Integrating pathological, clinical and genetic predictors to enhance survival

prognostication in patients with choroidal melanoma

Bertil Damato MD PhD1, Antonio Eleuteri PhD2, Anthony C Fisher PhD CSci2 Sarah E

Coupland MBBS PhD3 and Azzam FG Taktak PhD CSci2.

From the 1Ocular Oncology Service Royal Liverpool University Hospital, Prescot St,

Liverpool, L7 8XP and 2Department of Clinical Engineering, and 3 Department of

Pathology, Duncan Building, Royal Liverpool University Hospital, Daulby St, Liverpool

L7 8XP, UK.

Running Head: Modelling survival from choroidal melanoma

Keywords: Melanoma; Uveal Neoplasms; Disease-specific mortality Rate; Chromosome

Aberrations; Cytogenetic Analysis; Ophthalmology; Pathology

Correspondence to: Bertil Damato MD PhD FRCOphth, Ocular Oncology Service, Royal

Liverpool University Hospital, Prescot St, Liverpool L7 8XP, UK; Tel: +44 (0) 151

7063973; Fax: +44 (0) 151 7065436; e-mail: Bertil@damato.co.uk

Almost 50% of patients with uveal melanoma develop metastatic disease, which almost

always involves the liver. It is not known whether treatment of the ocular tumour prevents

metastasis and if so in whom. Most patients with metastatic disease die within a year of the

onset of symptoms and treatment only rarely seems to prolong life significantly. Studies on

systemic adjuvant therapy have not shown significant survival benefit.

As with other cancers, accurate prognostication is required, so that patients with a good

survival probability can be reassured and prevented from unnecessary and prolonged

screening for metastasis. Such intensive care would be reserved for high-risk patients,

thereby conserving resources. More reliable prognostication would also enhance prospects

for randomized studies evaluating whether systemic adjuvant therapy prevents or delays

metastatic disease.

Many factors are known to be predictive of metastatic disease, the most important being:

largest basal tumour diameter; ciliary body involvement; extraocular spread; epithelioid

melanoma cytomorphology; extravascular matrix patterns such as closed loops; high

mitotic count; and a variety of genetic abnormalities, particularly chromosome 3 loss,

chromosome 8q gain and class 2 gene expression profile.

The 7th edition of the TNM (Tumour, Node Metastasis) staging system for cancer
categorizes uveal melanomas according to: involvement of choroid, ciliary body and iris;

basal tumour diameter; tumour height and extraocular extension. A list of pathological risk

factors recommended for collection is included, together with a system for defining

histological grade according to melanoma cell type. There are no instructions or guidelines

for combining histological data with clinical and genetic predictive factors. As a result,

prognostication is inaccurate and only applicable to large groups of patients, not

individuals. Given the choice, the large majority of patients want to know their prognosis,

even if this is poor and unlikely to be improved by therapy. Ophthalmologists and

oncologists also depend on reliable prognostication because they tend to plan systemic

screening and other aspects of management on a case by case basis.

For several years, the authors have been developing methods of integrating pathological,

clinical and genetic data to enhance prognostic accuracy so that estimates of survival

probability are relevant to individual patients. These used neural networks because the

survival predictors correlated with each other and with metastasis in a non-linear fashion.

Two versions of the model were developed: one based only on clinical information; the

other also including histological and genetic factors. These generated two survival curves,

respectively showing the all-cause morality of our patients with uveal melanoma and of the

general population matched for age and sex so that metastatic mortality could be estimated

by comparing one curve with the other. This strategy avoided the need for diagnosis of

cause of death, which is known to be unreliable.

Validation studies showed our prognostic methods to perform adequately in patients treated

by local resection or enucleation; however, prediction was subsequently found to be

unreliable when only biopsy specimens were analysed, as in patients treated with

radiotherapy or phototherapy. This is because biopsy precluded mitotic counts and

assessment of extravascular matrix patterns and because the neural networks were unable

to compensate for the missing data. Such missing information has become more common

in recent years because of the way in which prognostic biopsy of irradiated melanomas has

become routine.

The aim of this study was to create a prognostic model that combined pathological, clinical

and genetic data, using imputation techniques to compensate for missing information.

PATIENTS AND METHODS

Patients

Patients were selected from the database of the Liverpool Ocular Oncology Centre for the

time period 1984 – 2009 if: (1) diagnosed with uveal melanoma, clinically or

histopathologically; (2) primarily treated by the first author (BD) or an associate at the

Tennent Institute of Ophthalmology, Glasgow, before January 1993 or at the Royal

Liverpool University Hospital between January 1993 and July 2006; and (3) resident in

mainland Britain. Patients were excluded because of: (1) bilateral melanoma; (2) missing
data regarding basal tumor dimension or anterior tumor extension; (3) iris or ciliary body

tumor not involving choroid; or (4) residence overseas, including Northern Ireland.

Clinical data

Pre-operative and follow-up assessments were performed as described previously.[Damato

et al., 2005] Briefly, the following features were defined by slit-lamp examination and

binocular indirect ophthalmoscopy: coronal tumor location; anterior and posterior tumor

margins; circumferential spread around disk margin, ciliary body, iris and angle; and

centripetal spread towards vitreous. Tumor basal dimensions and thickness were measured

with B-scan ultrasonography.

Pathological data

Tumor cell type and mitotic rate were determined by light microscopy using hematoxylin

and eosin staining.[Font et al., 2006] Closed connective tissue loops were identified using

sections stained with periodic-acid Schiff stain without counterstaining and viewed under a

green filter. Extraocular tumor spread was detected by slit-lamp examination,

ultrasonography, or pathological examination.

Genetic data

Between 1999 and 2007, chromosome 3 loss and chromosome 8 gains were identified by

fluorescent in-situ hybridization (FISH), which was performed as a clinical service to all

patients undergoing local resection or enucleation.[Damato et al., 2007;Pardue and Gall,

1975] Since 2007, genetic typing was based on multiplex ligation-dependent probe

amplification (MLPA).

Survival data

Information on every patient with newly-diagnosed uveal melanoma was sent to the

National Health Service (NHS) Cancer Registry, which automatically informed us of date

and cause of death, usually within three months of this event. Survival probabilities of the

general population were estimated using the UK Government Actuary’s Department (GAD)

interim life table figures for 2004 [[[[[[[?????]]]]]] (http://www.gad.gov.uk).

Data management

From 1984, clinical information was collected and computerized prospectively, using a

succession of customized databases. The tenets of the Helsinki Declaration were followed.

Institutional ethical committee approval for outcomes analysis was not required.

Mathematical methods

Modelling the data

The data were described mathematically using an Accelerated Failure Time model. This

predictive model was created by specifying an analytical functional expression that

described the relationships between the variables and survival. The formula developed for

this model, linking the survival time T to the vector of prognostic factors X was the

following:
 log(T ) = f ( X ) + se (1)

where e is a logistic random variable with scale s . The function f was a linear combination

of prognostic factors. Age and basal tumour diameter were continuous so that complex

interactions with time were anticipated; therefore, non-linear transformations were

introduced to achieve a better fit. With these continuous variables, the data were modeled

by fixed (nonparametric) nonlinear transformations, which did not depend on adaptable

parameters. The nonparametric transformations were achieved using restricted cubic

splines, which are a class of mathematical models. Mitotic count was treated differently,

being expanded nonlinearly by a combination of linear plus dummy factors (i.e., creating

non-uniform categories). This was necessary because of the sparsity of mitotic counts (e.g.,

few tumours with a count of, say, 5 per 40 high power fields as opposed to many with a

mitotic count of 1 per 40 high power fields). The model was further simplified by fixing a

parametric family for the distribution of events. In other words, survival times were

described mathematically by assuming a log-logistic distribution of the events.

Two versions of the model were created: one with clinical factors only and the other

including pathological and genetic data. The same mathematical methods were used in both

models.

Missing data

Values for missing data were estimated using the Alternating Conditional Expectations

algorithm, which essentially estimated each of the missing variables as a function of the

other variables. For example, if mitotic count was missing, this was estimated by modelling
its relationships with all the other baseline variables. In other words, if large, epithelioid

melanomas tended to have a higher mitotic count than small, spindle cell melanomas, then

the former kind of tumour would mathematically be given a higher mitotic score than the

latter. Mathematically speaking, this process approximated the joint distribution of the

baseline variables.

Bootstrap re-sampling was then used to estimate the model parameters. In other words, the

entire dataset was repeatedly and randomly split into training and test datasets, and the

model was then fitted to the training data and its performance tested using the test data.

This was done 200 times so that the statistical variability of the performance of the model

could be assessed, using the C index and residuals. Bayesian regularization was applied, to

reduce the chance of model overfitting the data. In other words, the model was simplified

and tailored to the complexity of the data so that predictions could be extrapolated to all

patients and not only relevant to those used to train the model.

Validation

Two validation measures are calculated, discrimination and calibration. Discrimination

described the ability of the model to rank the outcomes as a function of the prognostic

factors (e.g., checking that tumours with more malignant histological features were indeed

associated with a worse prognosis). Discrimination was expressed in terms of the c-index,

which is a mathematical way of generalising to censored data the diagnostic power of a test

and which is estimated from the area under the receiver operating characteristic (ROC)

curve. As a reminder, the ROC curve describes how sensitivity and specificity change as

the value of a test result is varied. For example, an ROC curve may describe the sensitivity
and specificity with which an increase in basal tumour diameter predicts metastatic death.

Calibration described the precision of the predictions (e.g., checking that approximately

50% of patients with a particular combination of risk factors actually survived five years

when such a survival probability was predicted). Calibration was measured by calculating

the censored prediction residuals, which for a “good” model should follow the log-logistic

distribution postulated in eq. (1).

Online interface

A web-based decision support system was developed using the Matlab Webserver toolbox

(The MathWorks, Inc, Natick, Massachusetts) and was hosted in:

www.ocularmelanomaonline.com. The first page of this website summarized the purpose of

the tool and provided links to our departmental website and Terms and Conditions. On

agreeing to proceed, the user was taken to the data entry screen, which displayed a form for

entry of the following data: age; gender; largest basal tumor diameter; anterior tumor

extension in relation to ora serrata; extraocular spread; years since treatment; epithelioid

cell type; presence of closed loops; mitotic rate; and monosomy 3. Survival curves showed

actuarial survival rates both for patients with choroidal melanoma and for the age-matched

general population (i.e. +/- five years) of the same gender (Figure 1). The patients’ survival

curve was shown with its 95% credibility interval (i.e. the probabilistic equivalent of the

‘frequentist’ 95% confidence interval). Survival was modeled for the first ten post-

operative years, except if cytogenetic data were used, in which case survival was predicted

for only seven years. The facility was provided to adjust the survival estimate if the patient

was known to be alive more than one year after treatment.

RESULTS

Cohort

The cohort comprised 3201 patients (1543 female; 1658 male) with a median age of 62.2

years (range, 12.4 – 96.9). The tumours were located in the left eye in 1603 patients

(50.1%) and the right eye in 1598 patients (49.9%). The anterior margin extended anterior

to the ora serrata in 843 eyes (26.3%) and involving the anterior chamber in 195 eyes

(6.1%). Posteriorly, the tumour extended post-equatorially in 2805 eyes 87.6%, involving

optic disc in 482 eyes (15.1%). Extraocular spread was detected in 55 cases (1.7%). The

median largest basal tumour diameter was 12.0 mm (range, 1.2-23.6) and the median

thickness was 4.7mm (range, 0.6-20.0).

Histological data were available for 1551 tumours, of which 973 (30.4%) contained

epithelioid cells. Closed loops were present in 49% of 960 tumours in which this feature

was assessed and the mitotic count per 40 high power fields exceeded 4 in 29.6% of 1409

tumours in which this examination was possible. Chromosome 3 loss was detected in 295

out of 646 tumours (45.7%) that underwent cytogenetic analysis.

A total of 1091 (34.1%) of patients had died, the certified cause of death being metastasis

from uveal melanoma in 622 patients. The follow-up time had a median of 5.3 years (range,

0.02 – 25.1), in surviving patients exceeding five years in 1236 patients, ten years in 698

patients and fifteen years in 301 patients.

Discrimination

Discrimination was expressed in terms of the c-index, which was 0.75 (95% confidence

interval [CI], 0.74 -0.76) for the clinical model and 0.79 (95% CI, 0.76 - 0.82) for the

cytogenetic model.

Calibration

Calibration followed the log-logistic distribution postulated in equation 1 (Fig. xx). With

both the clinical and cytogenetic models, agreement between ??? and ??? was excellent.
1.0
0.8
Survival Probability

0.6
0.4
0.2
0.0

-12.0 -8.8 -7.2 -5.6 -4.0 -2.4 -0.8 0.8 2.4 4.0

Residual
 1.0
0.8
Survival Probability

0.6
0.4
0.2
0.0

-15 -13 -11 -9 -7 -5 -3 -1 1 3 5

Residual

Comparison between modelled survival and Kaplan-Meier estimates

Figure xx shows the predicted and observed survival according to the estimated risk of

metastatic death at five years (i.e., low, medium or high), both for the clinical model (Fig

xxa) and the cytogenetic model (Fig xxb). All pairs of survival curves correlated well with

each other.