Future perspectives in melanoma research. Meeting report from the "Melanoma research: a bridge from Naples to the world. Napoli, December 5th-6th 2011".

Abstract

After more than 30 years, landmark progress has been made in the treatment of cancer, and melanoma in particular, with the success of new molecules such as ipilimumab, vemurafenib and active specific immunization.After the first congress in December 2010, the second edition of "Melanoma Research: a bridge from Naples to the World" meeting, organized by Paolo A. Ascierto (INT, Naples, Italy), Francesco M. Marincola (NIH, Bethesda, USA), and Nicola Mozzillo (INT, Naples, Italy) took place in Naples, on 5-6 December 2011. We have identified four new topics of discussion: Innovative Approaches in Prevention, Diagnosis and Surgical Treatment, New Pathways and Targets in Melanoma: An Update about Immunotherapy, and Combination Strategies. This international congress gathered more than 30 international faculty members and was focused on recent advances in melanoma molecular biology, immunology and therapy, and created an interactive atmosphere which stimulated discussion of new approaches and strategies in the field of melanoma.

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FUTURE PERSPECTIVES IN MELANOMA RESEARCH.
Meeting report from the “Melanoma Research: a
bridge from Naples to the World. Napoli,
December 5th–6th2011”
Ascierto et al.
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MEETING REPORTOpen Access
FUTURE PERSPECTIVES IN MELANOMA RESEARCH.
Meeting report from the “Melanoma Research: a
bridge from Naples to the World. Napoli,
December 5th–6th2011”
Paolo A Ascierto1,21*, Antonio M Grimaldi1,21, Brendan Curti2, Mark B Faries3, Soldano Ferrone4, Keith Flaherty5,
Bernard A Fox6, Thomas F Gajewski7, Jeffrey E Gershenwald8, Helen Gogas9, Kenneth Grossmann10,
Axel Hauschild11, F Stephen Hodi12, Richard Kefford13, John M Kirkwood14, Sancy Leachmann10, Michele Maio15,
Richard Marais16, Giuseppe Palmieri17, Donald L Morton3, Antoni Ribas18, David F Stroncek19, Rodney Stewart10,
Ena Wang20, Nicola Mozzillo1and Franco M Marincola20
Abstract
After more than 30 years, landmark progress has been made in the treatment of cancer, and melanoma in
particular, with the success of new molecules such as ipilimumab, vemurafenib and active specific immunization.
After the first congress in December 2010, the second edition of “Melanoma Research: a bridge from Naples to the
World” meeting, organized by Paolo A. Ascierto (INT, Naples, Italy), Francesco M. Marincola (NIH, Bethesda, USA), and
Nicola Mozzillo (INT, Naples, Italy) took place in Naples, on 5–6 December 2011. We have identified four new topics
of discussion: Innovative Approaches in Prevention, Diagnosis and Surgical Treatment, New Pathways and Targets in
Melanoma: An Update about Immunotherapy, and Combination Strategies.
This international congress gathered more than 30 international faculty members and was focused on recent
advances in melanoma molecular biology, immunology and therapy, and created an interactive atmosphere which
stimulated discussion of new approaches and strategies in the field of melanoma.
Introduction
This year, the Melanoma Research Bridge meeting was held
in Napoli on 5–6thDecember 2011 (Figure 1). The scientific
board selected four topics to be discussed during the two-
day meeting: Innovative approaches in prevention, diagnosis
and surgical treatment; New pathways and new targets in
melanoma: an update; Immunotherapy: new evidence;
Combination strategies.
The meeting started with a video lecture by Donald
Morton about the role of surgery after the new active sys-
temic medical therapy. Treatment of distant metastatic
melanoma is still inadequate, as there were no systemic
treatments with documented survival advantage until 2010/
2011 with the approval of ipilimumab and vemurafenib.
Prior to this, the 5-year median and overall survival for
stage IV melanoma was only 8–10 months and 2.3%,
respectively, while a meta-analysis by Korn et al. of all phase
II cooperative group trials suggested that no systemic ther-
apy evaluated in that setting was better than any other (that
is, no better than no treatment at all). Ipilimumab, Anti-
CTLA-4 Antibody, was tested in two phase III trials and
both showed a significant improvement in overall survival.
However, grade 3 or 4 toxicity was reported in 56.3% of
patients receiving ipilimumab, and the cost of the drug is
over $120,000.
Vemurafenib, a selective BRAF inhibitor, demonstrated
a survival benefit in one phase III trial (at 6 months OS
was 84% vs. 64% for dacarbazine). However, only 50% of
metastatic melanoma patients have the V600 BRAF mu-
tation and most responses are transient (90% of patients
* Correspondence: paolo.ascierto@gmail.com
1Department of Melanoma, Sarcoma, and Head and Neck Disease, Istituto
Nazionale Tumori Fondazione Pascale, Naples, Italy
21Unit of Medical Oncology and Innovative Therapy, Istituto Nazionale per lo
Studio e la Cura dei Tumori “Fondazione G. Pascale”, Via Mariano Semmola,
80131 Naples, Italy
Full list of author information is available at the end of the article
© 2012 Ascierto et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Ascierto et al. Journal of Translational Medicine 2012, 10:83
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progress within 9 months). New approaches to treatment
of metastatic melanoma are still needed.
Conventional logic is that surgical resection (locoregional
treatment) is not indicated with multiple metastases to
distant organ sites because such patients have widely disse-
minated melanoma. But multiple series indicate long-term
survival following resection of solitary distant metastases
for melanoma, and a new look at surgery for metastatic
melanoma is warranted. In fact 86% of patients presenting
with distant melanoma metastases have only 1–3 sites of
metastases in only 1 or 2 organs and only subsequently
develop widespread disease. This suggests that there may
be sequential progression or a metastatic cascade of disease
from one site to another.
The rationale for cytoreduction in metastatic cancer is
supported by: a) low morbidity and mortality (<1%) for
metastasectomy; b) improved radiographic staging allow-
ing for better selection; c) the observation that most
patients have 1–3 initial sites of disease; d) the fact that
the cost is less than many current medical therapies.
Also, biologic evidence of the metastatic cascade derived
from animal models suggests that metastases can
metastasize. Clinical case reports support this evidence,
and circulating tumor cell analyses have demonstrated a
marked reduction of circulating tumor cells after resec-
tion of metastatic disease.
All of this leads to the hypothesis that one consideration
for the initial treatment of metastatic melanomas is
complete resection. Data supporting this hypothesis in-
clude those derived from several phase II trials of adjuvant
immunotherapy after resection of stage IV melanoma. Sev-
eral cases were presented featuring patients with multiple
sites of disease resected over multiple years and then
enjoying prolonged disease-free survival (8-13+ years).
Sites of disease included lung, bowel, adrenal gland, and
brain. Overall survival of patients with stage IV disease
treated in this manner was 39% at 5 years and 30% at ten
years.
Post-surgical adjuvant immunotherapy has also been pur-
sued. A large randomized trial comparing a melanoma cell
line-based vaccine with placebo (both groups received adju-
vant BCG injections) in patients with resected stage IV
melanoma was performed. Patients were stratified by M1a
vs M1b/c and by the number of individual lesions (1, 2–3,
4–5). There was no difference in disease free survival (DFS)
(HR 0.91 favoring vaccine, p=0.418) or in overall survival
(OS) (HR 1.18 favoring placebo p=0.245). However, sur-
vival for both randomized groups was excellent (DFS: Me-
dian 8.3 mo vaccine, 7.2 mo placebo; DFS 5 yrs: 27.4%
vaccine, 20.9% placebo; OS: Median 31.5 mo vaccine, 38.7
mo placebo; OS 5 yrs: 39.6% vaccine, 44.9% placebo). These
excellent outcomes were seen for both M1a and M1b/c
patients and there was no difference between patients with
a solitary metastasis and those with 2–3 metastases. Even
among patients with 4–5 metastases there were long-term
survivors.
Among those patients whose disease recurs after initial
metastasectomy, there is also a role for re-resection. From
JWCI phase II data, 211 patients underwent initial metas-
tasectomy. Among these patients 131 had recurrence and
Figure 1 Majority of attendants of the Bridge meeting in Naples.
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were managed non-operatively (n=49), with complete
resection (n=40) or with incomplete resection (n=42).
Median survival (complete resection 17.4 months, incom-
plete resection 12.6 months, non-operative 5.6 months)
(5 yr survival: complete resection 19.3%, incomplete 6.5%,
non-operative 2.0%) suggest that patients may have bene-
fitted from resection. Similarly in the MMAIT IV Can-
vaxin vaccine trial, among 308 patients with recurrence,
154 were treated surgically and 154 were treated without
surgery. Median survival times (post-recurrence) were
better for the surgery group (26.5 months vs. 9.0 months)
as was the 5-year survival rate (26.5% vs. 7.0%).
Remarkably high survivals seen in phase II trials were
confirmed in the phase III, multicenter trials for resec-
tion with adjuvant BCG. The source of these good out-
comes is not clear and may be from patient selection or
the effectiveness of surgery with or without BCG as an
immune adjuvant. A new trial is underway to evaluate
these possibilities. The trial enrolls patients with resect-
able stage IV melanoma and stratifies by sites of metasta-
sis and number of metastatic lesions. Patients are
assigned to one of three arms: surgery alone, surgery+
BCG, and best medical therapy. Crossover is allowed at
the time of progression and the primary endpoint is
overall survival.
After Donald Morton’s lecture at the Bridge Congress
a discussion began on the role of BCG as an adjunct to
surgery alone. Among the topics discussed was the possi-
bility that biology is driving the more favorable outcome
of those patients, who are fully resectable for stage IV
disease, in contrast to those patients who are not fully
resectable and not considered for surgery because they
have disseminated stage IV disease. In contrast to Don-
ald Morton’s view, other oncologists argued that the dif-
ference in tumor biology (slow versus fast growing
disease) accounted for the difference in survival and not
the surgery. Published results of the two phase III trials
on Canvaxin are expected shortly, but they will not pro-
vide evidence that surgery plus BCG alone is better than
surgery plus BCG and Canvaxin. The results of surgery
alone might be favorable, but can, at least in part, be
explained by patient selection. The relative importance
of surgical treatment versus tumor biology and patient
selection remains controversial.
Innovative approaches in prevention, diagnosis and
surgical treatment
The meeting began with a discussion about the role
of melanoma genetic testing in prevention and early de-
tection. Melanoma-prone families comprise a minority of
patients, but they have the greatest risk of developing the
disease. Prevention and early detection play an essential
role. To educate people on the risks of photodamage and
melanoma, we must translate knowledge into changes in
behavior; this means understanding cognitive processes. In
a study at the Huntsmann Cancer Institute, the 52 patients
enrolled were divided into three categories: p16 positive
with a personal history of melanoma, p16 positive without
a personal history of melanoma, and p16 negative without
a history of melanoma. The study found that reporting of
p16 genetic test results was associated with significant im-
provement in the frequency of performance of self skin
examinations and a reduction in sunburns. Genetic test
reporting also improved compliance with annual total
body skin examinations by health care professionals in
the p16 positive group that had not had a melanoma.
Importantly, baseline compliance with these recommen-
dations was poor when counseling was based on familial
risk rather than on the genetic test report. These data
suggest that the process of genetic test reporting
enhances the patient’s ability to comply with prevention
and early detection recommendations. The development
of cognitive models that explain why genetic test report-
ing hasthispositiveeffect
generalizable and effective prevention education for
sporadic melanoma as well. Larger trials are needed to
further this effort.
Targetable chemoprevention pathways exist in melanoma
and are being exploited in high risk patients. One patho-
genetic mechanism for melanoma initiation is oxidative
stress and resultant DNA damage while immune evasion is
a mechanism in the promotion/progression phase. Based
on the success of ASA in a high-risk human model
(Lynch), a melanoma high-risk cohort is being recruited in
preparation for analogous prevention trials in melanoma.
Prevention has a greater potential impact than therapy on
cancer because it impacts both morbidity & mortality and
melanoma is an ideal cancer for prevention because it can
be readily identified and has a well-established environ-
mental cause. To summarize,, melanoma has targetable
pathways that can be assayed in accessible tissues using
relevant biomarkers in genetically characterized high-risk
study participants.
A candidate chemoprevention agent for melanoma is
sulforaphane. This agent has been isolated from broccoli
sprouts (some studies have demonstrated that crucifer-
ous vegetables are protective against cancer), and is an
active agent identified by classic medicinal chemistry
approaches with antioxidant activity. The antioxidant
effect is accomplished via activation of the Nrf-2/ARE
pathway and enhanced immunologic activity via STAT
activation, leading to potential reversal of immuno-eva-
sion. Predisposition pathways that are potentially target-
able with sulforaphane include MC1R and p16. MC1R
variants confer 2–4 fold increased risk for melanoma,
while p16 mutation carriers have about a 76% lifetime
risk for melanoma development. Importantly, p16 muta-
tion carriers who also have an MC1R variant are at even
may leadto more
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higher risk. A novel oxidative stress function for p16 has
been identified: RNAi knockdown of p16 leads to
increased oxidative stress that can be reversed by an anti-
oxidant and RNAi knockdown of p16 results in increased
oxidative DNA damage. Genetic epidemiology studies also
suggest that MC1R & p16 pathways may cooperate and this
effect may be accomplished in part by simultaneously
impacting oxidative stress pathways. Sulforaphane enhances
antioxidant gene expression in melanocytes and in human
epidermis ex-vivo and may effectively target oxidative stress,
by bypassing the molecular defects in these high-risk
groups. A Phase I/II chemoprevention trial of sulforaphane
is needed to validate efficacy in surrogate nevi.
The Congress included an interesting discussion about
the current concepts and future directions in melanoma
staging and prognosis beyond the American Joint Com-
mittee on Cancer (AJCC)/ (International Union for the
Control of Cancer (UICC) melanoma staging system.
Overall, in the most recent version of the AJCC melanoma
staging system (7thEdition) no major changes were
recommended for TNM and stage grouping criteria for
stages I, II and III melanoma. Earlier models were vali-
dated using an evidence-based approach and an AJCC
melanoma database comprising over 50,000 pts [1-3].
Highlights of revisions to the staging system include the
following: (1) mitotic rate (measured in mitoses/mm2
using the dermal “hot spot” approach) was identified as in-
dependent prognostic factor, and based on a threshold of
at least 1 mitosis/mm2, was included as a criterion for de-
fining T1b melanoma; (2) immunohistochemical detection
of nodal metastases is acceptable, and (3) there is no lower
limit to designate N+disease [1-3]. These changes were
also approved with Union for International Cancer Con-
trol (UICC) representation on the melanoma staging com-
mittee. In multivariate survival analyses in melanoma,
mitotic rate was the second most powerful independent
predictor of survival after tumor thickness [1,4]. Along
with microstaging of all primary melanomas, pathological
nodal staging for stage Ib-IIc melanoma helps to minimize
prognostic heterogeneity within stages and incorporate
sentinel lymph node assessment into the staging system
[1,3].
Survival data of 7,635 patients with metastatic melanoma
at distant sites (stage IV) sub-grouped by the site of meta-
static disease and serum lactate dehydrogenase (LDH)
levels were analyzed. As had been previously shown in earl-
ier, albeit smaller studies, patients with distant metastatic
disease only in the skin have a better survival than patients
with lung metastasis or visceral metastasis; patients with
lung metastasis also have a more favorable survival profile
than patients with other visceral disease[1,3]. Importantly,
patients with distant metastasis and elevated LDH levels
also have a poorer survival than patients with normal LDH
levels [1,3].
Limitations exist in traditional staging systems, and
include the following: (1) number of characteristics that
can be included - ie, patient, tumor, etc; (2) inability to
use continuous variables; (3) estimates of survival based
only on the time of diagnosis; and (4) TNM-based sta-
ging applies to large cohorts of patients, but is not truly
individualized [5]. To improve melanoma staging and
prognosis, it is evident that there is a need to develop
and integrate new statistical models and contemporary
analytic approaches that better inform using multiple
characteristics and continuous variables, enhanced ability
to combine evolving molecular features (eg, BRAF muta-
tional status, PTEN expression) to better estimate
cancer-specific survival in individual patient settings, and
conditional probability models that estimate survival
after treatment or at any time during follow-up [2,5-8].
Significant insight and clinical prognostic/predictive
capacity driven principally by clinicopathological evidence-
based risk-stratification are rapidly evolving. Tremendous
strides in our understanding of the molecular underpin-
nings and heterogeneity of melanoma are beginning to
enter current standard evaluation and management arena
[9]. It is anticipated that identification of clinically relevant
and “context-specific” biomarkers will facilitate staging
and outcome predictions in patients with melanoma.
An update on Multicenter Sentinel Lymph node Trial
(MSLT) Randomized MelanomaTrials was very interesting.
MSLT 1 compared immediate versus delayed complete
lymph node dissection for nodal metastases from melan-
oma >1.0 mm or≥Clark IV. Randomization (60:40) to
either wide local excision with sentinel lymph node biopsy
or wide local excision alone. Complete lymph node dissec-
tion was performed when nodal disease was diagnosed
(either by SLN involvement or by clinical recurrence).
Enrollment occurred from 1994–2002 and 2001 patients
were enrolled. At the time of data lock (6/30/2011) 961
patients had completed 10 years of follow up, 672 died or
have been lost to follow up and 210 remained on study.
The current ongoing trial is MSLT 2, which examines
whether complete lymph node dissection is necessary in
the setting of a positive SLN. In most cases (approximately
88–89%) no additional metastases are discovered at the
time of completion dissection. In addition the trial incorpo-
rates nodal ultrasound in follow up to facilitate early dis-
covery of recurrence. In addition, those patients with
involvement of non-sentinel nodes have very high systemic
recurrence risks and may not benefit from additional
prophylactic regional treatment. In MSLT2, patients with
sentinel lymph node involvement (either by standard path-
ology or by multimarker RT-PCR) are stratified by Breslow
thickness (>3.5 mm or <3.5 mm), site of sentinel lymph
node (SLN) procedure (MSLT center or non-MSLT center)
and degree of SLN involvement (pathology or RT-PCR)
and randomized 1:1 to either completion lymph node
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dissection (CNLD) or observation with ultrasound and
clinical examinations. Target accrual is 2000, and as of Oc-
tober 19, 2011 1,354 had been randomized. Enrollment is
taking place at 63 sites around the world. Regarding the
RT-PCR evaluation of samples from the trial, to date 1275
patients have had pathologically negative SLN screened by
multimarker RT-PCR. Among these 1275 patients, 407
(24.2%) were positive of which 225 (55.3%) agreed to be
randomized based on the PCR results and 188 (46.2%)
accepted their randomization assignment. At the most
recent meeting of the Data Safety Monitoring Board, it
was concluded that an achievable sample size of 300
would not be adequate to determine if CLND was bene-
ficialfor RT-PCRpositive
randomization based on RT-PCR was stopped. RT-PCR
positive patients will continue to be followed for survival
and prognostic information. The trial also evaluated
ultrasound screening prior to SLN biopsy. As it is cur-
rently practiced around the world, ultrasound did not
provide adequate sensitivity or specificity to be useful. This
screening ultrasound has now been dropped from the
trial.
After the presentation of the new data concerning the
surgical treatment, the discussion focused on the current
status of adjuvant treatment of melanoma patients and
the possible selection of patients who might benefit. The
aims of adjuvant therapy in high-risk melanoma are to
reduce the risk of relapse, increase survival, provide
treatment with tolerable safety profile. Interferon (IFN)
is the only approved agent for the adjuvant therapy of
melanoma. Patients may develop significant side effects
frequently necessitating dose reduction or discontinuation
of therapy. Mechanisms of action of IFN are to promote
proliferation and clonal expansion of CD4 and CD8 T
cells, to enhance antibody production of B cells, to in-
crease cytotoxic activity of natural killer cells (NK) and
CD8 T cells, and to have negative effects on the activation
and proliferation of T regulatory cells (Tregs). Anti-tumor
effects are anti-proliferative, anti-vascular, pro-apoptotic
activity and modulating the immune response (role un-
clear). As showed by the meta-analysis of Mocellin, IFN
benefits are analogous to other well established adjuvant
treatments like in breast, colorectal and ovarian cancers,
but no optimal IFN-α dose and/or treatment duration, or
a subset of patients was identified to be more responsive
to adjuvant therapy (4 months was the shorter duration of
IFN administration). Molecular profiles may help in identi-
fying patients who can benefit most from interferon adju-
vant therapy. Most trials evaluating IFN used Breslow
thickness and lymph node invasion for staging. This par-
ameter was used for subgroup analyses of randomized
control trials however the staging system was not identical
over time. Subgroup analyses are hypothesis generating.
Analyzing retrospectively the data of patients enrolled in
patients. Assuch
two adjuvant EORTC trials, 18952 and 18991, the cohort
of patients with ulcerated primary melanomas and micro-
scopic lymph node involvement benefited in terms of
replase free survival (RFS) and DMFS. This finding will
now be prospectively validated in a EORTC trial which is
enrolling patients with ulcerated melanomas.
In tissue studies performed in the context of a neoad-
juvant trial, clinical responders had significantly greater
increases in endotumoral CD11c+and CD3+ cells com-
pared with non responders. In addition, HDI was found
to up-regulate pSTAT1, whereas it down-regulates
pSTAT3 and total STAT3 levels in both tumor cells and
lymphocytes. Higher pSTAT1/pSTAT3 ratios in tumor
cells pretreatment were associated with longer overall
survival.
Pretreatment levels of proinflammatory cytokines (IL-
1β, IL-1α, IL-6, TNF-α) were found to be significantly
higher in the serum of patients with longer RFS values.
Molecular HLA typing of patients receiving adjuvant IFN
demonstrated that patients positive for HLA Cw*06 had a
better relapse free and overall survival. These findings
need to be prospectively validated in other adjuvant trials.
In 2013 the trial results of MAGE3 and Ipilimumab in
the adjuvant setting will be available. MAGE-A3 is a tumor
specific antigen. It is not expressed in normal cells, and it is
therefore a good target for immunotherapy. It was identi-
fied via screening with anti-tumor killer T-cells. It is easy to
detect in patients (RT-PCR on tumor tissue) and is present
in major tumor types (lung, bladder, liver, melanoma) in
early and advanced stages of a given disease and is poten-
tially associated with poor survival prognosis. Based on the
encouraging results of the phase II trial in metastatic
melanoma, as well as the results of the phase II trial in
adjuvant NSCLC and the high unmet medical need, a
phase III trial was initiated in adjuvant melanoma. This
phase III trial is called DERMA and has enrolled 1300
patients worldwide. To test Ipilimumab in the adjuvant set-
ting two trials were designed: the EORTC trial of Ipilimu-
mab vs placebo in stage III patients, that has completed
accrual, and the ECOG 1609 study of Ipilimumab vs high
dose interferon; the enrollment of this study started on
May 2011. For patients with BRAF mutations some trials
with BRAF inhibitors and/or combination with MEK inhi-
bitors are currently underway.
Data were reported on electrochemotherapy (ECT), a
new technology to treat melanoma patients. Electroche-
motherapy is a combination therapy performed by elec-
tric pulses in association with a chemotherapic agent,
generally bleomicin. The rationale underpinning this
procedure is that external electrical stimulations can
make cell membrane permeable to some molecules that
in normal conditions cannot cross the membrane and
penetrate into cells (electroporation). ECT is a method
consisting of the combination of intra-tumoral injection
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of cytotoxic agents with the application of intensive elec-
trical stimuli. Cliniporator is the device that permits the
delivery of electrical pulses for this purpose. The electric
pulses have high intensity (1000 V/cm), short duration
(100μs), and can be repeated (8 pulses/nodule). When
the electric pulses are applied to tumor cells, in 1500 ms,
hydrophilic molecules normally excluded by the cell
membrane, can enter inside the cytosol, by the formation
of hydrophilic channels, and in 3 minutes, hydrophilic
channels close and molecules migrate to nucleus. ECT
allows drugs to reach the DNA and increase cytotoxicity
(Bleomycin × 10,000 - Cisplatin × 80). ECT is performed
by needles of different types and sizes for different indi-
cations (dimensions of the nodules, localizations of the
nodules, etc).
In the ESOPE study, a phase II trial, electrochemotherapy,
compared with bleomicin, was shown to be significantly
more effective in metastatic tumour nodule treatment than
the drug as single agent or electric pulses alone. Nodule
complete response was confirmed by histological and
immunohistochemistry analysis. Higher response rates were
obtained in melanoma nodules. At the National Cancer
Institute in Naples tumor nodules from 86 patients with
different diagnosis were treated with ECT: 38 patients with
melanoma, 18 with basal cell carcinoma, 12 with Kaposi’s
Sarcoma, 9 with squamous cell carcinoma, 5 with breast
cancer, 2 with pancreatic cancer and 2 with bone metastasis.
A total of 126 ECT treatments were performed, distributed
as follows: in 38 patients with melanoma nodules, one or
more treatments; in 16 patients with basal cell carcinoma,
two or more treatments; in 7 patients with Kaposi’s
Sarcoma, three or more treatments; and in 3 patients with
squamous cell carcinoma, four or more treatments. ECT
can be curative, if it results in the disappearance of treated
nodules; palliative, if it stables disease and reduces pain;
hemostatic, if it stops bleeding, or neoadjuvant, if it reduces
the size of the disease that can then be surgically removed.
The most common side effects of ECT are erythema, elec-
trodes tattoo, erosion or ulceration with scaring, slight
oedema and pain.
ECT is a simple, safe, economic, highly effective and
cosmetic repeatable procedure with a short learning
phase, that improves the quality of life independent of
life expectancy.
New pathways and new targets in melanoma: an update
DNA methylation is known to control gene expression
of multiple pathways relevant to melanoma. Examples of
specific changes include hypermethylation of CDKN2A,
MGMT, and PTEN, and hypomethylation of key antigens
such as the Melanoma Antigen family (MAGE) loci and
NY-ESO-1. While methylation of promoters is governed
by DNA methyltransferases (DNMTs) the factors respon-
sible for demethylating DNA have only recently been
identified. Active demethylation has long been suspected
based on evidence such as the IL-2 promoter’s demethyla-
tion within 20 minutes after stimulation of naïve T-cells
in vitro. Recent work at the Huntsman Cancer Institute
has shown that a trio of proteins including activation
induced deaminase (AID), Gadd-45, and MBD-4 work in
concert to demethylate DNA in zebrafish embryos. These
factors may drive some of the abnormal methylation
patterns seen in melanoma, and may maintain cells in a
more stem-cell like state. In efforts to improve the thera-
peutic effectiveness of immune therapy, drugs targeting
the DNMTs have shown successful re-expression of
melanoma antigens in-vitro and in patients, and have
improved response rates to IL-2 therapy. Limitations of
currently available epigenetic modifiers include rela-
tively short half-lives, and concominant DNA damage
leading to cytopenias. In efforts to circumvent these pro-
blems, new di-nucleotide-based compounds designed at
Supergen have shown greater stability than previous
demethylating agents such as 5-Aza deoxycytidine and
show favorable pre-clinical toxicity profiles. As future
studies directed towards improving response rates in
immunotherapy, and circumventing drug resistance oc-
curring with targeted therapy will likely employ epigen-
etic modifiers, more stable compounds such as these
may be more desirable for combination studies in
melanoma.
Clinical and pre-clinical studies with molecular tar-
geted therapy reveals a dependence on MAPK signaling
for melanoma tumor growth and maintenance, and re-
activation of the MAPK pathway through direct and par-
allel pathways appears to be essential for mediating drug
resistance and tumor progression [10]. During neural
crest development the MAPK pathway controls a highly
conserved transcriptional response that involves repres-
sion of FOXD3 mRNA and protein, which in turn acti-
vatesMITFexpression
migration and differentiation [11-13]. This response
remains intact in melanoma cells, as inhibition of the
MAPK pathway causes re-expression of FOXD3, which
in turn causes cell cycle arrest, increased cell survival,
decreased migration, loss of differentiation markers
(pigment); properties consistent with a transient pro-
genitor state [14-16]. Indeed, MAPK-inhibited melan-
omacellsexpresshigher
progenitor/stem cell markers such as DCT and SOX10.
These studies suggest that inhibition of the MAPK path-
way causes a subset of melanoma cells to de-differenti-
ate into a multipotent cell population, which is more
resistant to cytotoxic apoptosis. Future in vivo studies
will be needed to determine the consequence of FOXD3
re-expression in melanoma cells after BRAF-inhibitor
treatment to determine 1) if FOXD3 is a useful bio-
marker for drug-dependent tumor regression and 2)
topromotemelanocyte
levelsofneuralcrest
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if melanoma cells de-differentiate. If BRAF-inhibitor
induced de-differentiate occurs, our knowledge of the em-
bryonic neural crest pathways that control melanocyte
development can be exploited to identify synthetic lethal
interactions that rely on FOXD3 re-expression and its role
in differentiation of other neural crest lineages, such as
glia, eg., biological therapies.
ERK has a pivotal role in melanoma because this path-
way is hyperactivated through gain-of-function mutations
in the majority of melanoma cases. Primarily, this is driven
by mutations in genes such as BRAF (44% of cases), NRAS
(22%), ERBB4 and cKIT (5% each). Some of these driver
oncogenes are validated therapeutic targets and in rando-
mized clinical trials, BRAF inhibitors can mediate extraor-
dinary responses [improved progression free survival (PFS)
and median OS], in patients with V600BRAF mutations.
Curiously however, a frequent side effect of BRAF inhibi-
tors is the induction of cutaneous squamous cell carcin-
omas (cuSCC), which is driven by a paradoxical activation
of the MAPK pathway in pre-cancerous keratinocytes that
carry oncogenic mutations in RAS genes. Surprisingly,
nilotinib, a selective Bcr/Abl tyrosine kinase inhibitor, also
drives paradoxical RAF activation and synergizes with
MEK inhibitors to kill drug-resistant CML cells. These
data highlight the importance of understanding the genetic
landscape of individual tumours and emphasize the poten-
tial of complete genome sequencing to provide better
understanding of human cancer.
The AMP activated protein kinase (AMPK) controls
energy homeostasis in cells by measuring AMP/ATP
ratios. In metabolic stress, AMPK restores energy bal-
ance by increasing energy production and blocking en-
ergy consuming. Intriguingly, whereas most cancer cells
are sensitive to the growth inhibitory effects of AMPK
activation, BRAF mutant melanoma cells are resistant to
AMPK through the action of the protein kinase RSK.
Furthermore, in vivo, AMPK activators drive the produc-
tion of VEGF-A in BRAF mutant melanoma cells and
the combination of metformin and VEGF signaling inhi-
bitors drive a synthetic interaction that blocks the
growth of BRAF mutant melanoma cells in vivo.
BRAF mutations are expressed in about 50% cutaneous
melanomas (20% continuous sun-exposure, 50–80% inter-
mittent sun-exposure), and in areas of high sun exposure,
like Australia, 80% mutations are V600E, and this kind of
mutation is present in about 90% of patients between 20
and 40 years-old. Vemurafenib and dabrafenib are two
powerful BRAF inhibitors that give a high response rate in a
very short time in BRAFV600 mutated melanoma patients
and have good activity even in brain metastases. About 50%
of mutated patients respond to BRAF inhibitors. In the
BRIM 3 trial vemurafenib had a PFS or 5.3 months, and in
the phase II BRIM 2 trial PFS was 6.7 months; the BREAK-
2 trial of dabrafenib showed even different PFS in patients
withV600E mutation and V600K, with an advantage for “E”
mutation (27.4 vs 19.7 weeks). The BRIM 3 trial showed an
important advantage even in overall survival with 83%
6 month survival for vemurafenib vs 63% 6 months survival
for dacarbazine. However, patients tend to relapse; about 5
patterns of relapse have been described, but generally (43%),
the progression is in new sites only, while in 21% it is in
pre-existing site only.
To continue BRAFi treatment beyond progressive disease
(PD) resulted in good outcomes in anecdotal reports;
prolonging therapy beyond PD could mean prolong sur-
vival (median survival beyond PD >9 vs 3 mths p=0.008),
but this type of strategy calls for a randomised discontinu-
ation trial. This effect may be due to a “tumour flare” on
BRAFi withdrawal, even after PD.
MEK inhibitors as single agents have activity against
mutated BRAF melanoma, unexposed to prior BRAF in-
hibitor therapy, but they won’t salvage BRAF inhibitor
resistance. A new combination of the MEK and BRAF
inhibitors trametinib and dabrafenib as first line therapy
for BRAF mutated melanoma patients is showing great
promise. In BRAFV600Ehuman melanoma xenograft
BRAFi+MEKi showed enhanced antitumor activity, with
more sustained tumor control than that seen either sin-
gle agent. This combination of BRAF and MEK inhibi-
tors is obtaining very good results in melanoma patients
naïve to prior anti-BRAF treatment, with about 5
complete responses, and a high tumor reduction rate.
83% of these 77 patients were ongoing at 30 weeks of
treatment, when the study was presented. However, even
this combination needs to be evaluated in new rando-
mized clinical trials.
Resistance to BRAF inhibitors is mediated by different
mechanisms as shown from about 60% of biopsies per-
formed in progressing lesions. Among these mechan-
isms the most reproducible in patient-derived samples
are secondary NRAS mutations, upregulation of RTKs
(PDGFRβ, IGF1R) and BRAF truncations. The mechan-
ism of resistance may predict for sensitivity to the
addition of secondary treatments such as growth factor
receptor inhibitors or PI3K/AKT/mTOR inhibitors.
Combining immunotherapy and BRAF targeted therapy is
possible; vemurafenib does not adversely affect the function
of human or murine lymphocytes; the combination of
vemurafenib with anti-CTLA4 immunotherapy is mediated
by improved intratumoral infiltration by activated lympho-
cytes in a fully syngeneic and immunocompetent mouse
model of BRAFV600Emutant melanoma; a phase 1 clinical
trial of a combination of vemurafenib and ipilimumab is
ongoing.
Immunotherapy: new evidence
The development of the first tumor antigen-specific
monoclonal antibodies dates back to the '70s. The
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characteristics of these reagents in terms of specificity, re-
producibility and availability in large amounts generated a
lot of hopes and enthusiasm about the clinical application
of immunotherapy for the treatment of malignant
diseases. Unexpectedly most if not all the clinical trials
yielded negative results. As a result the scientific commu-
nity became skeptical about the clinical usefulness of
tumor antigen-specific monoclonal antibodies to develop
immunotherapeutic strategies for the treatment of malig-
nant diseases. Things changed in 1997 when rituximab
and trastuzumab were approved by FDA for the treatment
of non Hodgkin lymphoma and breast cancer, respectively.
In the following years a growing number of tumor antigen-
specific monoclonal antibodies have been approved and
several of them have become part of the therapeutic arma-
mentarium used for the treatment of malignant diseases.
Among the many tumor antigens which are being
evaluated as potential targets of immunotherapy, the
membrane bound chondroitin sulphate protidoglycan 4
(CSPG4), which was initially named High Molecula
Weight-Melanoma Associated Antigen, certainly deserves
mention. This target is expressed with high density on the
cell membrane of many types of malignant cells. They in-
clude melanoma (~85%), glioma (~70%), triple negative
breast cancer (~50%), mesothelioma (~50) chordoma and
chondrosarcoma (~50%),
leukemic (~55%) lesions. Furthermore CSPG4 is upregu-
lated on activated pericytes in the tumor microenviron-
ment; as a result, CSPG4 immunotargeting may inhibit
neoangiogenesis in the tumor microenvironment and sup-
press growth of tumor cells, even if they do not express
CSPG4. In view of the postulated role played by cancer ini-
tiating cells in metastatic spread and in disease recurrence
it is noteworthy that CSPG4 is expressed on cancer initiat-
ing cells at least in melanoma, head and neck cancer and
breast cancer. Because of the interest in utilizing CSPG4
as a target of immunotherapy, it is noteworthy that this
antigen has a restricted distribution in normal tissues.
CSPG4-specific mAb have been found to be effective in
inhibiting the growth of human melanoma cells and their
metastatic spread in immunodeficient mice. This effect is
mediated by the inhibition of several signaling pathways
including the ERK and FAK pathways.
Another potential target of antibody-based immunother-
apy discussed at the meeting is glucose regulated protein of
94,000 daltons (Grp94). Grp94, a member of the Heat
shock protein (HSP) 90 family, is located in the endoplas-
mic reticulum of all mammalian cells. This chaperone is
essential for the conformational maturation of several pro-
teins that play key roles in transducing proliferative and
anti-apoptotic signals. These functional properties of mem-
bers of the HSP90 family have provided the rationale for
the clinical use of HSP90 inhibitors for the treatment of
malignant diseases with the expectation that the inhibition
andacutelymphoblastic
of its chaperone function induces the degradation of its
“client” proteins. Therapeutic effects have been observed.
However the clinical use of these inhibitors is hampered by
theassociatedsideeffects.
emphasize the need to develop strategies to overcome the
limitations. In this light the fully human mAb W9, which
was described at this meeting, is of great interest, since it
recognizes an extracellular epitope of Grp94. This epitope
is selectively expressed on malignant cells. mAb W9 inhi-
bits the proliferation of tumor cells; this effect is mediated
by the inhibition of several signaling pathways.
Ipilimumab improves survival in previously treated
metastatic melanoma patients (+/− CNS disease) com-
pared to gp100 peptide vaccine (HR=0.68), and in asso-
ciation with dacarbazine improves survival in untreated
patients with metastatic melanoma compared to dacar-
bazine alone (HR=0.72), with 10% high grade adverse
events. To improve on these results clinical investigators
are testing different strategies of therapy such as inte-
grating cancer vaccines and CTLA-4 antibody blockade.
Concurrent therapy with GM-CSF-based vaccines in
murine tumor models have revealed potent therapeutic
synergies, but associated with toxicity; moreover CTLA-4
Ab enhances immunologic memory responses. GVAX
(granulocyte-macrophage colony-stimulating factor [GM-
CSF] gene transduced irradiated melanoma vaccine cells)
offers the possibility that “host versus melanoma” immune
responses can be generated in melanoma patients. At the
Dana-Farber Cancer Institute, a trial of anti-CTLA-4
enrolled 14 stage IV melanoma patients pretreated with
GVAX, and treated them with 3 mg/kg ipilimumab every
2–3 months. In the 14 GVAX patients, this combination
obtained three partial responses, one partial response (near
CR) following DTIC and six stable disease with a median
duration of 30 months. Possible Mechanisms of action of
GM-CSF-based vaccination+CTLA-4 blockade can be
the expansion of primed anti-tumor immune effector cells;
this association allows CTLA-4 blockade to selectively
target anti-tumor effector cells (i.e. therapeutic index). In
attempts to simplify the therapeutic strategy of combining
GM-CSF biology with immune checkpoint blockade, the
Eastern Cooperative Oncology Group planned a Phase II
Trial of GM-CSF Protein Plus Ipilimumab in Patients with
Advanced Melanoma randomizing melanoma patients to
receive Ipilimumab 10 mg/kg induction/maintenance plus
GM-CSF 250 μg days 1–14 in a 21 day cycle or Ipilimumab
alone. The primary endpoint is overall survival.
Humoral responses to VEGF and angiopoietins have
been associated with clinical benefit in some patients re-
ceiving therapeutic vaccines. Importantly, VEGF has
known immune modulatory effects, specifically decreasing
dendritic cell maturation. Basing on these considerations,
started a phase I clinical trial with Ipilimumab plus bevaci-
zumab. Melanoma patients were first treated in two
Theseclinicalfindings
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cohorts, one treated with 10 mg/kg ipilimumab plus
7.5 mg/kg bevacizumab and another with 10 mg/kg ipili-
mumab plus 15 mg/kg bevacizumab, with induction of ipi-
limumab every 3 weeks × 4 cycles then every 3 months
maintenance, and a maintenance with Bevacizumab con-
tinued every 3 weeks. Of 22 patients treated to date, clin-
ical activity has been observed. CTLA-4 plus VEGF-A
blockade may have effects on both tumor immunity and
tumor vasculature. Randomized phase II and III trials will
be needed to discern the effect of the addition of VEGF-A
blockade to CLTLA-4 blockade.
Features of the tumor microenvironment could dominate
at the effector phase of the anti-tumor T cell response and
limit efficacy of current immunotherapies. Systematic ana-
lysis of the tumor microenvironment could identify a pre-
dictive biomarker profile associated with clinical response,
and also highlight new biologic barriers that need to be
overcome to optimize therapeutic efficacy of vaccines and
other immunotherapies. An “inflamed” gene expression pat-
tern of tumor microenvironment has been associated with
favorable clinical outcome to multiple vaccine platforms in
melanoma. Ipilimumab clinical responders also appear to
show an “inflamed” tumor gene expression profile. There-
fore, an inflammatory gene expression profile in metastatic
melanoma might have utility as a predictive biomarker for
response to vaccines and other immunotherapies. Post-
vaccination, increased CD8 transcripts combined with
decreased melanoma antigen transcripts in the tumor is a
pattern associated with clinical benefit. One major barrier
to effective immune-mediated tumor destruction is poor T
cell migration and the “non-inflamed” subset of patients.
Still, T cell migration into tumors appears to be necessary
but not sufficient for clinical response. Inflamed melanomas
containing CD8±T cells have highest expression of
immune inhibitory pathways including IDO (indolea-
mine-2, 3-dioxygenase)-induced tryptophan catabol-
ism, PD-L1engagement
extrinsic suppression by
and T cell anergy due to poor expression of B7
costimulatory ligands. The underlying mechanism
explaining “inflamed’ versus “non-inflamed” tumor
microenvironment are not yet understood. Possibil-
ities being explored include inter-patient heterogen-
eity at the level of oncogene pathway permutations
within the tumor cells, germline polymorphisms at
the level of the host, or differences in gut flora
commensal organisms, “Inflamed” tumors likely are
not rejected due to dominant immune suppressive
mechanisms (IDO, PD-L1, Tregs, Anergy), which are
all potential therapeutic targets. Increased PD-L1,
IDO and Tregs in the tumor site are driven by CD8
+T cells in the tumor microenvironment. Blockade
of these pathways is being explored in the clinic,
already with preliminary progress. A new set of
of
CD4+CD25+FoxP3+Tregs
PD-1onT cells,
surface markers driven by EGR2 may provide a
strategy for identifying
CD8+T cells from the tumor microenvironment and
LAG3and CRTAMare
targets.
Melanoma is definitely not a status quo, but an evolving
process included as part of an intracellular network of inter-
connections, influenced by several factors such as the gen-
etic basis of the individual subject, the genetics make up of
the disease and environmental factors. To understand the
immune mediated tumor rejection, a holistic approach that
capture the complexity entity of the given time and condi-
tion instead of focusing on single or limited parameters
should be considered, especially when the mechanism is
elusive. Transcriptome analysis of the tumor microenviron-
ment under a variety of immunotherapies has uncovered a
common gene expression pattern represented by activation
of key immune modulators such as IRF1, START1, T-bet,
IFNG and IL15; up regulation of effector molecules such as
GNLY, GZM and TIA accompanied by over expression of
CXCR3 and CCR5 with corresponding ligands (CXCL9-11,
CCL5). The impact of this same gene signature on the re-
sponse to anti-tumor immunotherapy are indicative of im-
mune mediated tissue destruction such as in autoimmune
disorders, acute infection clearance and transplant rejection
suggesting a converging mechanism independent of the
causal initiation. It is even more conceivable that this same
gene signature with consequent changes in the level of tran-
scription in tumors is increasingly important as a biomarker
associated with good prognosis and survival. Gene sets
found to be highly correlated with clinical response are the
Interferon-Gamma-pathway, AKT pathway, CCR5 pathway
and NKT pathway. Majority of effector function related
genes are down-regulated while proliferation and cell cycle
related genes are up regulated suggesting a phenotypic
defined immune cell subset in CR different from NR which
may be responsible for the potent effector function and pos-
sible mechanism of rejection. A prediction model developed
based on those significant genes can accurately predict
about 75% of melanoma patients clinical outcome under
adoptive TIL therapy, although, those data need to be
validated in an independent study. However, the down-
regulated genes could be result of the intrinsic genetics het-
erogenity (CCR5, CXCR3 and IRF5) of the patient which
has intrinsic impact to the tumor.
Genetic polymorphism, the essence of human hetero-
geneity, play an important role in diverse disease suscep-
tibility and impact the natural history of disease.
Polymorphism of IRF-5 appears to be a predictor of im-
mune responsiveness of melanoma metastases to adop-
tive therapy with TIL. The rs10954213 G allele, which is
protective against SLE, is the most predictive of non
responsiveness suggesting a correlation between auto-
immunity and melanoma immune responsiveness. The
intrinsicallydysfunctional
candidatetherapeutic
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expression profile of TIL classified according to AA vs
GG IRF5 rs10954213 (G>A) appears to be a borderline
predictor of immune responsiveness. The expression
profile of pre-treatment melanoma metastases classified
according to AA vs GG IRF5 rs10954213 (G>A)
appears to be a stronger predictor of immune respon-
siveness compared with TILs suggesting possible involve-
ment of tumor microenvironment. However, comparison
of melanoma cell lines derived from the pretreatment
melanoma lesions classified according to the AA vs GG
IRF5 rs10954213 (G>A) highlights a signature of genes
that differentiates the two genotypes clarified that the
genotype of the tumor cells itself make the difference
independent of micro environmental influences. The sig-
natures differentiating the two cell line genotypes
in vitro could predict of the responsiveness of melanoma
metastases in vivo suggesting that immune responsive-
ness is at least in part genetically determined. Thus, it
appears that immune responsiveness is at least in part
dependent on the genetic background of the host which
affects the biology of cancer cells primarily and secondarily
the immune responsiveness of tumors.
The major challenge for the field is how to monitor the
antitumor immune response for non-antigen-specific im-
munotherapy such as anti-CTLA4, anti-PD1 and IL-2 and
for antigen-specific immunotherapy since the fact that the
antigen is administered, doesn’t mean that immune system
“sees” only that specific antigen (epitope spreading). We
do not know which parameters of immune responses and
which assays used to assess these parameters are optimal
for efficacy analysis. There is a need for the development
and validation of tools to identify patients who can benefit
from a particular form of immunotherapy. The analysis of
single parameters alone may not provide sufficient insights
about complex immune system–tumor interactions. Com-
mon immunoassays do not take into account changes in
the differentiation of immune cells, in the antigenic profile
of tumors and responding T cells, in T-cell homing recep-
tors, or the complex analysis of responses to “private” anti-
gens or epitope spreading. The development of protein
arrays that contain 9000 human proteins are being used to
identify the generation of antibody responses following im-
munotherapy. Since production of IgG antibody responses
require CD4 help, identification of a new or increased IgG
antibody response following immunotherapy potentially
provides a surrogate for generation of an anti-tumor Tcell
response. This strategy is being employed by several
groups to characterize the immune response following im-
munotherapy and holds promise as a strategy to monitor
responses against a wide range of possible targets.
Tumor infiltrating lymphocyte (TIL) therapy has been
the cornerstone of adoptive cellular therapy of melanoma.
TIL therapy is changing and other adoptive cell therapies
are now available [17]. Recent improvements in TIL
therapy of melanoma include the use of lymphodepletion
recipient preparative regimens and more rapid TIL pro-
duction – young TIL [18]. The beneficial effects of
leukocyte depletion are likely due to the elimination of
Tregs and increased serum cytokine levels that result in
greater in vivo TIL persistence and expansion which have
resulted improved clinical outcomes [19]. The in vivo per-
sistence of young TIL is greater than classical TIL, but the
clinical benefits of young TIL therapy are still being
evaluated.
When TIL therapy is not possible because metastatic
tumor can’t be resected or TIL can’t be isolated from
resected tumor, genetically engineered autologous T cells
can be used for adoptive T cell therapy. Autologous T
cells that have been genetically engineered to express a
high affinity T cell receptor (TCR) specific for the cancer
testis antigen NY-ESO-1 were used to treat melanoma
and sarcoma [20]. Preliminary results of adoptive cell
therapy using T cells with genetically engineered TCRs
have been promising but TCRs are HLA-restricted, the
required vectors are expensive and gene transduction is
technical difficult. In the future, the use of autologous
naïve and stem cell-like memory T cells may further
enhance adoptive cell therapy using genetically engi-
neered T cells [21].
Culturing and expanding TIL for clinical therapy is
technically demanding, expensive and time consuming
which has restricted the clinical use of this therapy. Re-
cently, it has been found that TIL production can be
improved by using gas permeable G-Rex flasks for initial
TIL culture and rapid expansion [22]. The benefits of
this method of TIL production are lower final volume
and fewer flasks and no electronic or mechanical devices
are required.
Combination strategies
The rationale for adjuvant therapy lies in the greater
responsivness of micrometastatic and operable regional
disease (Stage IIIA-B), as compared to inoperable
advanced (stage IV) disease. Adjuvant therapy with IFN
reduces the hazard of relapse and mortality by 33%,
whereas multiple studies have shown response rates in
advanced stage IV disease that are in the range of ~16%
[23]. The presence of advanced inoperable disease has
immunomodulatory consequences that have been docu-
mented by Tatsumi and Storkus [24]. The objective re-
sponse rates observed with immunotherapies beginning
with IFNα have been to be inversely correlated with the
disease burden. The trials E1684, E1690, and E1694 show
how durable and significant the impact of IFN upon re-
lapse-free and overall survival. Three meta-analyses of
the aggregate of all trials that have been conducted with
IFNα confirm RFS and OS benefits of IFN [25-27]. How-
ever, it has not yet been estabilished what the optimal
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dose, route, and duration of IFN therapy are. All trials
conducted with IFNα show unequivocal and durable
benefits in terms of RFS but only two independent trials
have shown both RFS and OS impact, both of which uti-
lized IV induction at 20MU/m2followed by SC mainten-
ance IFN at 10MU/m2 for a full year of treatment.
Two trials, the Intergroup E1697 and Neoadjuvant
Trial UPCI 00–008 have tested the effects of one month
of IV IFNα2b. The phase III intergroup trial E1697 com-
pared 1 month of iv high-dose IFN vs. observation,
demonstrated the lack of durable benefit of the 1 month
treatment in mature data released in in stage IIB/IIIA
resected melanoma patients with futility analysis at 1155
patients [28]. The neoadjuvant trial UPCI 00–008 con-
ducted in patients with bulky lymph node metastatic
disease showed significant antitumor effects in 55% of
patients with stage IIIB-C disease assessed at 1 month,
as well as significant immunomodulatory effects in
patients receiving the 1 month iv high-dose regimen—so
we conclude that the one month regimen is active, but
that durable benefits of this agent require longer than
1 month of administration. The search for biomarkers that
correlate with antitumor benefits of IFN has been a critical
undertaking. Patients with the development of serological
or clinical signs of autoimmunity during HD-IFN derive
the greatest benefit in terms of PFS and OS [29]. But the
serum cytokine/chemokine profile can predict treatment
benefit with HDI: in fact, baseline pro-inflammatory cyto-
kine levels were found to predict 5-year relapse-free sur-
vival in patients treated with High-Dose IFNα. The
updated data from the EORTC 18991 trial showed benefit
from this 5 year Peg-IFN regimen that diminished at
7.6 years, compared with the earlier published analysis and
there is no significant impact upon DMFS or OS either
early or at 7.6 years maturity in this trial. Analyzing the
subgroup of with stage III N1 disease shows significant RFS
and DMFS impact in 2007, but at 7.6 years this is no longer
statistically significant; patients with stage III N2 showed
no benefit in any of the several endpoints, and patients
with primary tumor ulceration analyzed at the 7.6 year
time point show the greatest benefit of Peg IFN among
the subset of patients with Stage III N1 disease and
ulcerated primary tumors (median OS of peg IFN vs. ob-
servation: > 9 vs. 3.7 years).
New adjuvant strategies have been tested more recently,
but among mature phase III trials only HDI demonstrates
confirmed significant durable OS & RFS benefit at
>20 years (E1684/90/94). A variety of tumor cell vaccines
have been assessed giving largely disappointing results:
Canvaxin was shown to be ineffective and possibly detri-
mental in Ph III trials for both stage III and IV resectable
tumor; GMK, a ganglioside GM2 vaccine administered
with QS21 adjuvant conjugated to the KLH carrier, was in-
active and MAGE A-3 results are pending. Neither
GMCSF nor peptide vaccination improved OS or DFS
overall in the ECOG led intergroup US study E4697 (the
trends to benefit among Stage IV subjects will require
further study), and Anti-CTLA4 blocking mAbs (tested in
EORTC trial 18071, and US Intergroup trial E1609) will
not mature for some time. BRAF and MEK inhibitors are
planned for evaluation but these studies are not yet
launched.
Ipilimumab has been studied by Medarex-BMS in the
020 and 024 trials, each demonstrating significant durable
benefits in advanced unresectable patients with metastatic
melanoma—so the evaluation of this agent in the adjuvant
setting is reasonable, as already discussed: the larger ques-
tion that remains unanswered is which dosage of ipilimu-
mab will be most effective—as the FDA has approved the
dosage of 3 mg/kg but the EORTC 18071 trial has only
evaluated the dosage of 10 mg/kg, compared to placebo.
The US Intergroup trial E1609 has addressed this with
recent modifications that will evaluate both 10 mg/kg and
3 mg/kg vs the active standard of HDI.
The neoadjuvant setting has already been alluded to, as
it may give rapid and mechanistic answers regarding new
potential adjuvant therapies. Neoadjuvant High-Dose
IFN-α2b was studied in the trial UPCI 00–008 that
showed clinical responses at day 29 in 55% of patients,
and a molecular impact upon STAT3 with reduction of
the pSTAT3/STAT3 constitutively expressed in tumor
tissue. This study also showed modulation of IFNAR2
and increased expression of pSTAT1, and TAP2 in tumor
tissue. The immunologic impact upon CD3 T cell, and
DC responses to tumor (with increased CD3 T cell and
CD11c dendritic cell populations in tumor) provided the
strongest evidence of the immunomodulatory mechan-
ism of IFN adjuvant therapy. Neoadjuvant therapy with
Ipilimumab at 10 mg/kg has now been tested as pre-
sented by A. Tarhini, [30]. These interesting results mir-
ror results obtained with tremelimumab+HDI that have
recently been published in advanced melanoma [31]. A
current neoadjuvant trial of Ipilimumab 10 mg/kg or
3 mg/kg+HDI will also shed light on dose–response
effects of ipilimumab at the two different dosages, com-
bined with high-dose IFN.
The effects of immunotherapy in melanoma are observed
in the tail of the survival curves, with long term survivors,
while the major effects of targeted therapy for melanoma
occur in the initial splay of the curve with high response
rates. In patients with metastatic melanoma harboring
BRAF V600 mutation, vemurafenib has achieved striking
results in terms of PFS and OS. This agent has yet to be
evaluated in the adjuvant setting, but its effects in relation
to tumor debulking, increased T cell infiltrates in some
series, and possibly increased antigenicity and APC function
may translate to improved adjuvant therapeutic benefits:
however, the finite durability of benefits, and the absence of
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mature survival data in phase III trials qualify this assess-
ment. It may be that BRAF inhibitors are most useful as
partners in combination with IFN for the adjuvant therapy
of bulky disease, to capitalize upon immunomodulatory
functions of BRAF inhibitors, and to limit the necessary
interval of BRAF inhibitor therapy. Phase II data are needed
for IFN-BRAF combinations and this will be one area for
future exploration.
Adjuvant application of molecularly targeted therapy in
combination with immunomodulators offers opportunity
to magnify therapeutic impact of the immunotherapies,
and to obtain more durable benefits from the molecularly
targeted therapies. Whether agents that do not induce
durable CR or durable disease control in stage IV will have
benefits in the adjuvant arena is now testable.
In 2008, Korn performed a meta-analysis of phase II co-
operative group trials in metastatic stage IV melanoma
aimed at determining progression-free and overall survival
benchmarks for future phase II trials [32]. The results were
daunting, since only 25.5% of the patients treated in these
phase II studies were alive at 1 year (median PFS,
1.7 months; median OS, 6.2 months). From that time, his-
tory has however changed in regard to two new modalities,
due to the approval and the introduction into the clinics of
innovative new drugs. Until 2010, just two chemotherapeu-
tic agents were available for the treatment of metastatic
melanoma: Dacarbazine and (in Europe) Fotemustine and
(in the US) Aldesleukin. In 2011, Ipilimumab was approved
for both first and second lines in USA or solely for second
line in Europe and Vemurafenib was approved for first and
second lines inV600EBRAF mutated patients. Both the drugs
gave effective but different results (i.e. Ipilimumab, com-
pared with Dacarbazine, provides an important advantage
in OS but not in PFS, while Vemurafenib impacts on both
PFS and median OS), reflecting different mechanisms of
action and kinetics [33-35].
In this regard, new strategies for the therapy of melanoma
have used the combination of different drugs with different
mechanisms of action. Some examples of ongoing trials are:
a dose-escalation study of the combination of anti-PD1 and
Ipilimumab (NCT01024231) in subjects with unresectable
or metastatic melanoma; a study of RO5185426 and GDC-
0973 in patients with BRAF-mutation positive metastatic
melanoma; and a phase I/II Ipilimumab Vemurafenib com-
bination (NCT01400451). A fundamental differentiation for
prognosis and, above all, therapeutic effects is the distinc-
tion of all patients in two main subgroups: BRAF-mutated
and BRAF-wild-type. In patients withV600EBRAF mutation
and, thus, oncogenic activation of the MAPK pathway,
targets that can be hit are BRAF, MEK, and, probably,
ERK. Selective BRAF inhibitors are Vemurafenib and Dab-
rafenib. Both of them, compared with Dacarbazine,
obtained an advantage in response rates, PFS and OS;
however, a new BRAF inhibitor is now under evaluation,
LGX818 (Novartis), and new therapeutic strategies are on-
going in clinical trial, such as Vemurafenib+Surgery or
Radiotherapy in patients presenting progression during
therapy with Vemurafenib. At 2011 ASCO Meeting, Kim
showed how the treatment beyond progression with
Vemurafenib does impact on OS among BRAF-mutated
patients [36]. Another therapeutic target is MEK: there are
at least five MEK-selective inhibitors, and GSK1120212
(GSK) has been demonstrated to achieve better results in
BRAF-mutated patients non pre-treated with BRAF inhi-
bitors. The new strategy is to combine BRAF and MEK
inhibitors in first line therapy for BRAF-mutated patients.
At 2011 ASCO Meeting, a trial combining a BRAF inhibi-
tor (GSK2118436) and a MEK inhibitor (GSK1120212)
was presented; it showed high response rates with a very
good toxicity profile [37]. A similar ongoing trial is the
BRIM-7, based on the combination of Vemurafenib and a
MEK inhibitor (GDC-0973).
New possible combinations of multi-target drugs include
MEKi, ERKi, PI3Ki, and AKTi. Ongoing trials are repre-
sented by: Phase Ib Study of PI3 (Phosphoinositol3)-Kinase
Inhibitor BAY80-6946 with MEK (Mitogen-activated Pro-
tein Kinase) Inhibitor BAY86-9766 in Patients With
Advanced Cancer (NCT01392521) and “A Study to Investi-
gate Safety, Pharmacokinetics (PK) and Pharmacodynamics
(PD) of BKM120 Plus GSK1120212 in Selected Advanced
Solid Tumor Patients [NCT01155453]”. In the subset of
V600EBRAF-mutated population, the strategy of combining
chemotherapic agents and small molecules, such as Levati-
nib or PARP Inhibitors, was adopted in order to overcome
the hurdle of the less effective results of the chemotherapy.
In the BRAF-wild-type population, the principal strategy
proposed for treating such patients in the future is the
combination of chemotherapic agents and immunomodu-
lating monocolonal antibodies. The comparison between
the best overall response rate, disease control rate, and
duration of response of the three randomized phase II-III
studies with ipilimumab [38] showed how the combination
of Chemotherapy and Ipilimumab is superior to Ipilimumab
and Dacarbazine alone. The Phase II Study Combining Ipi-
limumab and Fotemustine in Patients with Metastatic
Melanoma [NIBIT-M1 Trial] indeed demonstrated the ad-
vantage of this combination [39].
In both previously treated and non treated metastatic
melanoma patients, albumin-bound paclitaxel (nab-pacli-
taxel) was well tolerated and showed a good activity in as-
sociation with Carboplatin (4 months progression free
survival,74%;median progression
5.8 months).
ImmunomodulatingmAbs+Anti-angiogenetic
pounds is another combination actually evalutated; as
presented by Hodi at 2011 ASCO Meeting, the associ-
ation of Ipilimumab with Bevacizumab gave interesting
results in a small cohort of melanoma patients [40].
freesurvival,
com-
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Furthermore, different immunomodulating antibodies
may be combined in clinical trials. Associating two
Immuno checkpoint blocking antibodies such as Ipilimu-
mab and sub-efficacious doses of anti-PD1 was demon-
strated to achieve a median reduction of the tumor
volume much higher than that obtained using higher
doses of the single antibodies in mouse models.
Finally, anti-CTLA-4 can be combined with either elec-
trochemotherapy, through association of suboptimal doses
of a chemotherapeutic agent - bleomicin or cisplatin - and
an electroporation performed by an electrical impulse
driven by a needle (interesting results were obtained at the
National Cancer Institute of Naples as well as in other
Institutions from different countries), or vaccination or T-
reg depletors (as in the experience based on the use of
Denileukin Diftitox).
Overall, several innovative weapons are available to
fight melanoma; our efforts will be aimed at assessing
the best strategy for the patients’ treatment. Surely, the
motto in melanoma therapy for next years will be: Com-
bine, Combine, Combine!
In patients with metastatic melanoma harboring V600
mutations GSK2118436 & GSK1120212 are both investi-
gational agents, and the present standard of care is
vemurafenib. Invemurafenib-refractory
BRAF V600Wild-Type patients, the standard-of-care is
either ipilimumab or high-dose IL-2 (in the U.S.) for
those who did not receive these agents first-line, or
chemotherapy for those who have received ipilimumab,
IL-2 and vemurafenib. Considering the future develop-
ment of investigational agents, possible phase III trial
designs must consider the acheiveably endpoints (overall
vs. progression-free survival) and the safety of the treat-
ment in relation to the magnitude of benefit being
sought. Using the example of GSK2118436 (BRAFi) and
GSK1120212 (MEKi) the most scientifically rigorous
control arm would be GSK2118436, whereas the conven-
tional regulatory comparator would be vemurafenib. If
the contribution of both agents to overall efficacy must
be determined, then an additional control arm with
GSK1120212 would be needed. Based on preliminary
data with this two drug combination, the safety of the
combination appears to be superior to either drug alone.
If so, one might consider a lower threshold of increased
efficacy to establish this combination as a new treatment
standard then would be the case if the combination were
more toxic than single agent therapy.
Unlike the example of GSK2118436 and GSK1120212,
not all targeted or immunologic agents nominated as poten-
tial melanoma therapeutics are going to have single-agent
activity; if synergistic, two agents should be active together
even when neither is alone. Given that very few of the po-
tential two drug combinations of investigational agents will
arise from within a single pharmaceutical company,
patients, or
combining investigational agents early in clinical develop-
ment involves significant risk-taking for the companies
involved. Presuming that neither agent has significant sin-
gle-agent activity, and independent approval may not be
possible; having the success of one companies agent depend
on the solvency of another company and willingness to in-
vest in continued development of an agent lacking single
agent activity calls for a greater degree of collaboration than
has previously been manifested in the pharmaceutical in-
dustry. There is a need for increased infrastructure and a
regulatory framework to facilitate investigational agents
being combined early in development (such as NCI Can-
cer Therapeutics Evaluation Program). Moreover, compan-
ies are currently disincentivized to allow investigational
agents to be combined with other investigational agents
has unique toxicities observed with such a combination
may hinder the development of each individual drug.
Incentives must be created for the pharmaceutical com-
panies to “contribute” agents into a pool of investigational
agents.
Even among proven drugs, one can find examples
where conflicting agendas might limit scientifically sup-
ported combination regimens. Treatment with a selective
inhibitor of BRAFV600Eincreases CD8+ T Cell infiltrate
in tumors of patients with metastatic melanoma. This is
likely a consequence of increased MDA expression with
selective BRAF inhibitors when MITF expression is dere-
pressed. These observations support the investigation of
BRAF inhibitor/immunotherapy combinations and ipili-
mumab is a plausible agent for this purpose. Given that
vemurafenib and ipilimumab are currently approved a
single agents in metastatic melanoma and the pharma-
ceutical companies that produce them are vying for
maximum market share, will the most scientifically
rigorous clinical investigations be undertaken to evaluate
this combination or inhibited out of concerns of new risks
that could be uncovered which could taint the perceived
safety profile of either agent?
Regulatory authorities must adapt to scientific under-
pinnings that drive the pursuit of combination therapies
and maintain an awareness of the unmet need for the pa-
tient population and the line of therapy being investi-
gated. Mechanism-of-action and clinical measures of
benefit dictate optimal endpoints for definitive trials.
Future advances will likely be limited by availability of
investigational drugs for novel/novel combinations.
Heritable changes in the expression of single genes or
patterns of genes not based on modifications of the
DNA sequence are methylation in C5 of cytosine within
CpG dinucleotides, hystone modifications and changes
in chromatin structure. Hypomethylation generally result
in gene expression while hypermethylation results in
gene silencing. Epigenetic modifications are generally
reversible pharmacologically as with Inhibitors of DNMT
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(e.g., 5-azacytidine, 5-aza-2′-deoxycytidine, Zebularine) or
Inhibitors of HDAC (e.g., TSA, depsipeptide, SAHA,....).
Epigenetically-regulatedTAA
MAGE-A1, -A2, -A3, -A4, -A6, -A10, MAGE-A12, BAGE,
GAGE1-6, SSX1-5, NY-ESO-1, HAGE, PRAME, RAGE-1,
etc. CTA expression is regulated by promoter methylation.
CTA expression in melanoma cells can be regulated by
DHA with a dose-dependent induction. Methylation sta-
tuses of melanoma cells may influence prognosis and (im-
mune-)response to therapy. LINE-1 is a surrogate marker
for global genomic methylation status, and, as shown by an
analysis of 42 stage IIIC melanoma patients about survival
according to LINE-1 methylation, hypermethylation is
related with a poorer prognosis and specific methylation
profiles associate with survival of stage IIIC melanoma
patients. Instead LINE-1 methylation correlates with the
number and level of expressed CTA.
The combination of IL-2 and standard doses of radiation
has been tested in metastatic melanoma, with the conclu-
sion that there is “. . . no apparent synergy in antitumor
effect”. Stereotactic body radiation therapy (SBRT) is to-
tally different from “conventional” radiation, because it
uses multiple beams from multiple directions, achieving a
higher dose to the tumor, lower dose to surrounding nor-
mal tissue and tumor motion (due to respiration) is taken
into account using “4D planning” (a movie loop of CT
images to determine treatment volume).
The rationale for testing SBRT+IL-2 is that high dose
per fraction radiation, in contrast to standard dose fractions,
can augment immune responses in murine tumor models
by lowering intratumoral Treg,increasing CD8 T-cell infil-
tration into the tumor, inducing antigen release, releasing
Damage-AssociatedMolecular
HMGB1 and up-regulating MHC class 1, B7.1 and Fas/
CD95. IL-2 can induce clinically meaningful immune
responses in (a minority of (ORR ~15–16%)) patients with
metastatic melanoma and renal cancer.
A phase I dose-escalation study of SBRT was per-
formed in patients with widely metastatic melanoma to
determine the maximum tolerated dose of SBRT when
used in conjunction with high-dose IL-2. The study mea-
sured the local control of SBRT-treated lesions, esti-
mated the overall tumor response, and to monitored
toxicities. Exploratory studies of immune responses on
peripheral blood mononuclear cells were also performed
using polychromatic flow cytometry.
5 out of 7 patients with melanoma had objective
regression. All SBRT-treated lesions regressed and there
were some responds in lesions not treated with SBRT.
There were no dose-limiting toxicities from SBRT and the
IL-2 toxicities were those anticipated. All 5 patients had a
complete regression of melanoma by PET imaging,
although minor residual imaging abnormalities persisted
on CT in 4 of these patients.
in humancancerare
Patterns (DAMPs),
Responding patients showed increased proliferation at
baseline and after IL-2 of CD4+ T cells with activated
TEM
phenotype (CD25+FoxP3-Ki67+CCR7-CD45RA-
CD27+CD28+/−) and CD8+ T cells with early TEM
phenotype(Ki67+CD25-CCR7-CD45RA+CD27+CD28+).
There was no change in proliferation of Tregcomparing
responders and non-responders.
Competing interests
PAA participated to Advisory Board from Bristol Myers Squibb, MSD,
Roche-Genentech, GSK, Celgene, Amgen, Medimmune, and Novartis and
received honoraria from Brystol Myers Squibb, MSD and Roche-Genentech.
AMG has no competing interest. BC Prometheus Pharmaceuticals: grant
support and speakers bureau. MBF has no competing interests. SF has no
competing interests. KF Consultant: Roche/Genentech, GlaxoSmithKline. BAF
has no competing interests. TG consultant for GSK-Bio, Incyte, BMS, Roche-
Genentech, and Eisai. JEG has no competing interest. HG has an Advisor role
in MSD compensated. KG has participated on Advisory Board for BMS, and
has received honoraria from BMS. AH consultancies, paid presentations or
financial trial support from: BMS, Boehringer Ingelheim, Celgene, Eisai, GSK,
IGEA, MSD, Novartis, Roche-Genentech. SH has served as a nonpaid
consultant to BMS and Genentech, and received clinical trial support from
BMS and Genentech. RK Institutional reimbursement for Advisory Boards and
conference travel from GSK and Roche. JMK is a consultant to GSKbio, and
has participated in advisory boards for Novartis, Merck, and GSK. SL serves on
an Advisory Board for Myriad Genetics Laboratories. MM Advisory Boards
from BMS, Roche and GSK. RM receives research support from Novartis, and
has consulted for Roche. He is eligible to income from drugs that are
commercialized by the Institute of Cancer Research through the “Rewards to
Inventors Scheme”. DLM has no competing interest. GP has no competing
interest. AR participated to Advisory Board from Amgen, Bristol Myers Squibb,
Celgene, Roche-Genentech, GSK, Merck, Millennium, Novartis and
Prometheus and received honoraria from these companies. DFS is a co-
inventor on a patent related to the growth of TIL in gas permeable flasks. RS
has no competing interest. EW has no competing interest. NM has no
competing interest. FMM has no competing interest.
Acknowledgement
The meeting was supported by Fondazione Melanoma Onlus and the
Society of ImmunoTherapy of Cancer (SITC). A special thanks to Collage
Congressi of Napoli for their support and cooperation in organizing the
meeting and to Michael Hoetzel for providing us the group picture from the
meeting.
Author details
1Department of Melanoma, Sarcoma, and Head and Neck Disease, Istituto
Nazionale Tumori Fondazione Pascale, Naples, Italy.2Earle A. Chiles Research
Institute, Providence Cancer Center, Portland, OR, USA.3Department of
Melanoma Research, John Wayne Cancer Institute at Saint John’s Health
Center, Santa Monica, CA, USA.4University of Pittsburgh Cancer Institute,
Pittsburgh, PA, USA.5Massachusetts General Hospital Cancer Center, Boston,
MA, USA.6Laboratory of Molecular and Tumor Immunology, Robert W. Franz
Cancer Research Center, Earle A. Chiles Research Institute, Providence
Portland Medical Center, and Department of Molecular Microbiology and
Immunology, Oregon Health and Science University, Portland, OR, USA.
7University of Chicago, Chicago, IL, USA.8Department of Surgical Oncology,
The University of Texas MD Anderson Cancer Center, Houston, TX, USA.9First
Department of Medicine, Athens Medical School, University of Athens,
Athens, Greece.10Huntsman Cancer Institute, University of Utah, Melanoma
and Cutaneous Oncology Program, Salt Lake City, UT, USA.11Department of
Dermatology, University of Kiel, Kiel, Germany.12Department of Medical
Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.13Westmead
Institute for Cancer Research, Westmead Millennium Institute and Melanoma
Institute Australia, University of Sydney, Sydney, NSW, Australia.14Division of
Hematology/Oncology, Departments of Medicine, Dermatology, and
Translational Science, University of Pittsburgh School of Medicine and
Melanoma Program of the Pittsburgh Cancer Institute, Pittsburgh, PA, USA.
15Medical Oncology and Immunotherapy, Department of Oncology,
University Hospital of Siena, Istituto Toscano Tumori, Siena, Italy.16Molecular
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