Review Not peer-reviewed version

Efficacy of Pembrolizumab in

Advanced Melanoma: A Narrative

Review

*
Giulio Rizzetto , Edoardo De Simoni , Elisa Molinelli , Annamaria Offidani , Oriana Simonetti

Posted Date: 26 June 2023

doi: 10.20944/preprints202306.1811.v1

Keywords: advanced melanoma; pembrolizumab; immunotherapy

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Review

Efficacy of pembrolizumab in advanced melanoma: a

narrative review
Giulio Rizzetto 1, Edoardo De Simoni 1, Elisa Molinelli 1, Annamaria Offidani 1, and Oriana
Simonetti 1*
1 Clinic of Dermatology, Department of Clinical and Molecular Sciences, Polytechnic University of Marche,
60126, Ancona, Italy; [email protected] (G.R.); [email protected] (E.M.);
[email protected] (A.O.) [email protected] (OS)
* Correspondence: [email protected]; Tel.: +39-071-596-3494

Abstract: (1) Introduction: pembrolizumab showed to increase survival in patients with metastatic
melanoma, Considering the numerous oncoming studies, we decided to conduct a narrative review
of the latest efficacy evidence regarding the use of pembrolizumab, alone or in combination, in pa-
tients with metastatic melanoma. (2) Methods: A search was conducted on Pubmed using "pem-
brolizumab," and "metastatic melanoma" as keywords, considering studies from 2022 onward. (3)
results: we reviewed pembrolizumab and associations, cost-effectiveness, virus, advanced acral
melanoma, long-term outcomes, real-life data, biomarkers, obesity, and vaccines (4) Conclusions:
pembrolizumab is a fundamental option in the therapy of metastatic melanoma. However, a certain
group of patients do not respond and therefore new combination options need to be evaluated. In
particular, the use of vaccines tailored to tumor epitopes could represent a breakthrough in the
treatment of resistant forms. Further studies with larger sample numbers are needed to confirm
preliminary results.

Keywords: advanced melanoma; pembrolizumab; immunotherapy

1. Introduction
Melanoma is one of the most problematic malignancies for public health, with a progressive
increase in new diagnoses. In addition, about 20% of patients may develop an advanced (unresec-
table/metastatic) form [1]. However, the possibility of evaluating new markers for lymph node in-
volvement [2,3], and new therapeutic regimens for metastatic melanoma allowed a substantial reduc-
tion in mortality [4]. Immunologic check-point inhibitors (ICIs), particularly ipilimumab, pembroli-
zumab, and nivolumab, permitted a revolution of the approach to the metastatic patient, inducing a
relevant increase in survival [5-7]. Ipilimumab, an inhibitor of Cytotoxic T-lymphocyte-associated
protein-4 (CTLA-4), in monotherapy allowed to increase the 10-year survival of metastatic patients
from 10% to 22% [8].
The programmed death (PD-1) inhibitors, nivolumab and pembrolizumab, also showed to in-
crease survival in metastatic patients [9-13]. In three clinical trials in patients with advanced mela-
noma (KEYNOTE-001, KEYNOTE-002, and KEYNOTE-006), therapy with pembrolizumab alone re-
sulted in 30-40% complete clinical response [9,11,14-17]. Specifically, in the KEYNOTE-001 study on
advanced melanoma, pembrolizumab monotherapy resulted in a 3-year overall survival (OS) of 41%
in patients previously treated with ipilimumab and 45% in therapy-naïve patients [13]. New studies
are currently emerging in the literature regarding the efficacy of pembrolizumab in patients with
metastatic melanoma, even considering the long-term efficacy in real-life.
Pembrolizumab is a humanized IgG4/kappa anti PD-1 monoclonal antibody produced in ham-
ster ovarian cells. The PD-1 receptor is a negative regulator of T-cell activity that has been shown to

© 2023 by the author(s). Distributed under a Creative Commons CC BY license.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 June 2023 doi:10.20944/preprints202306.1811.v1

be involved in the control of T-cell immune responses. KEYTRUDA potentiates T-cell responses, in-
cluding antitumor responses, by blocking PD-1 binding to PD-L1 and PD-L2, which are expressed on
antigen-presenting cells and may be expressed by tumors or other cells in the tumor microenviron-
ment [18]. According to current indications, pembrolizumab is indicated as monotherapy in the treat-
ment of adults and adolescents > 12 years of age with unresectable or metastatic melanoma. In addi-
tion, pembrolizumab as monotherapy is indicated in the adjuvant treatment of adults and adolescents
> 12 years old with melanoma stage IIB, IIC, or III and who had complete tumor resection [18].
A recent meta-analysis [19] compared the use of pembrolizumab with conventional chemother-
apy drugs for the treatment of advanced forms of melanoma, showing promising results of pembroli-
zumab (pembrolizumab monotherapy group 2 mg/kg with OS 13.4 months, 10 mg/kg group OS 14.7
months, chemotherapy group OS 11.0 months). However, there were insufficient data to confirm sta-
tistical significance.
In cutaneous melanoma, in addition to tumor mutahional burden (TMB), MHC-II expression,
plodia, heterogeneity, and tumor purity were also associated with response to pembrolizumab. Spe-
cifically, MHC-II expression on tumor cells was found to be predictive of response to anti-PD1, hy-
pothesizing that they represented a subset of tumors capable of stimulating CD4 T-cells or CD8 T-
cells. In support of this, MHC-II transcriptomics was correlated with the expression of CD4, PRF1,
and GZMA (cytolytic molecules) [20].
Considering the numerous studies being published, we decided to conduct a narrative review
of the literature reporting the latest efficacy evidence regarding the use of pembrolizumab, alone or
in combination, in patients with metastatic melanoma.

2. Materials and Methods

A search was conducted on Pubmed using "pembrolizumab," and "metastatic melanoma" as
keywords. Only English language studies were included in the narrative review. Studies from 2022
onward were included as a time limit. The purpose is to summarize the latest evidence produced
regarding the efficacy of pembrolizumab in the patient with metastatic melanoma. Studies that did
not evaluate the efficacy of pembrolizumab or that used a neoadjuvant/adjuvant setting and case
reports were excluded.

3. Results
From our review of the literature on Pubmed, a total of 143 articles were found. After excluding
studies that did not meet the search criteria, a total of 20 studies were reviewed.

3.1. Pembrolizumab and associations

In a multicenter, double-blind phase III study, the combination of pembrolizumab and tali-
mogene laherparepvec (T-VEC) versus placebo and pembrolizumab was evaluated in 692 patients
with metastatic/unresectable melanoma who were naive for anti PD-1. In both groups pembroli-
zumab was administered at 200 mg once every 3 weeks. T-VEC and pembrolizumab did not signifi-
cantly improve progression-free survival (PFS) and OS compared with the placebo/pembrolizumab
group. Thus, in this study, it appears that conbination therapy does not bring improvement in the
parameters considered [21].
A recent phase I study evaluated the combination of pembrolizumab, vemurafenib, and cobi-
metinib as first-line in BRAF V600E / K mutated BRAF metastatic patients. There were 2 groups
treated with pembrolizumab / vemurafenib and pembrolizumab / vemurafenib / cobimetinib, respec-
tively. The median OS was 23.8 months for vemurafenib / pembrolizumab, while the triple combina-
tion did not allow calculation of OS as it was discontinued due to the occurrence of side effects, lead-
ing to the closure of the study [22]. BRAF inhibitors (BRAFi) appear to be able to modulate the tumor
microenvironment (TME). In fact, administration of a BRAFi in a BRAF-mutant melanoma model
resulted in increased expression of CD40 ligand and interferon-gamma on tumor-infiltrating CD4+
lymphocytes (TILs) on the one hand, and decreased T-reg and myeloid cells on the other, suggesting
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 June 2023 doi:10.20944/preprints202306.1811.v1

antitumor modulation of the TME [22]. Furthermore, an increase in T-cell exhaustion markers TIM-3
and PD1 was correlated with BRAFi in patients with metastatic melanoma [22]. However, the triple
therapy proposed in this study did not allowed the progression of the trial due to the significant
adverse events, as dermatitis (n=8), hepatitis (n=1), arthralgias (n=1), and QTc prolongation (n=1).
Despite the failure of previous combinations, a phase Ib/II study of 24 patients with metastatic
melanoma evaluated the efficacy of pembrolizumab and all-trans retinoic acid (ATRA), achieving an
overall response rate of 71%, of which 50% were in complete response, and a 1-year OS was 80%. The
aim of this study was to target circulating myeloid-derived suppressor cells (MDSCs), seeking to
enhance the action of immunotherapy. The promising results open the way for further studies and
represents an interesting new therapeutic option [23]. MDSCs are cells characterized by high immune
function. Being immature cells, MDSCs can change following exposure to agents such as ATRA, a
derivative of vit. A. Specifically, ATRA appears to reduce both the frequency and functions of MDSCs
through activation of ERK1/2, upregulation of glutathione synthase, and increased glutathione. The
latter reduces ROS production and consequently results in terminal differentiation of MDSCs. Thus,
it is hypothesized that combining Pembrolizumab with ATRA could reduce the frequency of MDSCs
and improve the efficacy of pembrolizumab, particularly by reducing polymorphonucleate-MDSCs
[23].
Finally, in a phase Ib study of patients with metastatic melanoma, the association between pem-
brolizumab (200 mg IV every 3 weeks) and escalating doses of IL-2 (6,000 or 60,000 or 600,000 IU/kg
IV bolus every 8 hours up to 14 doses per cycle) was evaluated in cohorts of 3 patients. Nine patients
were included, obtaining a partial response in 1 (11%), previously treated with pembrolizumab, with
high doses of IL-2. Another patient showed a stable condition after 4.5 years of follow-up, even
though he had not pembrolizumab treatment prior to the study. No patient showed dose-limiting
toxicities. For this reason, treatment with pembrolizumab and IL-2 could be done, especially in those
patients who did not respond to pembrolizumab therapy alone. However, evaluation on a larger
sample is needed [24]. (Tab.1)

Table 1. Efficacy of pembrolizumab in association: summary of reviewed studies on metastatic/unre-

sectable melanoma.

Study Type of study treatments results

T-VEC/pembrolizumab not sig-
double-blind phase III nificantly improve PFS and OS
P/T-VEC
Chesney JA et study compared with placebo/pem-
vs
al. 2023 [20] brolizumab
P/placebo
692 patients anti PD-1
naive
median OS was 23.8 months
P/ vemurafenib
P/vermurafenib
vs
phase I study
Shaikh SS et al. P/ vemurafenib
P/ vemurafenib /cobimetinib
2022 [21] /cobimetinib
discontinued for sides
BRAF V600E/K meta-
static patients
overall response rate 71%, of
phase Ib/II study
P/ATRA which 50% complete response
24 patients
Tobin RP et al 1-year OS was 80%.
2023 [22]
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 June 2023 doi:10.20944/preprints202306.1811.v1

P/ escalating doses partial response 1 (11%), with

of IL-2 (6,000 or high doses IL-2
60,000 or 600,000 1 patient stable condition after
Silk AW et al phase Ib study
IU/kg IV bolus 4.5 years of FU
2023 [23] 9 patients
every 8 hours up No dose-limiting toxicities.
to 14 doses per cy- Evaluation on a larger sample
cle) is needed
Coxsackievirus
Silk AW et al. phase 1b study objective response in 47%, com-
A21 (V937) intra-
2023 [25] 36 patients plete response in 22% of cases
tumorally + P
Pembroli-
Phase II clinical trial
Long GV et al. zumab/EVX-01
KEYNOTE-D36 ongoing
[41] (neoepitope vac-
cine)
After 18 months, RFS was
KEYNOTE-942 clinical 78.6%
mRNA-4157/pem-
trial (Combination group) vs 62.2%
brolizumab (107)
completely resected, (pembrolizumab alone). In ad-
or pembrolizumab
high-risk melanoma dition, the combination group
alone (50)
157 patients showed a reduction in the risk
of recurrence or death by 44%
* Abbreviations: talimogene laherparepvec (T-VEC), progression-free survival (PFS), overall survival
(OS), Pembrolizumab (P), all-trans retinoic acid (ATRA), recurrence free survival (RFS.

3.2. Pembrolizumab cost-effectiveness

A recent study based on a partitioned-survival model with a 1-week cycle length and a 20-year
base-case time horizon evaluated the cost-effectiveness of pembrolizumab versus paclitaxel and car-
boplatin as first-line in patients with metastatic or unresectable melanoma. Considering that an in-
crease of 2.63 life-years and an increase of 2.24 quality-adjusted life-years was estimated, pembroli-
zumab emerges as an effective treatment option in metastatic patients as first-line and also cost-ef-
fective, not exceeding the total cost of treatment the 3 times the per capita gross domestic Chinese
[25]. This confirms that first-line therapy with pembrolizumab is not only an effective but also an
economically viable option in countries with a per capita product similar to China.

3.3. Pembrolizumab and virus

Coxsackievirus A21 (V937) is an oncolytic virus that has been shown to be effective against met-
astatic melanoma. Oncolytic viruses are believed to act against tumours through two distinct mech-
anisms: direct lysis of tumour cells and activation of a specific anti-tumour immune response. Cox-
sackievirus is an RNA virus, which can cause mild upper respiratory symptoms, and V937 is a se-
lected, genetically unmodified, oncolytic strain that can enter cells by binding to intracellular adhe-
sion molecule-1 (ICAM-1) and decay-accelerating factor (DAF) receptors. Both of these receptors are
specifically expressed on the surface of melanoma cells. From a pharmacodynamic point of view,
oncolytic viruses result in increased production of interferon, CD8+ T cells and expression of pro-
grammed death ligand 1 (PD-L1) in the tumour microenvironment, as well as reduced populations
of suppressor T cells. For this reason, it was assumed that they could improve responses when com-
bined with inhibitors inhibiting PD-1/PD1-L or cytotoxic T lymphocyte antigen 4 (CTLA-4) [26].
In a recent phase 1b study of 36 patients Coxsackievirus A21 (V937) was administered intra-
tumorally in combination with pembrolizumab, achieving an objective response in 47% of cases, and
a complete response in 22% of cases. Interestingly, the density of intratumoral CD3+CD8- T cells was
lower in responders, suggesting that the combination of Coxsackievirus A21 (V937) and pembroli-
zumab might help overcome the limitations of a nonimmunologically "active" tumor environment
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 June 2023 doi:10.20944/preprints202306.1811.v1

[26]. Surprisingly, responses were not correlated with an increase in viral signalling proteins (RIG-I,
TLR7 and TLR8), viral entry proteins (ICAM-1 and DAF), PD-L1, nor was an inflamed tumour mi-
croenvironment found. In fact, the levels of CD3+CD8- infiltrating T lymphocytes in the pre-treat-
ment tumour samples were actually lower in responders than in non-responders. One hypothesis
that might explain this is that the pre-treatment tumour samples of responders include fewer regula-
tory cells [26]. Another hypothesis that may explain how fewer tumour-infiltrating lymphocytes may
favour a better response is that fewer immune cells were immediately available in the tumour to fight
Coxsackievirus infection, resulting in more robust viral replication and an increased circulating viral
load. A significant increase of IP-10/CXCL10 and a smaller increase of MDC/CCL22 was also ob-
served in the serum of patients. CXCL10 plays an important function in the recruitment of antitumour
T-cells in melanoma. Furthermore, CXCL10 via the CXCR3 receptor promotes the migration of lym-
phocytes to dendritic cells, which is necessary for the response to PD-1 blockade [27]. Clinically, it
has been shown that elevated pre-treatment CXCL9 and CXCL10 levels correlate with response to
anti-PD-(L)1 therapy in patients with non-small cell lung cancer , and CXCL9 and CXCL10 increase
in the first months of treatment in melanoma patients responding to PD-1 inhibitor therapy. Finally,
elevated pre-treatment CXCL9 and CXCL10 levels appear to correlate with response to anti-PD1 ther-
apy in patients with non-small cell lung cancer [28], similarly we find an increase in CXCL9 and
CXCL10 in the first months of treatment with PD-1 inhibitors in responders with melanoma.
In the light of these data, it is hypothesized that V937 infection increases the production of inter-
feron-gamma and CXCL10 by immune cells in the tumor microenvironment, particularly macro-
phages, which promote the response to anti-PD-1 by properly guiding activated lymphocytes to den-
dritic cells [26].
In our opinion, the use of oncolytic viruses could be a promising solution, helping to overcome
tumour immune escape mechanisms.
In a pilot study, the efficacy of intratumourally injected ONCOS-102, an oncolytic adenovirus
expressing human GM-CSF, was evaluated in combination with pembrolizumab. 21 patients with
advanced melanoma with disease progression after treatment with pembrolizumab were enrolled.
The treatment was well-tolerated, with few adverse effects (pyrexia, nausea, chills), and no dose-
limiting toxicities, being objectively effective in reducing the size of one or more non-injected metas-
tases, highlighting the systemic action of the treatment. Serial analyses were also performed on tu-
mours injected with ONCOS-102 using RNA expression and immunofluorescence, showing the cor-
relation between immune cell level and persistence and patient outcome [29].

3.4. Pembrolizumab and advanced acral melanoma

A retrospective cohort study evaluated the efficacy in 325 patients with unresectable acral mel-
anoma of PD-1 inhibitors, including pembrolizumab, in combination or not with ipilimumab. In-
cluded were anti-PD-1 184 (57%), anti-PD-1/ipilimumab 59 (18%), and 82 (25%) ipilimumab alone.
The objective response rate (ORR) was significantly higher in the anti PD-1/ipilumumab combination
group (43%) than anti PD-1 alone (26%) and ipilimumab alone (15%), respectively. However, anti
PD-1/ipilimumab did not lead to a significant difference in OS and PFS compared with anti PD-1
alone [30]. For this reason, further trials considering the acral melanoma subgroup are needed to
evaluate the best treatment approach.

3.5. Pembrolizumab, long-term outcomes and real-life data

Regarding long-term outcomes, 5-year OS data for pembrolizumab in metastatic melanoma
have been published, with reference to the KEYNOTE-001 studies [31] e KEYNOTE-006 [32]. The
results were comparable to those of nivolumab, showing a 5-year OS of 41% and 43% respectively in
the two trials [33].
In a study of 103 Chinese patients with metastatic melanoma, 3-year follow-up was performed
after failure of first treatment and second treatment with pembrolizumab 2 mg/kg every 3 weeks for
≤35 cycles. The median OS was 13.2 months and the 36-month OS rate was 22.3%. It is interesting that
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 June 2023 doi:10.20944/preprints202306.1811.v1

median OS data by melanoma subtype are reported, specifically 14.8 months for acral, with a signif-
icant difference between acral PD-L1-positive disease (median OS 22.8 months) and acral PD-L1-neg-
ative disease (8.4 months). In addition, pembrolizumab therapy as a second-line therapy was shown
to have good tolerability [34]. These studies suggest that a good therapeutic response is maintained
even in long-term follow-up, partly due to the tolerability of the treatment.
In a retrospective study on 132 patients with metastatic melanoma treated with ICIs, including
pembrolizumab, [35] Perez et al. report that 34.8% achieved a complete clinical response, while PFS
was 97.5% at 1 year and 94.7% at 3 years after treatment discontinuation. Thus, this real-life study
suggests that discontinuation of ICIs in those metastatic patients with complete remission of disease
is feasible, keeping the clinical condition stable at 3 years. However, it is also necessary to evaluate
these aspects in studies with a larger sample size.
In another national retrospective study of 5097 New Zealand patients with metastatic melanoma
treated with ICIs, including pembrolizumab, a 1-year OS of 72% was reported, while the 2-year OS
was 60%, confirming data from previous clinical trials [36].
Finally, a recent study of 1037 patients with metastatic melanoma compared the efficacy of
nivolumab and pembrolizumab. Both have approval for the treatment of metastatic melanoma, but
the choice between the two is often left to the clinician. The results of this study showed that the
median OS was 17.4 months for pembrolizumab and 20 months for nivolumab, while the estimated
OS at 2 and 3 years was 42/34% for pembrolizumab and 47/37% with nivolumab. The differences in
OS, PFS, and overall response were not statistically significant in naive patients with metastatic mel-
anoma, confirming the clinician's role in treatment choice [37].

3.6. Pembrolizumab and biomarkers

Possible new markers of therapeutic response to pembrolizumab recently emerged in a study of
46 metastatic melanoma patients. CD103 expressed on CD8+ T lymphocytes is associated with a com-
plete response to pembrolizumab with statistical significance when the density (p=0.04) and propor-
tions (p=0.012) of this T-cell population increased. Improved survival correlated with the increased
proportion of CD8+ CD103+ T-cells (P = 0.0085) and also with reduced expression of periplakin (P =
0.012) and periplakin + SOX10 (P = 0.0012) [38]. This preliminary study highlights that the role of
certain cellular markers could help select those metastatic patients most promising to respond to
pembrolizumab monotherapy, however further studies on a larger sample are needed.
In a study of 68 metastatic melanoma patients, tryptophan and glucose metabolism was evalu-
ated using C11-labeled α-methyl tryptophan (C11-AMT) and fluorodeoxyglucose (FDG) PET imag-
ing of tumor lesions. In addition, the expression of tryptophan-metabolizing enzymes (TMEs; TPH1,
TPH2, TDO2, IDO1) and the tryptophan transporter, LAT1, were evaluated at immunohistochemis-
try. In this study, it was found that elevated tryptophan metabolism in metastatic melanoma is a
predictor of poor response to pembrolizumab [39].
Interestingly, some soluble biomarkers have also been proposed. In a recent study of 41 patients
with metastatic melanoma, plasma levels of soluble PD-1 (sPD-1), soluble programmed cell death
ligand 1 (sPD-L1), butyrophilins (BTNs) 2A1, 3A1, and body mass index (BMI) were evaluated as
predictors of efficacy of anti PD-1 therapy. Low levels of sPD-1 plus high levels of sBTN2A1 were
associated with a better overall response rate. In addition, patients with BMI ⩾ 25 and sPD-1 < 11.24
ng/ml had longer time to treatment failure (p < 0.001) [40].
Regarding imaging tecnique, another study used positron emission tomography (PET) with zir-
conium-89 (89Zr)-labeled pembrolizumab prior to pembrolizumab treatment to evaluate the predic-
tive ability of response to anti PD-1. In 11 patients with metastatic melanoma 89Zr-pembrolizumab
tumor uptake was calculated, obtaining a statistically significant direct correlation with clinical re-
sponse, OS, and PFS. Although this method is interesting in the landscape of pembrolizumab efficacy
markers, the small sample size requires further studies to confirm its clinical utility [41]. (Tab.2)
Texture analysis features at contrast medium CT of melanoma metastases also appear to be a
predictor of response to treatment with anti PD-1. In a study of 127 metastases, 3 items were identified
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 June 2023 doi:10.20944/preprints202306.1811.v1

as independent predictors of response to therapy: skewness at fine texture scale, skewness at medium
texture scale, and variation of entropy at fine texture scale. Although further studies are needed to
confirm their validity, CT methods may also be an aid in assessing response to anti PD-1 immuno-
therapy [42].

Table 2. Pembrolizumab and biomarkers: summary of reviewed studies on metastatic/unresectable

melanoma.

Study Type of study Biomarkers results

increased density/proportions
CD103 CD103 CD8+ T associated with
complete response to P

preliminary study
Edmonds NL et Reduced expression of
al. 2022 [34] Periplakin periplakin and periplakin +
SOX10 associated with im-
periplakin + SOX10 proved survival during P
46 patients

C11-AMT
elevated tryptophan metabo-
FDG PET
Oldan JD et al. Prospective clinical trial lism predictor of poor response
tryptophan-metabolizing
2023 [35] to P.
enzymes
68 patients
Low levels of sPD-1 + high lev-
els of sBTN2A1 associated with
sPD-1
Prospective cohort study a better overall response rate.
sPD-L1
41 patients patients with BMI ⩾ 25 and
Incorvaia L. et BTNs 2A1, 3A1
sPD-1 < 11.24 ng/ml had longer
al 2023 [36] time to treatment failure.

89Zr-P tumor uptake signifi-

Kok IC et al Prospective study
(89Zr)-labeled P PET cant direct correlate with clini-
2022 [37] 11 patients
cal response, OS, and PFS
3 items predictors of favorable
response to P
Texture analysis features 1. skewness at FTS,
Bonnin A et al. Retrospective study
at contrast medium CT of 2. skewness at MTS,
2022 [38] 127 metastases
melanoma metastases 3. variation of entropy at
FTS

* Abbreviations: Pembrolizumab (P), C11-labeled α-methyl tryptophan (C11-AMT), fluorodeoxyglu-

cose (FDG), tryptophan-metabolizing enzymes (TMEs; TPH1, TPH2, TDO2, IDO1), soluble PD-1
(sPD-1), soluble programmed cell death ligand 1 (sPD-L1), butyrophilins (BTNs), zirconium-89 (89Zr),
progression-free survival (PFS), overall survival (OS), fine texture scale (FTS), Medium texture scale.

3.7. Pembrolizumab and obesity

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 June 2023 doi:10.20944/preprints202306.1811.v1

Although obesity is a major risk factor for cancer mortality [43], an "obesity paradox" emerges
as obesity correlates with better clinical outcomes in patients with metastatic melanoma, especially
when treated with anti PD-1. In a retrospective study of 266 patients with metastatic melanoma
treated with pembrolizumab or nivolumab, it emerges that obesity had a protective role, specifically
a 10 cm2/m2 increase in visceral fat index (VFI) was associated with longer overall survival after
adjusting for covariates, but this was not maintained when adjusted for systemic immune-inflamma-
tion index. Thus, the prognostic role of visceral obesity is conditional on inflammatory status. Obesity
appears to determine an increase in PD-1 expression, induces T-cells to release more PD-1 protein,
and increases the secretion of adiponectin and leptin from adipose tissue. These alterations result in
increased T-cell depletion and dysfunction, which promote tumour progression. On the other hand,
this provides a better understanding of the relationship between obesity and ICIs, as these agents
remove the inhibitory signals of T-cell activation and promote an effective anti-tumour response. [44]

3.8. Pembrolizumab and vaccines

The phase II KEYNOTE-D36 clinical trial will evaluate the efficacy and safety of the combination
of pembrolizumab and EVX-01, a personalized neoepitope vaccine, in patients with metastatic or
unresectable melanoma. EVX-01 consists of multiple peptides that represent a neoepitope expressed
only in the patient's specific tumor, allowing a tumor-specific immune response [45].
Regarding the use of vaccines in combination with pembrolizumab, data were recently pre-
sented from a KEYNOTE-942 clinical trial in patients with completely resected, high-risk melanoma.
Patients were treated with mRNA-4157/pembrolizumab (107) or pembrolizumab alone (50). 9 doses
of mRNA-4157 (1mg) were administered every 3 weeks, while pembrolizumab 200mg was adminis-
tered intravenously for 18 cycles. After 18 months, recurrence free survival (RFS) was 78.6% in the
combination group versus 62.2% in the group with pembrolizumab alone. In addition, the combina-
tion group showed a reduction in the risk of recurrence or death by 44% (HR = 0.561; 95% CI: (0.309,
1.017). These results are very promising, highlighting the therapeutic possibilities of the combination
of adjuvant standard of care and a vaccine tailored to specific tumor neoantigens, however, a phase
III study with larger sample size is needed to confirm the results [46,47]. In particular, this mRNA
vaccine encodes up to 34 tumour neo-antigens and is produced based on the specific mutation se-
quence of the patient's tumour. Once administered into the body, the neo-antigen sequences encoded
by RNA are translated endogenously and subjected to natural antigen processing and presentation
by antigen-presentig cells, a crucial step in adaptive immunity.

4. Discussion
Current therapeutic options in advanced melanoma include PD-1 inhibitors associated or not
with CTLA-4 or lymphocyte-activating gene (LAG)-3 inhibitors, and in cases of BRAF V600E/K mu-
tated BRAF inhibitors, called targeted therapy [11,48]. However, the DREAMseq study showed that
immunotherapy followed by target therapy in case of progression increased OS of 20% compared
with reversing the two treatments [49]. This is because BRAF inhibitors are supposed to modulate
the tumor immune microenvironment by increasing interferon gamma production on intratumoral
CD4+ T lymphocytes and to increase CD8+ tumor-infiltrating lymphocytes (TILs) [50,51].
Currently, guidelines suggest in patients with metastatic melanoma, regardless of BRAF status,
the use of an ICIs with anti PD-1, in association or not with anti-CTLA4, allowing long-lasting re-
sponses and long-time survival in responders [52].
Specifically, clinical trials of ICIs in patients with metastatic melanoma report a 5-year OS of up
to 60% and a 5-year ongoing response rate of up to 62% [49,53]. However, about 40-60% of treated
patients do not respond to ICIs, and about 30% of treated patients relapse in the following 2 years
[7,11,54-56].
Therefore, the use of vaccines in combination with pembrolizumab could be a solution for all
those patients who do not respond to immunotherapy due to immune escape. Clinical trial data show
that 50 to 64% of melanoma patients, even if on immunotherapy, will experience disease progression
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 June 2023 doi:10.20944/preprints202306.1811.v1

after 1 to 5 years [48]. The mechanisms of resistance may be related to several aspects. Reduced ex-
pression of molecular targets, particularly PD-L1, on neoplastic cells results in primary tumor re-
sistance to pembrolizumab [57, 58]
Other causes of resistance to immunotherapy are low neoantigen load on tumor cells, allowing
them to overcome immune surveillance [59], and immunosuppression brought about by the tumor
microenvironment [60-63].
Another aspect to be considered is the use of markers that could help predict response to pem-
brolizumab, thus allowing selection of patients who might benefit from association with other treat-
ments, such as personalized incoming vaccinations. In our opinion, the use of texture analysis fea-
tures to contrast medium CT of melanoma metastases could be a method that could be easily inte-
grated into the staging of the metastatic patient, however, further studies are needed to confirm its
usefulness [39].
Furthermore, evaluation of the tumor microenvironment, in addition to being useful in assessing
the prognosis of melanoma [64-68], also appears to be able to give insight into the efficacy of pem-
brolizumab therapy [35], although the data need further confirmation on larger samples.
It is important to notiche that therapy with pembrolizumab can be combined with BRAF inhib-
itors. The BRAF V600 mutation is found in approximately 35-50% of metastatic melanomas, and is
associated with increased disease aggressiveness and reduced survival [69,70]. A recent multicen-
ter, double-blind phase III trial (STARBOARD) of 624 patients with BRAF V600E/K metastatic mela-
noma compared the efficacy of triple therapy (encorafenib, binimetinib and pembrolizumab) versus
placebo plus PD-1 monotherapy (pembrolizumab). However, data from this study are not currently
available.

5. Conclusions
In conclusion, pembrolizumab represents a fundamental option in the therapy of metastatic mel-
anoma, as confirmed by data in real-life and in long-term follow-up. However, a certain proportion
of patients do not respond to this therapy and therefore new combination options need to be evalu-
ated. Among the new treatments, the use of vaccinations tailored to tumor epitopes could represent
a breakthrough in the treatment of resistant forms, but further studies with larger sample numbers
are needed to confirm preliminary results.
Supplementary Materials: not available

Author Contributions: Conceptualization, G.R., O.S. and A.O..; methodology, E.M..;.; validation, A.O. and E.D.;
data curation, E.D.; writing—original draft preparation, G.R..; writing—review and editing, O.S:.; All authors
have read and agreed to the published version of the manuscript.

Funding: This research received no external funding

Institutional Review Board Statement: Not applicable

Informed Consent Statement: Not applicable

Data Availability Statement: Not applicable

Conflicts of Interest: The authors declare no conflict of interest

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