Lung Cancer 142 (2020) 47–50

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Lung Cancer
journal homepage: www.elsevier.com/locate/lungcan

Case report

Impressive clinical response to anti-PD-1 therapy in epithelioid
mesothelioma with high clonal PD-L1 expression and EML4-ALK
rearrangement

T

Giuseppe Brontea,*, Angelo Delmontea, Marco Angelo Burgioa, Alberto Verlicchia,
Maurizio Puccettib, Sara Bravaccinia, Paola Craveroa, Maria Maddalena Tumedeia, Danila Dianoa,
Giulio Rossic, Paola Ulivia, Giovanni Martinellia, Lucio Crinòa
a
b
c

Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy
Azienda Unità Sanitaria Locale (AUSL) Imola, Imola, Italy
AUSL Romagna, Ospedale Santa Maria delle Croci, Ravenna, Italy

A R T I C LE I N FO

A B S T R A C T

Keywords:
Mesothelioma
PD-L1
Immunotherapy
Immune checkpoint inhibitors
EML4-ALK

Objectives: Treatment options for malignant pleural mesothelioma (MPM) are limited but some studies on immune checkpoint inhibitors (ICIs) in MPM have reported antitumor activity. Very little is known about immunerelated predictive factors.
Materials and methods: Here we report the case of a 45-year-old woman presenting with dyspnea and evidence of
pleural effusion. She was diagnosed with malignant epithelioid pleural mesothelioma with brain metastasis and
peritoneal carcinosis, refractory to initial standard chemotherapy treatment. Because of high PDL1 expression
(100 %), she was treated with the anti-PD1 agent, pembrolizumab.
Results: Chemotherapy with cisplatin and pemetrexed was started, imaging studies showing stable disease after
3 treatment cycles. The patient underwent pleural decortication but rapidly progressed and thus started chemotherapy with carboplatin and gemcitabine. After 2 cycles she experienced seizures caused by a brain metastasis. This secondary lesion was surgically removed and confirmed as a metastasis from mesothelioma.
Samples from both the primary tumor and the metastasis were molecularly characterized, the pleural sample
proving ALK-positive and the brain sample, ALK-negative. PD-L1 was positive in 10 % of tumor cells in the
pleural biopsy and 100 % in the brain lesion. Next generation sequencing analysis was negative for both samples.
It was decided to start alectinib. Disease progression (peritoneal carcinosis and liver metastases) was documented after one month followed by complete bowel obstruction and recurrence in the site of the brain surgery.
Alectinib was stopped and supportive care begun with parenteral nutrition via nasogastric tube. Pembrolizumab
was started and after 15 days the patient’s condition had significantly improved, enabling recanalization and
restoration of enteral nutrition. Imaging displayed complete response of the brain metastasis, peritoneal carcinosis, bone lesions and mediastinal nodal metastases. A partial response was documented in the pleural and
pulmonary nodules, with stable liver metastases. The patient is still undergoing immunotherapy and has no
cancer-related symptoms.
Conclusions: Our findings indicate that the use of immunotherapy in MPM warrants further investigation.
Furthermore, the impressive clinical response obtained by our patient suggests that immune checkpoint inhibitors could help in the management of the disease after the failure of other treatments.

Abbreviations: MPM, malignant pleural mesothelioma; OS, overall survival; PD-L1, programmed cell death ligand-1; CTLA-4, cytotoxic T lymphocyte-associated
protein 4; 18FDG-PET/CT, 18F-fluorodeoxyglucose positron emission tomography/computed tomography; MRI, magnetic resonance imaging; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; NGS, next generation sequencing; ALK, anaplastic lymphoma kinase; EML4, echinoderm microtubuleassociated protein-like 4; MET, mesenchymal-to-epithelial transition
⁎
Corresponding author at: Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Via P. Maroncelli 40,
47014, Meldola, Italy.
E-mail address: giuseppe.bronte@irst.emr.it (G. Bronte).
https://doi.org/10.1016/j.lungcan.2020.02.006
Received 12 September 2019; Received in revised form 6 February 2020; Accepted 12 February 2020
0169-5002/ © 2020 Elsevier B.V. All rights reserved.

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Fig. 1. Hematoxylin & eosin (H&E), ALK and PD-L1 expression analyzed by immunohistochemistry in tumor tissue from the pleura and brain metastasis (10x
magnification).

1. Introduction

ALK translocation, a known oncogene driver, was found in tumor tissue.

Malignant pleural mesothelioma (MPM) is a rare, aggressive tumor
originating from mesothelial cells. It is associated with exposure to
asbestos, mainly occupational, rarely environmental or domestic.
Diagnosis is usually made at an advanced stage because symptoms are
non-specific and late. The management of this tumor is challenging and
outcome is poor. Therapeutic strategies are mainly based on surgery
with curative intent or palliative cytotoxic chemotherapy. Few patients
are candidates for surgery which must necessarily take into account
numerous factors (i.e. histology, tumor stage, performance status, nutritional status, functional parameters for thoracic surgery). For the last
15 years pemetrexed plus cisplatin or carboplatin has been the standard
first-line chemotherapy regimen for MPM, obtaining a median overall
survival (OS) of ≤ 16 months [1]. Radiotherapy is generally reserved
for pain relief when the chest wall is infiltrated. Vinorelbine and
gemcitabine are sometimes used as second-or-more-line treatments,
obtaining poor disease control (< 30 %) and a median OS of < 6
months [2]. Thus, there are currently no further approved treatment
lines.
Several studies have reported that the programmed cell death ligand-1 (PD-L1), an immune checkpoint that inhibits T-cell function, is
expressed in 18–28 % of mesothelioma cells and associated with poor
prognosis [3]. The first immune checkpoint inhibitor studied was tremelimumab, which targets the cytotoxic T lymphocyte-associated protein 4 (CTLA-4). In MPM patients who progressed on first-line platinumbased chemotherapy, tremelimumab achieved 31 % disease control
with a median progression-free survival (PFS) of 6.2 months (95 %
confidence interval [95 %CI] 1.3–11.1) and a median OS of 10.7
months (95 %CI 0.0–21.9). However, a randomized trial of tremelimumab vs. placebo did not highlight any benefit from anti-CTLA-4 inhibition. Anti-PD1 and anti-PD-L1 antibodies have also been studied. In
a phase 1b trial of the anti-PD-1 antibody pembrolizumab after failure
of first-line treatment, patients with PD-L1 > 1% showed a 20 % objective response and 52 % stable disease with a median response
duration of 12 months (95 % CI 3.7-not reached) and good treatment
tolerability [4]. Similar results in terms of response have been obtained
with the anti-PD-1 nivolumab [5]. These findings suggest that treatment with immune checkpoint inhibitors can become a standard approach in MPM patients soon.
We present an unusual case of a female patient with MPM who
experienced an exceptional response to pembrolizumab. Of note, she
did not benefit from oncogene-directed targeted therapy, even though

2. Case presentation
The patient is a 45-year-old woman and former light smoker (2
packs/year) who was in good health until November 2017 when she
developed exertional dyspnea. Imaging studies showed evidence of
pleural effusion. Thoracentesis and videothoracoscopy furnished a diagnosis of malignant epithelioid pleural mesothelioma. Systemic chemotherapy was begun with the standard regimen of cisplatin and pemetrexed. Imaging evaluation showed stable disease after 3 cycles of
chemotherapy. In March 2018 the patient underwent pleural decortication, with confirmation of the previous histological diagnosis of
malignant epithelioid pleural mesothelioma on the basis of morphology
and immunohistochemistry (CK5, calretinin, positive WT1, negative
CK7). Radiation therapy was considered but decided against because of
the presence of parenchymal lung nodules, suggesting that the disease
was in progression. This was confirmed by 18F-fluorodeoxyglucose
positron-emission tomography and computed tomography (18FDG-PET/
CT), which also revealed extension of the disease to the bone (skull,
spine, rib, hip). For this reason a new line of systemic chemotherapy
was started with carboplatin and gemcitabine. After 2 treatment cycles
the patient experienced neurologic symptoms (i.e. seizures) and underwent brain magnetic resonance imaging (MRI) that revealed a single
left temporoparietal brain metastasis. In August 2018 the secondary
lesion was surgically removed and histological examination confirmed
a diagnosis of metastasis from malignant epithelioid pleural mesothelioma. Samples from the primary tumor and metastasis were analyzed
for molecular gene alterations and protein expression. ALK expression
was evaluated by immunohistochemistry (IHC) using anti-ALK D5F3
antibody clone (Roche Ventana, Medical Systems, Tucson, AZ) on a
BenchMark XT platform, while ALK translocation was assessed by
fluorescence in situ hybridization (FISH) using ALK Break Apart FISH
Probe (Abbott Molecular, Inc. Abbot Park, IL, USA). PD-L1 expression
was determined by IHC using antibody clone SP263 (Roche Ventana)
and gene profiling by next generation sequencing (NGS) using the
Myriapod NGS-IL 56 G Onco panel (Diatech Pharmacogenetics, Jesi,
Italy). The pleural sample was ALK-positive by IHC, and echinoderm
microtubule-associated protein-like 4 – anaplastic lymphoma kinase
(EML4-ALK) translocation was confirmed by fluorescence in situ hybridization (FISH) in 18 % of tumor cells. Conversely, the sample from
the brain lesion was negative for ALK expression (Fig. 1). IHC analysis
of PD-L1 expression in both primary tumor and metastasis showed
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G. Bronte, et al.

Fig. 2. Assessment of brain metastasis (red circle) via MRI and peritoneal carcinosis (red arrows) via abdominal CT scan before and after 3 months of pembrolizumab.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 3. Histologic examination of the liver (L)
metastasis from malignant epithelioid mesothelioma shows 2 different clones (M1 and
M2) with negative BAP1 staining in tumor cells
(M1a and M2a; of note the normal positive
nuclear staining in dispersed inflammatory
cells) but discrepant expression of PD-L1
(strongly positive in M1b and only occasionally
expressed in M2b) and ALK (negative in M1c
and strongly positive in M2c).

begun with parenteral nutrition via nasogastric tube. In October 2018
pembrolizumab 2 mg/kg every 21 days was started. After 15 days the
patient’s conditions had improved enough to re-start enteral nutrition.
Re-evaluation by 18FDG-PET/CT, thoracic and abdominal CT with iodinated contrast media and brain MRI showed a complete response of
the brain metastasis, peritoneal carcinosis, bone lesions and mediastinal
nodal metastases (Fig. 2). A partial response was documented in the
pleural and pulmonary nodules and stable disease in the liver

evidence of PD-L1 immunopositivity in 10 % of tumor cells in the
pleural biopsy and 100 % in the brain lesion (Fig. 1). NGS analysis did
not reveal any mutations of those included in the panel. These findings
prompted us to start treatment with alectinib 600 mg twice daily but
after one month a whole-body CT scan documented peritoneal carcinosis and 2 subglissonian liver metastases, leading to the development
of complete bowel obstruction. Brain MRI showed a recurrence in the
site of the brain surgery. Alectinib was stopped and supportive care
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response we observed supports the hypothesis that immune checkpoint
inhibitors could provide a valid therapeutic alternative, even after the
failure of previous treatments. The variable positivity of PD-L1 and ALK
in the primary tumor and metastasis indicates wide intra-patient tumor
heterogeneity which can substantially influence the biological and
clinical behavior of the disease.

metastases. The latter had been biopsied, revealing the presence of a
double clonal cell population showing ALK translocation in 18 % of
tumor cells and PD-L1 expression in 70 % of tumor cells (Fig. 3). The
patient is still undergoing immunotherapy and has no cancer-related
symptoms. The residual liver disease appears to be the only remaining
neoplastic component. At the beginning of April 2019 we treated the
liver lesions with radiofrequency thermoablation.

Authors’ contributions
3. Discussion
GB and LC drafted the manuscript, prepared the figures and performed the final revision of the manuscript. AD, MAB, AV and PV extracted the clinical data from clinical records and revised the manuscript. MP, SB, MMT and GR were responsible for the histological
examination and IHC analysis, prepared the figures and revised the
manuscript. PU performed the genotyping analysis and revised the
manuscript. DD prepared the radiological figure and revised the
manuscript. GM revised the manuscript for important intellectual
content. All authors read and approved the final versions of the
manuscript for submission.

The limited therapeutic options for MPM often lead clinicians to
look for actionable molecular alterations, as normally done for other
malignancies. NGS analysis offers the opportunity of detecting numerous potential molecular targets and predictive factors. Some studies
have focused on identifying genetic alterations in MPM, but the majority of these are not actionable therapeutic targets. Alterations in
crizotinib-targeted genes, such as mesenchymal-to-epithelial transition
(MET), anaplastic lymphoma kinase (ALK) and c-ros proto-oncogene
(ROS1) have rarely been found in patients with pleural mesothelioma
[6]. However, Hung et al. detected ALK translocations in 13 % of patients with peritoneal mesothelioma [7].
The frequency of brain metastases in MPM is around 5%, with
prognosis clearly poorer in these patients than in those with no brain
involvement (median OS 6.5 vs.11.0 months, respectively, p = 0.037)
[8]. Our patient showed 2 unusual characteristics, i.e. EML4-ALK positivity in the pleura and the presence of brain metastasis. Moreover,
the brain metastasis expressed PD-L1 in 100 % of tumor cells, unlike the
pleural tumor tissue where PD-L1 was positive but significantly lower.
In addition, brain tumor tissue was negative for EML4-ALK translocation. These findings suggest that the disease developed 2 different
genotypic and/or phenotypic forms, even though the histology was the
same.
The presence of 2 different clones was confirmed by IHC performed
on the tissue sample from a liver metastasis (Fig. 3). However, immunotherapy with pembrolizumab succeeded in obtaining a wide
clinical response with complete remission of the peritoneal carcinosis.
Such an outcome endorses the findings of the previously-mentioned
clinical trials on immune checkpoint inhibitors. The use of single-agent
salvage immunotherapy (i.e. pembrolizumab, nivolumab or avelumab)
achieved response rates ranging from 10 % to 30 % [9]. Interestingly,
patients who were treated with pembrolizumab after initial disease
progression obtained a partial response [10]. Complete regression of
the bulky abdominal disease in our patient is an impetus for the investigation of the biological features of mesothelioma that might help
to explain this impressive result. Although high PD-L1 expression may
not have been responsible for the outcome, it helped to identify the coexistence of two clonal populations of mesothelioma cells.
The composition of the immune cells in the tumor microenvironment and the expression of immune checkpoints and their ligands in the
tumor cells has a strong intra-patient and inter-patient heterogeneity.
Some studies are exploring other immune checkpoints (e.g. TIM-3, LAG3) which can help to explain the variability in response to immunotherapy [11].
The concept of the immunophenotypic heterogeneity of mesothelioma warrants further exploration in an attempt to find new therapeutic strategies to improve the outlook of patients with this disease.

Funding
None.
Declaration of Competing Interest
None declared.
Acknowledgements
The authors thank Gráinne Tierney for editorial assistance.
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4. Conclusions
This case report highlights the need for further investigation into the
use of immunotherapy in patients with MPM. The impressive clinical

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