Interim analysis of survival in a prospective, multi-center registry cohort of cutaneous melanoma tested with a prognostic 31-gene expression profile test
Hsueh et al. Journal of Hematology & Oncology
Interim analysis of survival in a prospective, multi-center registry cohort of cutaneous melanoma tested with a prognostic 31-gene expression profile test
Eddy C. Hsueh 2
James R. DeBloom 1
Jonathan Lee 0
Jeffrey J. Sussman 6
Kyle R. Covington 5
Brooke Middlebrook 5
Clare Johnson 5
Robert W. Cook 5
Craig L. Slingluff Jr 4
Kelly M. McMasters 3
0 Northside Melanoma and Sarcoma Specialists of Georgia , Atlanta, GA , USA
1 South Carolina Skin Cancer Center , Greenville, SC , USA
2 Dept. of Surgery, St. Louis University , St. Louis, MO , USA
3 Dept. of Surgical Oncology, James Graham Brown Cancer Center, University of Louisville School of Medicine , Louisville, KY , USA
4 Dept. of Surgery and Cancer Center, University of Virginia School of Medicine , Charlottesville, VA , USA
5 Castle Biosciences, Inc. , 820 S. Friendswood Drive Suite 201, Friendswood, TX , USA
6 Dept. of Surgery, University of Cincinnati Cancer Institute , Cincinnati, OH , USA
Background: A 31-gene expression profile (GEP) test that provides risk classification of cutaneous melanoma (CM) patients has been validated in several retrospective studies. The objective of the reported study was a prospective evaluation of the GEP performance in patients enrolled in two clinical registries. Methods: Three-hundred twenty two CM patients enrolled in the EXPAND (NCT02355587) and INTEGRATE (NCT02355574) registries met the criteria of age ≥ 16 years, successful GEP result and ≥1 follow-up visit for inclusion in this interim analysis. Primary endpoints were recurrence-free (RFS), distant metastasis-free (DMFS), and overall survival (OS). Results: Median follow-up was 1.5 years for event-free patients. Median age for subjects was 58 years (range 18-87) and median Breslow thickness was 1.2 mm (range 0.2-12.0). Eighty-eight percent (282/322) of cases had stage I/II disease and 74% (237/322) had a SLN biopsy. Seventy-seven percent (248/322) had class 1 molecular profiles. 1.5-year RFS, DMFS, and OS rates were 97 vs. 77%, 99 vs. 89%, and 99 vs. 92% for class 1 vs. class 2, respectively (p < 0.0001 for each). Multivariate Cox regression showed Breslow thickness, mitotic rate, and GEP class to significantly predict recurrence (p < 0.01), while tumor thickness was the only significant predictor of distant metastasis and overall survival in this interim analysis. Conclusions: Interim analysis of patient outcomes from a combined prospective cohort supports the 31-gene GEP's ability to stratify early-stage CM patients into two groups with significantly different metastatic risk. RFS outcomes in this real-world cohort are consistent with previously published analyses with retrospective specimens. GEP testing complements current clinicopathologic features and increases identification of high-risk patients. Trial registration: ClinicalTrials.gov, NCT02355574 and NCT02355587
Gene expression profiling; DecisionDx-Melanoma; Cutaneous melanoma; Metastasis; Prognosis; Staging
The majority of cutaneous melanoma (CM) patients are
diagnosed with early-stage (AJCC stage I or II) disease
and are considered to have a favorable prognosis [
However, two-thirds of all melanoma deaths occur in
patients initially included in this “low-risk” group, which
indicates that metastatic risk is underestimated for a
substantial number of early-stage patients [
recent study reporting data from the Surveillance,
Epidemiology, and End Results (SEER) program reported
that the mortality rate for cutaneous melanoma is
increasing faster than the incidence rate, highlighting the
need for additional prognostic tools to supplement
standard clinicopathologic factors and improve
identification of high-risk disease [
]. More vigilant follow-up
for high-risk melanoma patients, including the addition
of imaging and increasing clinical assessment frequency,
leads to earlier detection of asymptomatic distant
metastatic disease, when tumor burden is lower, and when
surgical approaches as well as contemporary therapies
have greater benefit [
]. Distinguishing those CM
patients with high-risk tumor biology from those who
are categorized as low risk by TNM staging alone is
therefore a clinically important goal.
A prognostic gene expression profile (GEP) test
(DecisionDx-Melanoma, Castle Biosciences, Inc.) that
evaluates 31 gene targets expressed in the primary
melanoma tumor to provide a binary classification of low
(class 1) or high (class 2) risk of metastasis within 5 years
of the initial diagnosis has been previously reported [
The test assesses the expression of three control genes,
four genes with proven prognostic utility for uveal
melanoma tumors [
], and 24 gene targets previously
reported to be differentially expressed in metastatic
compared to primary tumors [
]. Performance of
the test has been evaluated in several retrospective
validation studies, showing that it accurately prognosticates
survival independent of clinical or pathologic assessment
12, 21, 22
], and though it was originally developed
to identify the distant metastasis risk associated with
stage I and II melanoma tumors, the prognostic
capability of the test has recently been reported in patients with
stage III disease. In order to prospectively evaluate
outcomes for a real-world representative cohort of CM
patients, two multi-center registry studies (EXPAND
and INTEGRATE) were initiated. Here, we present an
interim analysis of the survival outcomes for 322
patients enrolled in these registries.
Patients were enrolled in one of two prospective studies,
EXPAND and INTEGRATE (Clinicaltrials.gov
identifiers: NCT02355587 and NCT02355574). The protocols
have identical enrollment criteria and aims, including (1)
the tracking of outcomes of patients for whom GEP
testing was completed, and (2) documentation of the clinical
application of test results. The studies differ only in
clinical data collection requirements based upon the
administrative capabilities of the contributing centers. While a
combined analysis of the two studies was originally
planned based upon the similarity of protocols and aims,
an interim analysis of this cohort was not included in
the initial study design. Based upon the 1.1-year median
time to recurrence observed for class 2 patients in
previous validation studies, an unplanned interim analysis of
this combined cohort of patients is warranted and
expected to answer the hypothesis that class 1 and class 2
risk groups will have significantly different
recurrencefree (RFS), distant metastasis-free (DMFS), and overall
survival (OS) rates [
12, 21, 22
Eleven US dermatologic and surgical centers
participated after receiving protocol approval from their
Institutional Review Boards. The number and diversity of
centers were selected to reduce bias arising from single
centers. Physicians enrolled patients with a diagnosis of
cutaneous melanoma who were ≥16 years of age and
had a successful GEP test result. The GEP test was
performed in a central CAP/CLIA laboratory using standard,
published protocols as previously described [
Sample size calculations were originally developed from
survival outcomes observed in a limited, retrospective
analysis of the GEP test, with assumptions of 0.05 for alpha
and a stringent power of 0.95. Based upon the rapid,
significant separation of risk profiles for class 1 and class 2
cohorts reported in expanded analyses of the test since the
inception of the studies (January, 2014), recruitment was
discontinued prior to accrual of the patient numbers
specified within the protocols. Clinical follow-up will continue
through 5-year post-diagnosis for each patient (n = 322).
At the time of the initial patient evaluation, prior to
GEP testing, the treating physician assessed each
patient’s baseline data, including Breslow thickness (BT),
ulceration status, T stage and mitotic rate. The majority
of centers reported mitotic rate in mm2; for those that
did not, high-powered fields (HPF) were converted to
mm2 according to a conversion rate of 4 HPF per mm2
specified by the AJCC v7 guidelines [
]. BT and
mitotic rate were used as continuous variables while
ulceration, SLN status and GEP class result were treated as
categorical variables. All data were entered into a secure
case report form and were abstracted and assembled
independently of statistical analysis. Last censor date for
clinical data was December 30, 2016.
Accuracy of the test was measured by sensitivity or
specificity. The former was measured as the number of
cases with an event [recurrence (including in transit and
regional nodal recurrences), distant metastasis or death]
that were identified as class 2, while specificity was the
number of cases that did not have a documented
event that were classified as low-risk class 1 that did
not have a documented event. Primary survival
endpoints of RFS (time to regional or distant metastasis),
DMFS (time to any metastatic event beyond the
regional nodal basin), and OS (time from diagnosis to
documented death of any cause) were assessed using
Kaplan-Meier and Cox regression analyses. For
multivariate analyses, only complete cases were considered.
In 26 cases, ulceration, BT and/or mitotic rate were
not known or specified and these cases were excluded
*n = number of samples
**All percentages are reflective of the indicated group out of the total number of samples in that column
***p values reflect differences between class 1 and 2 patients and were calculated using chi-squared or F tests as appropriate
from multivariate analyses. Cases without a
documented SLN biopsy result were categorized as
As an interim analysis at year 3 of an expected 5-year
study, the critical alpha level (p value) was established at
0.01. Statistical associations were evaluated using the F
test, chi-squared test, T test, and ANOVA, where
appropriate. Statistical analyses were performed in R version
3.3.2 (University of Auckland, NZ).
Clinical and demographical comparison between
A total of 335 patients were enrolled in the studies at the
last censor date. Of these, 322 patients had completed at
least one follow-up visit and were available for inclusion
in this interim analysis. A GEP class 1 profile was
observed in 77% (248 of 322) of samples. Table 1 provides
demographic variables for the entire cohort, stratified by
GEP class. The median age of subjects was 58 years (range
18–87 years) and the median Breslow thickness was
1.2 mm (range 0.2–12.0 mm). Within the group, 88% (282
of 322) of the cases had stage I or II disease, and SLN
biopsy was performed for 74% (237 of 322) of the subjects.
A class 2 profile was observed in 23% (74 of 322) of cases,
and was associated with older age, male gender, higher
Breslow thickness, ulceration, advanced clinical stage, and
positive SLN status (all p < 0.01). However, only half of
class 2 patients had ulcerated tumors and a quarter of
them had a positive SLN biopsy. Mitotic rate, primary
tumor location, and location of first recurrence were not
associated with a class 2 profile.
Interim analysis of outcomes
At 1.5 years of follow-up, regional metastasis was the
first site of recurrence in 52% (13 of 25) of cases, and
77% (10 of 13) of those were identified as high-risk class
2. Similarly, for the 12 patients for whom the site of first
recurrence was a distant metastasis, 83% (10 of 12) were
identified as class 2.
The GEP test was the most sensitive prognostic factor
for all endpoints assessed, including SLN biopsy and
ulceration, with a high-risk class 2 result showing 80%
sensitivity for recurrences, 83% for distant metastases,
and 73% for death (Table 2). Overall, recurrence rates
were 2% (5 of 248) for class 1 versus 27% (20 of 74) for
class 2 patients, distant metastasis rates were 1% (2 of
248) versus 14% (10 of 74), and overall survival rates
were 1% (3 of 248) versus 11% (8 of 74), respectively.
Median recurrence event times for class 2 patients in
prior retrospective studies is 1.1 years after diagnosis
12, 21, 22
]. With a median follow-up time for
nonevent samples that exceeds 1.5 years for all endpoints,
interim analysis of outcomes in this population is
possible. The GEP test was significantly associated with
recurrence (p < 0.001), distant metastasis (p < 0.001),
and death (p < 0.001). The 1.5-year RFS, DMFS and OS
rates for class 1 versus class 2 were 97% (95–100%)
versus 77% (67–87%), 99% (97–100%) versus 87% (81–
96%), and 99% (97%–100%) versus 92% (86–99%),
respectively (Fig. 1). The GEP test was also significantly
associated with each endpoint analyzed in the group of
subjects with stage I or II melanoma (n = 282,
Additional file 1: Figure S1).
Multivariate Cox regression modeling (n = 296
complete cases) showed that BT, mitotic rate, and GEP
class were independent predictors of RFS outcome (Table
3; p < 0.01, BT – HR: 1.43 [95% confidence interval (CI)
1.18–1.73], mitotic rate – HR: 1.05 [95% CI 1.01–1.08],
GEP – HR: 7.15 [95% CI 1.99–25.8]). For RFS, node status
was not significant in these interim analyses at an alpha of
0.01 [p = 0.035, HR: 2.46 (95% CI 1.07–5.68)]. For DMFS
and OS, only Breslow thickness was significant [p < 0.001,
Additional file 2: Table S1].
*n = number of samples
**p values reflect differences between high and low-risk groups for each prognostic factor (χ2 test)
Comparison to retrospective cohorts
Table 4 summarizes the outcomes data for this
prospective trial and previously reported retrospective analyses.
The original report of the GEP test performance
included 104 samples, with median follow-up time of
8.2 years for non-event samples and 1.7 years for
samples with an event (Table 4) [
]. The 1.5-year RFS
rates were 98% (95% CI 95–100%) for class 1 and 70%
(95% CI 58–85%) for class 2. The most recent validation
dataset included 523 stage I-III long-term follow-up
]. In this dataset, median follow-up time for
cases without an event was 7.2 years and median time to
a recurrence event was 1.2 years. For all cumulative
cases that have been reported in retrospective validations
(total for all cases: n = 782), the median time to a
recurrence event was 1.3 years (1.1 years for the class 2
cohort alone). The 1.5-year RFS rates were 95% (95% CI
93–97%) for class 1 cases and 67% (95% CI 62–73%) for
class 2 cases. While the median follow-up time is shorter
for this prospective analysis, with 1.5 years of follow-up
for cases without an event and 1.0 year for cases with an
event, the observed RFS rates of 97% for class 1 and 77%
for class 2 are consistent with 1.5-year event rates in
prior retrospective datasets (Table 4, Fig. 2).
An important unmet clinical need in melanoma is the
accurate identification of early-stage patients who harbor
a higher risk of developing advanced disease. While
Breslow thickness and ulceration are strongly associated
with metastasis and outcome, and sentinel lymph node
status is a proven prognostic tool that identifies a subset
of the patients with high-risk disease, two of three
patients who die from melanoma are originally diagnosed
with stage I or II tumors [
]. As these clinicopathologic
staging features are used to drive management decisions,
molecular tools that supplement and improve current
methods of prognostication should have significant
Class 2 1.5-year RFS
Percent (95% CI)
To improve upon the identification of high-risk stage I
and II patients, a gene expression profile test was
developed to provide prognostic information based on the
expression of 31 genes in the primary melanoma tumor
]. A limitation of this interim analysis could be the
impact of the short follow-up time. However, consistent
with previous reports, the GEP test was a significant
predictor of RFS, DMFS, and OS, in both the larger cohort
of 322 patients (Fig. 1), and in 282 stage I and II CM
cases (Additional file 1: Figure S1). The risk of
metastasis and death was significantly higher for patients with a
class 2 result compared to class 1 patients. The GEP
accurately identified 80% of the recurrences detected
during the study, and only 2% of the patients who were
predicted to be low risk (class 1) by the test developed
recurrent disease. Although multivariate analysis showed
BT to be the only independent prognostic factor for
DMFS and OS in this cohort, this is likely to be due to
the limited number of events for these two outcomes. In
all prior studies the GEP has shown independent
prognostic value along with Breslow thickness and SLN
In this study, 83% (10 of 12) of patients who developed
distant metastases were identified as high risk by the GEP
test, compared to only 50% (6 of 12) who had a
SLNpositive result. Additionally, we found that the median
time to recurrence for those who developed advanced
disease was only 1 year. National melanoma guidelines
specify that patient management should be tailored to an
individual’s probability of recurrence. Frequent follow-up
and intensified surveillance with imaging is recommended
for patients with high-risk clinicopathologic features,
while management of low-risk patients is generally
restricted to clinical follow-up at 3–12 month intervals [
A growing body of evidence supports cross-sectional
radiographic imaging as the most effective method for
detecting asymptomatic distant metastatic disease in patients
with stage II and III melanoma [
7, 10, 11
]. In this study, 6
of 12 patients with distant metastasis were originally
diagnosed with stage II disease. Of those, five were identified
as class 2, including two patients with stage IIA tumors.
While direct evidence of a benefit from surveillance has
not been published, considering the rapid time to event
observed in this interim analysis, and the accuracy of risk
prediction by the GEP test, increased surveillance with
imaging for class 2 patients might be useful, especially in
those patients who would not be offered surveillance
options based on stage.
The advantages of molecular testing for enhanced
prognosis are well documented for other diseases,
including breast cancer and ocular melanoma [
Consistent with previous studies, the results from this
prospective analysis indicate that the 31-gene GEP
test for melanoma, in combination with standard
clinicopathologic factors, can strengthen risk
determination and improve patient management. Better
risk prediction is particularly critical when considering
the recent advances in therapeutics for melanoma and
the evidence supporting better efficacy of
contemporary therapies if treatment is administered when tumor
burden is low [
This constitutes the first report of performance for the
prognostic 31-gene GEP test in a prospective population
of patients with cutaneous melanoma. In concordance
with prior retrospective studies, the test showed robust
ability to predict recurrence, distant metastasis and
death. While this report encompasses only an interim
analysis and, therefore, it is expected that additional
events will accrue in this population, the strong
statistical association with outcomes even at this early time
point provides assurance of the test’s prognostic value.
Additional file 1: Figure S1. Survival outcomes for stage I/II patients
with molecular classification by the 31-gene expression profile test. A)
Recurrence-free survival, B) distant metastasis-free survival, and C) overall
survival for Class 1 and Class 2 subjects with stage I or stage II disease
(n = 282). (TIFF 192 kb)
Additional file 2: Table S1. Cox regression analysis for distant
metastasis-free (DMFS) and overall survival (OS) in the 322-subject cohort.
(DOCX 12 kb)
AJCC: American Joint Committee on Cancer; ANOVA: Analysis of variance;
CAP/CLIA: College of American Pathologists/Clinical Laboratory Improvement
Amendments; CI: Confidence interval; CM: Cutaneous melanoma;
DMFS: Distant metastasis-free survival; GEP: Gene expression profile;
OS: Overall survival; RFS: Recurrence-free survival; SEER: Surveillance,
Epidemiology, and End Results; SLN: Sentinel lymph node
The authors wish to thank the physicians and clinical staff at each of the
contributing institutions, including Martin Fleming, MD, Adam Berger, MD,
Elizabeth Liotta, MD, Abdallah Khourdaji, MD, and Charles St. Hill, MD for
their contributions to the study. We would also like to recognize the
significant contributions to clinical data review and tissue processing by Jeff
Wilkinson, PhD, Natalie Lassen, PhD, John Stone, PhD, and Kristen
This study was partially sponsored by Castle Biosciences, Inc., which provided
financial compensation to those centers contributing melanoma tissue to
Availability of data and materials
The dataset analyzed during the current study is not publicly available due
to ongoing accrual of patient data.
RWC, CJ, and ECS conceptualized the study. RWC validated the study. KRC,
RWC, and BM formally analyzed the study. KRC, RWC, and BM wrote the
original draft. ECH, JRD, JL, JJS, KRC, BM, CJ, RWC, CLS, and KMM wrote,
reviewed, and edited the manuscript. RWC, BM, and KRC contributed to the
visuals of the study. RWC supervised the study. All authors have read and
approved this manuscript.
Ethics approval and consent to participate
The current study was approved by the following Institutional Review
– Thomas Jefferson Office of Human Research Institutional Review
– University of Virginia Institutional Review Board for Health Sciences
– University of Louisville Human Subjects Protection Program
– The University of Tennessee Health Science Center IRB
– Western Institutional Review Board (Northside Hospital, St. Louis
University, University of Cincinnati, Fairmont Dermatology, Elizabeth
Liotta, MD, LLC, South Carolina Skin Cancer Center, University of
Nevada School of Medicine)
Written informed patient consent was obtained from study participants
before enrollment in accordance with the Declaration of Helsinki.
Consent for publication
ECH and JJS are on Castle Biosciences’ Speaker Bureau. ECH, JJS, JL, and CLS
have received honoraria for advisory board participation. KRC, BM, CJ, and
RWC are employees of Castle Biosciences, Inc. and hold stock in the
company. All remaining authors declare that they have no competing
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