile , disposable scalpel , deparaffinized in xylene , and processed for rna isolation with the ambion recoverall total nucleic acid isolation kit ( life technologies corporation , grand island , ny ) .
all rna was assessed for quantity and quality using the nanodrop 1000 system ( life technologies corporation ) and the agilent bioanalyzer 2100 and then converted to cdna ( applied biosystems high capacity cdna reverse transcription kit ; life technologies corporation ) .
each cdna sample underwent a 14-cycle preamplification step and was then diluted 20-fold in tris - edta buffer .
fifty microliters of each diluted sample was mixed with 50  l of 2x taqman gene expression master mix ( life technologies corporation ) and loaded onto a custom high - throughput microfluidics gene card containing primers specific for 12 class - discriminating genes and three endogenous control genes .
each sample was run in triplicate on an applied biosystems ht7900 instrument ( life technologies corporation ) .
delta  ct values were calculated by subtracting the mean ct of each discriminating gene triplicate from the geometric mean of the three endogenous control genes ' mean ct values .
molecular class assignments were determined by comparing the 12 discriminating gene ct values from each sample to a well - characterized , proprietary um training set of low - risk class 1 and high - risk class 2 geps using a support vector machine- ( svm- ) learning algorithm .
the predicted confidence is indicated by the discriminant score , which is inversely proportional to the proximity of the sample to the hyperplane established between class 1 and class 2 training set samples .
surveillance regimens were not prespecified but instead were independently decided upon by each participating physician utilizing the decisiondx - um result and documented as part of the registry data entry .
the intensity of surveillance for each patient was categorized based upon a previously reported study   and determined by the frequency of imaging ( ultrasound , pet / ct , or mri ) and liver function tests ( lfts ) that a patient received in addition to their regular eye examination follow - up .
a high - intensity schedule was characterized by imaging and/or lfts every 36 months , whereas a low - intensity schedule was characterized by annual imaging and/or lfts .
the prospective , multicenter clear study was designed to assess ( a ) the management of patients according to their decisiondx - um results ( clinical utility ) and ( b ) their documented development of metastatic disease ( clinical validity ) .
seventy patients have been enrolled from four centers across the us ( table 1 ) .
thirty - seven ( 53% ) were class 1 and 33 ( 47% ) were class 2 .
none of the patients who consented to be in the registry study had technical failures with gep testing .
of the class 1 patients , 30 ( 81% ) were class 1a , while 7 ( 19% ) were class 1b .
consistent with their high - risk gep , 12 ( 36% ) class 2 patients experienced a metastasis , whereas only 2 ( 5% ) class 1 patients experienced a metastasis ( p = 0.002 by fisher 's exact test ) with a median follow - up of 2.38 years .
the median time to metastasis for class 2 patients was 1.4 years ( table 2 ) .
class 2 patients had a significantly worse 3-year metastasis - free survival ( mfs ) rate of 63% ( 95% confidence interval = 43%83% ) compared to 100% in class 1 patients ( log rank test p = 0.003 ) ( figure 1 )
. the median rate of metastasis was 3.78 years for class 2 patients , while the median was not reached for the class 1 patients .
the majority of the metastases were localized in the liver , but metastases were also found in the lungs , brain , and bone ( table 2 ) . of note ,
one of the class 1 patients who experienced metastasis had a large tumor , while the other patient did not undergo treatment of their primary tumor .
nine out of the 12 class 2 patients who ultimately had metastases were treated by enucleation , while 2 were treated with plaque radiotherapy , and one was treated by transpupillary thermotherapy ( ttt ) ( table 2 ) .
tumor nodal metastasis ( tnm ) staging by ajcc includes tumor diameter , thickness , ciliary body involvement , and extraocular extension of the tumor . in this cohort ,
neither largest basal diameter nor ciliary body involvement performed as statistically significant prognostic markers ( figure 2 ) .
while tumor thickness did provide significant stratification of the patients , gep showed stronger prognostic significance in kaplan - meier and multivariate analysis ( table 3 ) , similar to previous studies [ 2 , 28 ] .
one primary objective of clear was to document clinical management differences that are implemented for class 1 compared to class 2 patients .
of the 37 class 1 patients , the majority ( n = 30 ) were treated with low - intensity follow - up ( imaging and/or lfts every year ) , while all 33 class 2 patients were treated with high - intensity follow - up ( imaging and/or lfts every 36 months ) ( figure 3 ) .
two of the class 1 patients who received high - intensity surveillance had intermediate risk class 1b results .
only 4 out of the 37 ( 11% ) class 1 patients were referred to medical oncology . of note ,
two of these referred class 1 patients had ciliary body involvement , one of whom also had a large ( 22  mm diameter ) tumor and displayed loss of chromosome 3 .
therefore , it is possible that the presence of these clinical features contributed to their medical oncology referrals . due to their high - risk disease , 6 out of 33 ( 18% ) of class 2 patients
were referred to medical oncology and 8 class 2 patients ( 24% ) were referred to adjuvant clinical trials .
importantly , four class 2 patients went on to receive systemic adjuvant therapy ; three patients received combinatorial chemotherapy ( tamoxifen , sunitinib , and cisplatin ) within a clinical trial , while one received ivig immunotherapy .
three of the four patients remained metastasis - free at last follow - up ( mean follow - up : 4.48 years ; median follow - up : 4.02 years ) .
no class 1 patients were referred to clinical trials or had systemic adjuvant therapy . taken together
, these results indicate that class 2 patients that have imaging and lfts are managed by medical oncology and are offered clinical trial participation significantly more often than class 1 patients ( fisher 's exact test for intensity of surveillance p < 0.0001 ; for medical oncology / clinical trial referral p = 0.04 ; table 1 ; figure 3 ) .
the national comprehensive cancer network ( nccn ) has emphasized the critical importance that all participants in the medical management of cancer patients , including the patients themselves , are provided with timely , reliable , and actionable education regarding molecular testing for diagnosis and treatment . as
molecular biomarker tests are increasingly available for diagnostic , prognostic , and therapeutic purposes , there is a growing need for transparency in reporting clinical validity and utility to ensure confidence in the accuracy and clinical impact of their results [ 6 , 29 ] .
previous studies have reported the validity and utility of decisiondx - um to provide highly accurate prognostic information to guide individualized management of um patients [ 2 , 5 , 30 ] .
herein , we report the initial results of the clear prospective registry study that confirms the clinical utility of decisiondx - um , since it became clinically available . as this trial is ongoing , a final analysis of 5-year survival rates
the nccn recommends that the clinical utility of a biomarker test should be determined in a prospective clinical trial .
the clear results represent a second independent , prospective , multicenter study making the test unique , not only in um , but in comparison to the majority of high - complexity advanced diagnostic tests .
the multicenter coog study validated decisiondx - um 's ability to accurately predict patient outcomes , reporting that metastatic events were observed in only 1% of class 1 cases versus 26% of class 2 cases after 50 months of follow - up time ( median follow - up was 17.4 months ) .
similarly , interim results from our prospective registry study to track decisiondx - um patients indicated a low - risk of metastasis for class 1 patients compared to class 2 patients ( 5% versus 36% , resp . ; median follow - up of 27.3 months ) . taken together
, the results from the clear registry add to the compelling evidence - based clinical validity of decisiondx - um .
as has been demonstrated in other cancers for which prognostic and predictive testing are employed , tailoring a treatment strategy according to patient 's individual tumor biology offers the potential for improved quality - of - life and more efficient utilization of healthcare resources [ 3134 ] .
the clinical utility of decisiondx - um was initially reported following a review of medicare medical records that indicated the test significantly impacted patient management plans , resulting in less aggressive management of class 1 versus class 2 patients , and similar management impact results were observed from blinded surveys of ocular oncologists .
the clear results also demonstrate that the clinical management of class 2 patients is associated with significantly higher surveillance intensity , including more frequent imaging , lfts , and referral to medical oncology compared to class 1 patients .
the higher - intensity surveillance for class 2 patients is consistent with the goal of potentially identifying metastases earlier , thus permitting intervention , while the patient is asymptomatic and likely more amenable to treatment(s ) .
conversely , unnecessary surveillance can potentially be avoided for patients in whom extraocular recurrence of disease is unlikely .
it should be noted that it is possible , based on a class 1 result , for a patient who ultimately experiences metastasis to receive lower frequency management , thus potentially delaying the identification of metastatic disease by a few months ( i.e. , at 12 months versus 36 months with annual versus more frequent surveillance , resp . ) . however , given the consistency of the mfs rates between the coog study and our data presented here ( table 4 ) , we expect metastasis would occur in a small minority of class 1 patients .
furthermore , the small percentage of class 1 patients who experienced metastasis ( 5% ) that we report here is in line with previously reported metastasis rates reported for um patients who are identified as low - risk [ 21 , 35 , 36 ] and for breast and colon cancer patients classified as low - risk by other tests [ 37 , 38 ] .
the data reported in this study are important for um because numerous clinical , pathological , and genetic characteristics of the primary tumor have been proposed as being significantly prognostic for um metastasis , yet these methods have achieved neither adequate nccn level of evidence for clinical utility nor clinical validity . based upon multiple prospective and retrospective studies published to date ,
given its robust ability to identify high - risk patients , rational intervention trials to identify effective adjuvant therapies have been initiated [ 3942 ] .
four class 2 patients within this cohort pursued adjuvant treatment for their high - risk disease ; three of whom were metastasis - free at last follow - up , underscoring the importance of making more clinical trials accessible for high - risk um patients .
the decision to enroll class 2 patients in clinical trials is directly related to the level of evidence for metastatic propensity that has been reported for the test .
the continued clinical performance and utility demonstrated in this study contributes to the high level of evidence regarding the clinical validity and utility of the decisiondx - um test .
this study demonstrates that the 15-gene expression assay decisiondx - um continues to accurately predict metastatic risk for um patients , thus enhancing the molecular test 's established clinical validity .
furthermore , this is the first prospective analysis of clinical utility of the assay , and this study demonstrates that the test results are being used to guide decision - making for physicians and patients in the clinic .
 uveal melanoma management is challenging due to its metastatic propensity . 
 decisiondx - um is a prospectively validated molecular test that interrogates primary tumor biology to provide objective information about metastatic potential that can be used in determining appropriate patient care . to evaluate the continued clinical validity and utility of decisiondx - um , beginning march 2010 , 
 70 patients were enrolled in a prospective , multicenter , irb - approved study to document patient management differences and clinical outcomes associated with low - risk class 1 and high - risk class 2 results indicated by decisiondx - um testing . 
 thirty - seven patients in the prospective study were class 1 and 33 were class 2 . 
 class 1 patients had 100% 3-year metastasis - free survival compared to 63% for class 2 ( log rank test p = 0.003 ) with 27.3 median follow - up months in this interim analysis . 
 class 2 patients received significantly higher - intensity monitoring and more oncology / clinical trial referrals compared to class 1 patients ( fisher 's exact test p = 2.1  1013 and p = 0.04 , resp . ) . 
 the results of this study provide additional , prospective evidence in an independent cohort of patients that class 1 and class 2 patients are managed according to the differential metastatic risk indicated by decisiondx - um . 
 the trial is registered with clinical application of decisiondx - um gene expression assay results ( nct02376920 ) . 