Inter- and Intra-Patient Heterogeneity of Response and Progression to Targeted Therapy in Metastatic Melanoma

Abstract

MAPK inhibitors (MAPKi) are active in BRAF-mutant metastatic melanoma patients, but the extent of response and progression-free survival (PFS) is variable, and complete responses are rare. We sought to examine the patterns of response and progression in patients treated with targeted therapy.
MAPKi-naïve patients treated with combined dabrafenib and trametinib had all metastases ≥5 mm (lymph nodes ≥15 mm in short axis) visible on computed tomography measured at baseline and throughout treatment.
24 patients had 135 measured metastases (median 4.5/patient, median diameter 16 mm). Time to best response (median 5.5 mo, range 1.7-20.1 mo), and the degree of best response (median -70%, range +9 to -100%) varied amongst patients. 17% of patients achieved complete response (CR), whereas 53% of metastases underwent CR, including 42% ≥10 mm. Metastases that underwent CR were smaller than non-CR metastases (median 11 vs 20 mm, P<0.001). PFS was variable among patients (median 8.2 mo, range 2.6-18.3 mo), and 50% of patients had disease progression in new metastases only. Only 1% (1/71) of CR-metastases subsequently progressed. Twelve-month overall survival was poorer in those with a more heterogeneous initial response to therapy than less heterogeneous (67% vs 93%, P = 0.009).
Melanoma response and progression with MAPKi displays marked inter- and intra-patient heterogeneity. Most metastases undergo complete response, yet only a small proportion of patients achieve an overall complete response. Similarly, disease progression often occurs only in a subset of the tumor burden, and often in new metastases alone. Clinical heterogeneity, likely reflecting molecular heterogeneity, remains a barrier to the effective treatment of melanoma patients.

Full text

Inter- and Intra-Patient Heterogeneity of Response and
Progression to Targeted Therapy in Metastatic
Melanoma
Alexander M. Menzies
1,2
*, Lauren E. Haydu
1,2
, Matteo S. Carlino
1,2,3,5
, Mary W. F. Azer
3
, Peter J. A. Carr
2,4
,
Richard F. Kefford
1,2,3,5
, Georgina V. Long
1,2
1Melanoma Institute Australia, Sydney, Australia, 2The University of Sydney, Sydney, Australia, 3Westmead Hospital, Crown Princess Mary Cancer Centre, Sydney,
Australia, 4Westmead Hospital, Department of Radiology, Sydney, Australia, 5Westmead Institute for Cancer Research, Westmead, Australia
Abstract
Background:
MAPK inhibitors (MAPKi) are active in BRAF-mutant metastatic melanoma patients, but the extent of response
and progression-free survival (PFS) is variable, and complete responses are rare. We sought to examine the patterns of
response and progression in patients treated with targeted therapy.
Methods:
MAPKi-naı
¨ve patients treated with combined dabrafenib and trametinib had all metastases $5 mm (lymph nodes
$15 mm in short axis) visible on computed tomography measured at baseline and throughout treatment.
Results:
24 patients had 135 measured metastases (median 4.5/patient, median diameter 16 mm). Time to best response
(median 5.5 mo, range 1.7–20.1 mo), and the degree of best response (median 270%, range +9to2100%) varied amongst
patients. 17% of patients achieved complete response (CR), whereas 53% of metastases underwent CR, including 42%
$10 mm. Metastases that underwent CR were smaller than non-CR metastases (median 11 vs 20 mm, P,0.001). PFS was
variable among patients (median 8.2 mo, range 2.6–18.3 mo), and 50% of patients had disease progression in new
metastases only. Only 1% (1/71) of CR-metastases subsequently progressed. Twelve-month overall survival was poorer in
those with a more heterogeneous initial response to therapy than less heterogeneous (67% vs 93%, P=0.009).
Conclusion:
Melanoma response and progression with MAPKi displays marked inter- and intra-patient heterogeneity. Most
metastases undergo complete response, yet only a small proportion of patients achieve an overall complete response.
Similarly, disease progression often occurs only in a subset of the tumor burden, and often in new metastases alone. Clinical
heterogeneity, likely reflecting molecular heterogeneity, remains a barrier to the effective treatment of melanoma patients.
Citation: Menzies AM, Haydu LE, Carlino MS, Azer MWF, Carr PJA, et al. (2014) Inter- and Intra-Patient Heterogeneity of Response and Progression to Targeted
Therapy in Metastatic Melanoma. PLoS ONE 9(1): e85004. doi:10.1371/journal.pone.0085004
Editor: Keiran Smalley, The Moffitt Cancer Center & Research Institute, United States of America
Received October 30, 2013; Accepted November 27, 2013; Published January 6, 2014
Copyright: ß2014 Menzies et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Program Grants from the National Health and Medical Research Council of Australia (NHMRC), Cancer Institute NSW,
Australian Cancer Research Foundation, the Melanoma Foundation of the University of Sydney and Melanoma Institute Australia. GlaxoSmithKline funded the
clinical trial from which these data were obtained. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have read the journal’s policy and have the following conflicts: AMM – Roche (H & T), GlaxoSmithKline (T). RFK – Roche (C),
GlaxoSmithKline (C), Novartis (C). GVL – Roche (C, T & H), GlaxoSmithKline (C), Novartis (C). None to declare for the remaining authors. There are no employment or
leadership positions, no stock ownership, no expert testimony. Note: (C) = Consultant advisor, (H) Honoraria and (T) Travel support for conference attendance. This
does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: alexander.menzies@sydney.edu.au
Introduction
Molecular heterogeneity exists in all cancers [1,2], particularly
melanoma [3–5]. Genetic divergence occurs during clonal
evolution, resulting in inter- and intra-tumoral molecular hetero-
geneity within patients [3,6,7]. Certain driver genetic aberrations
exist in all tumor cells within an individual, but several others exist
in subclones, conferring varying degrees of drug resistance [2].
Intrinsic resistance mechanisms present in subclones of the overall
tumor burden diminish the initial response to systemic treatment,
and these and acquired mechanisms result in disease progression.
Ultimately the presence or development of these mechanisms
influence the initial response to systemic treatment, time to
progression, and overall survival. The influence and heterogeneity
of the tumor micro-environment is also increasingly understood to
play a role in tumor cell heterogeneity and treatment outcome [8].
Clinically, inter- and intra-patient molecular heterogeneity is
manifest by the variable responses observed between and within
patients treated with targeted therapies. BRAF inhibitors, used as
single agents or in combination with MEK inhibitors, are active in
most patients with metastatic melanoma, but the extent of response
and time to progression are variable between patients, and complete
responses are uncommon [9–11]. Patterns of disease progression are
also variable, with existing metastases progressing or new metastases
developing at the same time as ongoing response in other metastases
[12,13]. The terms ‘‘mixed response’’ and ‘‘isolated progression’’
are now used commonly, however these terms have not yet been
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accurately defined, and there is little known as to the prevalence or
predictors of these phenomena, nor the clinical outcomes of patients
with these patterns of response and progression.
We therefore sought to examine the patterns of response and
progression to targeted therapy by measuring every metastasis
$5 mm via computed tomography (CT) in a cohort of patients
with metastatic melanoma treated with combined BRAF and
MEK inhibitors.
Patients and Methods
Patients and Treatment
All MAPK inhibitor naı
¨ve BRAF-mutant metastatic melanoma
patients treated with dabrafenib and trametinib (CombiDT) on
parts B–D of the BRF113220 Phase 1/2 [11] trial
(NCT01072175) at Westmead Hospital in association with
Melanoma Institute Australia were included for analysis. The
collection and analysis of clinical data was approved by the
Westmead and Royal Prince Alfred Hospitals Human Research
Ethics Committees (Protocol No. X11-0023 and HREC/11/
RPAH/32) and written informed consent was obtained from each
patient. Patients received a range of doses of dabrafenib and
trametinib. Patient demographic and disease characteristic data at
trial entry were collected.
Disease Assessments
CT scans of 3 mm slice thickness were performed at baseline
and then every 8 weeks as per the clinical trial protocol. In
Table 1. Patient demographics and clinical characteristics.
Feature All patients
Uniform Response
at First Scan*
Mixed Response
at First Scan*
P
-value
#
N%N%N%
Number of patients 24 100 15 62 9 38 –
Age (years)
Median 51 – 57 – 42 – 0.290
Range 29–78 – 28–77 – 38–74 –
Sex
Male 13 54 8 53 5 56 0.625
Female 11 46 7 47 4 44
BRAF genotype
V600E 20 85 13 87 7 78 0.486
V600K 4 15 2 13 2 22
ECOG PS
0 19 79 11 73 8 89 0.360
1521427111
AJCC Stage
M1a 5 21 3 20 2 22 0.418
‘
M1b 5 21 4 27 1 11
M1c 14 58 8 53 6 67
Baseline LDH
,16ULN 19 79 13 87 6 67 0.255
.16ULN 5 21 2 13 3 33
Drug doses (Dab/Tra)
300/2 8 33 6 40 2 22 Not Tested
300/1.5 1 4 0 0 1 11
300/1 4 17 2 13 2 22
300/0 then 300/2 at PD 2 8 2 13 0 0
150
,
/2 8 33 4 27 4 44
300
,
/2 141700
Dab with 2 mg Tra 17 71 11 73 6 67 0.539
Dab with ,2 mg Tra 7 29 4 27 3 33
Abbreviations: ECOG PS, Eastern Cooperative Oncology Group Performance Status; AJCC, American Joint Committee on Cancer; LDH, lactate dehydrogenase; ULN,
upper limit of normal; Dab, dabrafenib total daily dose; Tra, trametinib daily dose; PD, progressive disease.
,
hydroxymethylcellulose dabrafenib preparation.
‘
testing M1a & M1b versus M1c.
*Uniform response: $80% of metastases with a complete or partial response and no progressing or new metastases. Mixed response: ,80% of metastases with a
complete or partial response, or the presence of any progressing or new metastases.
#
testing uniform versus mixed response cohorts.
doi:10.1371/journal.pone.0085004.t001
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addition to the RECIST v1.1 assessments [14] conducted
prospectively as part of the clinical trial, a more detailed radiologic
assessment of every metastasis $5 mm diameter in long axis
(lymph nodes $15 mm in short axis) visible on CT was performed
on every scan. This was referred to as the ‘‘ALL metastasis’’
assessment, and was conducted retrospectively, blinded to the
RECIST assessment and clinical data. Measurements were made
on each scan to the nearest millimeter using the IntelePACSß
computer software program.
RECIST data were used only as a comparison to the ALL
metastasis assessment data to assess for concordance of these
measures for best overall response, time to best response (TTBR),
and progression-free survival (PFS) (see supplementary methods).
The patient’s overall response at each time point was
determined using similar criteria as RECIST [14], but included
all metastases $5 mm to calculate the sum of diameters (SoD).
Disease progression was defined as the development of new
metastases and/or a $20% and $5 mm increase in the sum of
diameters of all metastases from nadir.
In addition, a response was recorded for each individual
measured metastasis at each time point and classified as complete
response (CR, disappearance or to less than 10 mm for a lymph
node), partial response (PR, $30% reduction), stable disease (SD,
neither CR/PR/PD) or progressive disease (PD, $5 mm and
$20% growth).
At first radiologic assessment, for this study, a uniform response
was predefined as $80% of metastases having a complete or
partial response with no progressing or new metastases. A mixed
response was defined as ,80% of metastases having a complete or
partial response, or the presence of any progressing or new
metastases.
Statistical Analysis
Patient demographic and clinical features were tested for
association with uniform versus mixed response at first scan using
the Fisher’s Exact Test, Pearson’s x
2
, and/or the Mann Whitney
U test as appropriate. Overall survival (OS) and PFS were
calculated from the date of commencement of targeted therapies
to the date of last follow-up or date of progression, respectively.
Univariate time to event analyses were conducted with the
Kaplan-Meier method together with the Log Rank test for
comparison of categorical covariates, and with the Cox propor-
tional hazards method for continuous covariates. Multivariate
overall survival was conducted with the Cox proportional hazards
method. When comparing the two assessment methods (RECIST
and ALL metastasis), best overall response was deemed concor-
dant if there was #10% difference in the percentage degree of best
response and also within the same response category. Time to best
response and progression-free survival were concordant if they
occurred at the same time (on the same scan) by both measures.
All statistical analyses were conducted with IBM SPSS Statistic
v21.
Results
Patient Demographics and Disease Characteristics
Twenty-four patients were included for analysis. The patient
population was typical for patients with BRAF-mutant metastatic
melanoma; the median age of patients was 51 years, 54% of
patients were men, 85% of patients had the V600E genotype, and
58% of patients had stage M1c melanoma (Table 1). All patients
were MAPK inhibitor naı
¨ve. Although several dosing regimens
were administered, 71% of patients were treated with trametinib
at the recommended part two dose of 2 mg daily in combination
with dabrafenib from trial commencement (Table 1). Two patients
received dabrafenib monotherapy until disease progression, after
which 2 mg daily trametinib was added.
Baseline Disease Assessments
135 metastases from the 24 patients were included for
assessment (median 4.5 per patient, range 1–18), substantially
more than included as RECIST targets (N=56, median 2 per
patient, range 1–5) (Table 2). The median diameter of metastases
was the same as RECIST targets (16 mm), but ranged from a
minimum 5 mm rather than 10 mm. Seventy-six percent (N=102)
of metastases were $10 mm, and 46 (45%) of these had not been
Table 2. Baseline disease assessments by examining RECIST
targets versus ALL metastases.
RECIST targets ALL metastases
Total 56 135
Diameter (mm)
Median 16 16
Range 10–108 5–108
Number $10 mm 56 102
Number per patient
Median 2 4.5
Range 1–5 1–18
Sum of Diameters (mm)
Median 48 100
Range 10–174 11–317
Site of metastases (n, %)
SQ 13, 23% 43, 32%
Lymph node 10, 18% 15, 11%
Lung 16, 29% 48, 36%
Liver 12, 21% 24, 18%
Gastrointestinal* 5, 9% 5, 4%
Abbreviations: SQ, subcutaneous and soft tissue.
*Gastrointestinal sites include adrenal (N=3), small bowel (N= 1), pancreas
(N=1).
doi:10.1371/journal.pone.0085004.t002
Figure 1. The proportions of categories of response a) by
patients (
N
= 24), b) by metastases (
N
= 135). Abbreviations: CR,
complete response; PR, partial response; SD, stable disease; PD,
progressive disease.
doi:10.1371/journal.pone.0085004.g001
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included as RECIST targets. Most frequent sites of disease
included lung and subcutaneous/soft tissue (SQ) (36% and 32%
respectively) (Table 2).
Overall Patient Response
The majority of patients had a response to treatment. When all
metastases $5 mm were measured, 17% (N= 4) of patients had a
complete response and 75% (N= 18) had a partial response to
treatment (Figure 1a). No patients had progressive disease as best
response. The median time to best response was diverse (median
5.5 months, range 1.7 to 20.1 months), and there was variability in
the degree of response at first assessment (median change 249%,
range +9to295%), the kinetics of response (% change over time),
and the degree of best response (median change 270%, range +9
to 2100) within the patient population (Figure 2). The degree of
best overall response by ALL metastasis and RECIST assessment
measures was concordant in 19/24 (79%) patients (Figure 3), the
category of response was concordant in 20/24 (83%) patients, and
TTBR was concordant in 17/24 (71%) patients.
Individual Metastasis Response
Ninety-three percent (126/135) of metastases had some
reduction in size with treatment and 84.5% (114/135) had either
a complete or partial response. Only 2.2% (3/135) of metastases
demonstrated progressive disease at first assessment, all within the
same patient. Importantly, 52.6% (N= 71) of metastases had a
complete response (Figure 1b, Figure 4). Of 102 metastases
$10 mm diameter, 42% (43/102) had a complete response, and
41% (23/56) RECIST target metastases had complete response.
The median TTBR for all metastases was 12.1 weeks (range
7.3–87.6 weeks) (Table 3). Compared with subcutaneous and soft
tissue metastases (median 8.3 weeks), median TTBR was
significantly longer for lymph nodes (30.3 weeks, P=0.009) and
liver metastases (31.7 weeks, P= 0.038), but not significantly
different for lung metastases (8.0 weeks, P= 0.076). TTBR was
significantly shorter as metastases decreased in size (HR = 0.98,
95% CI 0.96–0.998, P= 0.030), and the degree of response at first
scan correlated with the degree of best response (R
2
= 0.6613,
p,0.001) (Figure 5).
There was no significant difference in the rate of complete
response by disease site (P.0.05) (Table 3). Metastases that had a
complete response were significantly smaller compared with
metastases that had PD/SD/PR (median 11 mm vs 20 mm,
P,0.001). This factor remained significant when stratifying by
disease sites for lung, liver, and SQ metastases (all P,0.05), but
not for lymph nodes (N= 15).
Plots of the response of individual metastases over time within
individual patients (Figure 6) demonstrated the marked variability
in the degree of first response and best response, the kinetics of
Figure 2. Inter-patient heterogeneneity of response and progression with CombiDT. Plot of the percent change in the sum of diameters
of all metastases $5 mm within an individual patient compared to baseline at various time points during treatment with CombiDT until disease
progression. Each line represents an individual patient. Abbreviations: E, disease progressing due to existing lesions; N, new lesions; N+E, new and
existing lesions; OR, ongoing response without progression; T, treatment ceased due to toxicity.
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Figure 3. The degree of overall best response for each patient by RECIST and ALL metastasis disease assessments.
doi:10.1371/journal.pone.0085004.g003
Figure 4. The best response of each individual metastasis within each patient. Abbrevations: CR, complete response; PR, partial response;
SD, stable disease; PD, progressive disease.
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response, and the time to best response for each individual
metastasis.
Sixty-two percent (15/24) of patients had a uniform response at
first assessment, and 38% (9/24) of patients had a mixed response.
Patient demographics, disease characteristics and CombiDT doses
received were similar in the two groups (Table 1). The two patients
that received dabrafenib monotherapy until disease progression
had a uniform response to treatment.
Patterns of Disease Progression
At the time of analysis 18 (75%) patients had disease progression
(PD) (Figure 2). Median PFS was 8.2 months (range 2.6 to 18.3
months). PFS was highly concordant by ALL metastasis and
RECIST assessment methods (14/18, 77% of patients). Fifty
percent of patients progressed in new metastases only, 44% in
existing metastases only, and 6% in both new and existing
metastases simultaneously. There was no dominant site of disease
progression, but four (22%) patients with no prior history of brain
metastases progressed in new metastases in the brain. At time of
Figure 5. Correlation of the response of individual metastases at first scan versus best response (
N
= 135).
doi:10.1371/journal.pone.0085004.g005
Table 3. Factors influencing individual metastasis response to treament; time to best response by metastasis site, and the effect of
metastasis site and size on response.
Site of
metastasis
Median Time to Best
Response (Range) Weeks CR PR/SD/PD
P
-value*
N
Median Size
(Range) mm
N
Median Size
(Range) mm
All 12.1 (7.3–87.6) 71 11 (5–44) 64 20 (5–108) ,0.001
SQ 8.3 (7.6–56.3) 24 10 (7–30) 19 20 (10–98) ,0.001
LN 30.3 (7.7–87.6) 7 22 (15–31) 8 21 (17–48) 0.38
Lung 8.0 (7.3–63.9) 27 9 (5–44) 21 15 (5–44) 0.036
Liver 31.7 (7.7–56.0) 10 18 (7–27) 14 31 (16–47) 0.006
*P-value for comparison of median size of lesions with CR versus non-CR, Mann Whitney U test.
Abbreviations: CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; SQ, subcutaneous and soft tissue; LN, lymph node.
doi:10.1371/journal.pone.0085004.t003
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Figure 6. Intra-patient heterogeneneity of response and progression with CombiDT. Example plots of the percent change in the diameter
of individual metastases within four patients (a-d) compared to baseline at various time points during treatment until overall disease progression. The
degree and kinetics of response of individual metastases vary within a patient. Similarly, progression often occurs only in a subset of the overall
tumour burden. Patient D had disease progression in new lesions only.
doi:10.1371/journal.pone.0085004.g006
Figure 7. Intra-patient heterogeneity of disease progression. The number and type of metastases progressing at time of disease progression.
doi:10.1371/journal.pone.0085004.g007
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PD, the median proportion of metastases progressing in an
individual compared to the total tumor burden ever (including all
metastases at baseline and new metastases) was 49% (range 6 to
100%) (Figure 7). Only one metastasis that underwent complete
response subsequently progressed (1.4%, 1/71).
Survival Analyses
The burden of disease at baseline (SoD of ALL metastasis) and
the degree of overall response at first scan did not correlate with
PFS, 12-month survival or OS (Table 4). The type of initial
response (uniform versus mixed) similarly did not correlate with
PFS. 12-month and OS, however was significantly inferior for
mixed responders (67% and median 14.2 months) compared with
uniform responders (93% and median not reached, P= 0.009), a
result which remained significant when adjusting for baseline
disease burden (HR = 5.1, 95% CI: 1.2–21.1, P= 0.025).
Discussion
This is the first systematic study of patterns of clinical response
and progression to MAPK targeted therapy in all assessable
individual metastases in patients with metastatic melanoma,
demonstrating that melanoma response and progression is
heterogeneous between and within patients. Most individual
metastases undergo a complete response to treatment, yet only a
small proportion of patients achieve an overall complete response.
Disease progression is similarly heterogeneous, both in timing and
nature. Many patients have disease progression in a subset of their
overall tumor burden, and often in new metastases only.
Metastases that initially undergo complete response with treatment
seldom subsequently progress, and a more heterogeneous initial
response to treatment is associated with shorter overall survival.
Results of this study are strengthened by the detailed clinical
assessment of patients on the most highly active targeted therapy
in melanoma [11], the use of a standard modality for disease
assessment (3 mm slice thickness CT) at predetermined specified
time points, inclusion of every metastasis visible and measurable
on CT scan ($5mmor$15 for lymph nodes), and an assessment
of every individual metastasis across every time point from baseline
prior to treatment until disease progression. The inclusion of all
metastases as targets for assessment, as opposed to the maximum 5
target metastases for RECIST (and maximum 2 in any one organ)
provided a more detailed assessment, with increased ability to
assess for intra-patient heterogeneity. This question has been
previously addressed in studies of
18
F-labelled fluorodeoxyglucose
positron emission tomography (PET) metabolic response to single
agent BRAF inhibitors at day 15, with varying results, one study
examining 5 target metastases and observing a homogeneous
response [15], while the other examined every metastasis and
observed heterogeneity [16].
In this study, most metastases achieved a complete response
with treatment. These metastases were located at any body site,
and tended to be smaller than those that did not undergo complete
response, however, some metastases several centimeters in
diameter still had complete response. The reasons why smaller
metastases have a higher complete response rate may be because
they have to shrink less to become clinically occult, however, the
observation that these metastases seldom subsequently progress
perhaps supports alternative hypotheses, for example, they
undergo a more effective secondary immune response [17,18],
or contain less molecular or microenvironmental heterogeneity,
with less resistant tumor subclones. This observation warrants
further research, particularly as larger metastases may be
amenable for resection prior to therapy, and adjuvant trials for
occult metastatic disease are in progress.
Despite heterogeneity observed in the degree and timing of best
overall response amongst patients, most metastases undergo the
majority of tumor shrinkage by 3 months of treatment. Metastases
that have not undergone meaningful initial clinical response (e.g.
persisting local symptoms) by 3 months may therefore warrant
treatment with local therapy (surgery, radiotherapy). Furthermore,
in selected patients where the vast majority of metastases have
undergone complete response, remaining metastases could be
treated locally to render the patient free of overt disease. The
observation that the majority of tumor response occurs early
during treatment also suggests that additional systemic therapies
(e.g. immunotherapy) should be incorporated early in the course of
MAPK inhibitor treatment. Translational data demonstrating
early immune cell infiltration into tumors soon after treatment
commencement (as early as day 3) further supports this, and may
indicate that immunotherapies should be combined from the start
of MAPK inhibitor treatment [17,18].
Disease progression occurred at varying time points among the
patient cohort, and there was a high rate of disease progression
due to the emergence of new metastases. Often, patients
progressed in only a few metastases, with the remainder of disease
under treatment control. In this instance, disease progression may
therefore not equate to overt treatment failure, and local treatment
(e.g. surgery, radiotherapy) may be delivered to progressing
metastases with systemic treatment continued for ongoing clinical
benefit to the remainder of drug-sensitive disease [12,13,19]. This
approach may be more beneficial than a switch to immunotherapy
(e.g. ipilimumab), as little efficacy has been observed in this setting
[20,21], likely at least in part due to the release of MAPK
inhibition, whereas relative ongoing MAPK inhibition still occurs
in resistant tumors with continued MAPK inhibitor treatment
[22].
In this study cohort, a mixed response at first assessment
correlated with shorter overall survival, but not progression-free
survival. This result was likely influenced by small number of
patients, the doses of therapy received, and the fact that many
patients progressed in new metastases alone. Subsequent treat-
ments may have also influenced overall survival. Despite this,
however, this finding warrants validation in future studies, as
biomarkers to predict treatment outcome are scant, and the
method of categorizing response in this study could be reproduced
without additional procedures such as PET.
The clinical heterogeneity of tumor response and progression
demonstrated in this study likely reflects underlying molecular
heterogeneity. The majority of the melanoma burden in patients is
Table 4. Univariate progression-free and overall survival.
Outcome Factor
P
-value
PFS Baseline SoD 0.101
Percent Response at First Scan 0.084
Uniform versus Mixed Response at First Scan* 0.124
OS Baseline SoD 0.349
Percent Response at First Scan 0.105
Uniform versus Mixed Response at First Scan* 0.009
Abbreviations: SoD, sum of diameters.
*Uniform response: $80% of metastases with a complete or partial response
and no progressing or new metastases. Mixed response: ,80% of metastases
with a complete or partial response, or the presence of any progressing or new
metastases.
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sensitive to MAPK inhibition, however, a varying proportion of
primarily resistant subclones exist at baseline, and resistance may
also be acquired during treatment. This heterogeneity complicates
clinical management, confounds biopsy driven biomarker re-
search, and remains a barrier to the effective treatment of
melanoma patients, including the deployment of biopsy-driven
adaptive clinical trial design. A broader multi-targeted treatment
approach from the outset (e.g, MAPK and PI3K inhibitors) may
improve response rates and prolong survival, but will likely face
the same problem of clonal drug resistance and treatment failure.
Combinations of MAPK inhibitors and novel immunotherapies
(e.g. PD-1 antibodies) may provide more complete and durable
responses.
Acknowledgments
We acknowledge the assistance of Arthur Clements, Clara Lee, Matthew
Chan, Vicky Wegener, Rebecca Hinshelwood, Amie Cho, Katherine
Carson, Joanna Jackson, Andrea Del Pilar Forero Velandia, Jacob
Cunningham, and Kiran Patel (GSK).
Author Contributions
Conceived and designed the experiments: AMM GVL. Performed the
experiments: AMM. Analyzed the data: AMM LEH GVL. Contributed
reagents/materials/analysis tools: PJAC RFK GVL. Wrote the paper:
AMM LEH MSC MWFA PJAC RFK GVL.
References
1. Meacham CE, Morrison SJ (2013) Tumour heterogeneity and cancer cell
plasticity. Nature 501: 328–337.
2. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, et al. (2013)
Cancer genome landscapes. Science 339: 1546–1558.
3. Yancovitz M, Litterman A, Yoon J, Ng E, Shapiro RL, et al. (2012) Intra- and
inter-tumor heterogeneity of BRAF(V600E))mutations in primary and metastatic
melanoma. PLoS ONE 7: e29336.
4. Takata M, Morita R, Takehara K (2000) Clonal heterogeneity in sporadic
melanomas as revealed by loss-of-heterozygosity analysis. Int J Cancer 85: 492–
497.
5. Lin J, Goto Y, Murata H, Sakaizawa K, Uchiyama A, et al. (2011) Polyclonality
of BRAF mutations in primary melanoma and the selection of mutant alleles
during progression. British Journal of Cancer 104: 464–468.
6. Katona TM, Jones TD, Wang M, Eble JN, Billings SD, et al. (2007) Genetically
heterogeneous and clonally unrelated metastases may arise in patients with
cutaneous melanoma. Am J Surg Pathol 31: 1029–1037.
7. Wilmott JS, Tembe V, Howle JR, Sharma R, Thompson JF, et al. (2012)
Intratumoral Molecular Heterogeneity in a BRAF-Mutant, BRAF Inhibitor-
Resistant Melanoma: A Case Illustrating the Challenges for Personalized
Medicine. Molecular Cancer Therapeutics 11: 2704–2708.
8. Junttila MR, de Sauvage FJ (2013) Influence of tumour micro-environment
heterogeneity on therapeutic response. Nature 501: 346–354.
9. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, et al. (2011)
Improved survival with vemurafenib in melanoma with BRAF V600E mutation.
New England Journal of Medicine 364: 2507–2516.
10. Hausc hild A, Grob JJ, Demidov LV, Jouary T, Gutzmer R, et al. (2012)
Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label,
phase 3 randomised controlled trial. Lancet 380: 358–365.
11. Flahert y KT, Infante JR, Daud A, Gonzalez R, Kefford RF, et al. (2012)
Combined BRAF and MEK inhibition in melanoma with BRAF V600
mutations. New England Journal of Medicine 367: 1694–1703.
12. Kim KB, Flaherty KT, Chapman PB, Sosman JA, Ribas A, et al. (2011) Pattern
and outcome of disease progression in phase I study of vemurafenib in patients
with metastatic melanoma (MM). Journal of Clinical Oncology 29: (abstract
8519).
13. Azer MF, Menzies AM, Haydu LE, Kefford RF, Long GV (2013) Patterns of
Response and Progression in Patients with BRAF-mutant Melanoma Metastatic
to the Brain treated with Dabrafenib. Cancer: in press.
14. Eisenhau er EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, et al. (2009)
New response evaluation criteria in solid tumours: revised RECIST guideline
(version 1.1). European Journal of Cancer 45: 228–247.
15. McArthur GA, Puzanov I, Amaravadi R, Ribas A, Chapman P, et al. (2012)
Marked, homogeneous, and early [18F]fluorodeoxyglucose-positron emission
tomography responses to vemurafenib in BRAF-mutant advanced melanoma.
Journal of Clinical Oncology 30: 1628–1634.
16. Carlino MS, Saunders CA, Haydu LE, Menzies AM, Martin Curtis C Jr, et al.
(2013) (18)F-labelled fluorodeoxyglucose-positron emission tomography (FDG-
PET) heterogeneity of response is prognostic in dabrafenib treated BRAF
mutant metastatic melanoma. European Journal of Cancer 49: 395–402.
17. Wilmott JS, Long GV, Howle JR, Haydu LE, Sharma RN, et al. (2012) Selective
BRAF inhibitors induce marked T-cell infiltration into human metastatic
melanoma. Clinical Cancer Research 18: 1386–1394.
18. Frederick DT, Piris A, Cogdill AP, Cooper ZA, Lezcano C, et al. (2013) BRAF
Inhibition Is Associated with Enhanced Melanoma Antigen Expression and a
More Favorable Tumor Microenvironment in Patients with Metastatic
Melanoma. Clinical Cancer Research 19: 1225–1231.
19. Chan M, Haydu L, Menzies AM, Azer MWF, Klein O, et al. (2013) Clinical
characteristics and survival of BRAF-mutant metastatic melanoma patients
treated with BRAF inhibitor dabrafenib or vemurafenib beyond disease
progression. Journal of Clinical Oncology 31: (abstract 9062).
20. Ackerman A, Klein O, McDermott DF, Lawrence DP, Gunturi A, et al. (2013)
Outcomes of patients with metastatic melanoma treated with immunotherapy
prior to or after BRAF inhibitors. Cancer: in press.
21. Ascierto PA, Simeone E, Giannarelli D, Grimaldi AM, Romano A, et al. (2012)
Sequencing of BRAF inhibitors and ipilimumab in patients with metastatic
melanoma: a possible algorithm for clinical use. Journal of Translational
Medicine 10: 107.
22. Carlino MS, Gowrishankar K, Saunders CA, Pupo GM, Snoyman S, et al.
(2013) Antiproliferative effects of continued mitogen-activated protein kinase
pathway inhibition following acquired resistance to BRAF and/or MEK
inhibition in melanoma. Molecular Cancer Therapeutics 12: 1332–1342.
Heterogeneity of Tumour Response in Melanoma
PLOS ONE | www.plosone.org 9 January 2014 | Volume 9 | Issue 1 | e85004