Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration in the Diagnosis of Hilar and Mediastinal Lymph Node Metastases of Melanoma
José-Fernando VAL-BERNAL1 , María MARTINO2, Elena YLLERA3, Félix ROMAY4, María SÁNCHEZ-ARES5, Ihab Abdulkader NALLIB5
1Pathology Unit, Medical and Surgical Sciences Department, University of Cantabria and IDIVAL Research Institute, SANTANDER, SPAIN
2Anatomical Pathology Service, Marqués de Valdecilla University Hospital, University of Cantabria and IDIVAL Research Institute, SANTANDER, SPAIN
3Radiodiagnostic Service, Marqués de Valdecilla University Hospital, SANTANDER, SPAIN
4Neumology Service, Marqués de Valdecilla University Hospital, SANTANDER, SPAIN
5Anatomical Pathology Service, University Hospital, Santiago de Compostela, SPAIN
Keywords: Melanoma, Metastasis, Bronchoscopy, Cytology, Immunohistochemistry
Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive technique for investigating
hilar and mediastinal lymphadenopathy. This study reports eleven cases in which EBUS-TBNA was used to assess mediastinal and hilar lymph
nodes for the presence of metastatic melanoma.
Material and Method: A retrospective study was performed of all patients who had a history of melanoma and underwent EBUS-TBNA to assess
hilar or mediastinal lymphadenopathy for the presence of metastatic melanoma. In seven cases, molecular analysis to detect mutations in the
BRAF gene was also used.
Results: Eight patients had been diagnosed with malignant melanoma in the past (mean 54.4 months, range 18 to 115 months) while in the other
three this tumor was primarily diagnosed in the staging phase. The male-female ratio was 6:5, and the mean age was 60.3 years (range 42 to 88
years). The mean hilar or mediastinal lymph node size detected with computed tomography was 3.0 cm (range 1.1 to 8.1 cm). Eight (72.7%)
cases had metastases to the lung associated with metastases in the mediastinal lymph nodes. In four (50%) of these cases, the lung metastasis was
solitary. Three (27.3%) cases had metastases in the mediastinal lymph nodes in absence of lung metastases. Metastatic melanoma was diagnosed
by cytology and confirmed by cell block study with immunohistochemistry in all cases. BRAF mutations were detected in two (28.6%) of seven
Conclusion: Cytology and tissue samples obtained from EBUS-TBNA are adequate to detect metastatic melanoma and permit in some cases the
determination of biomarkers and identify the presence or absence of mutations in the BRAF gene. The procedure is safe, fast, and precise for the
staging of melanoma.
Primary malignant melanoma (MM) is a tumor that most
often originates in the skin but can appear in multiple sites
where melanocytes are present. This tumor accounts for
5.2% of all cancers and is now regarded as the fifth most
common cancer in men and the sixth most common
cancer in women in the western world 1
. In addition, the
incidence of this tumor is increasing in white populations
worldwide. The median age at diagnosis is 57 years 2
MM has a high malignant potentiality. Metastatic spread may
arise from small tumors 3. MM is known to disseminate
virtually to all the organs. However, initial spread occurs to
regional lymph nodes with subsequent extension to deep nodes including mediastinal nodes. Therefore, intrathoracic
lymph node metastases are frequent. Thus, Das Gupta and
Brasfield 4 found metastatic MM in mediastinal nodes in
69 (55%) out of 125 patients studied at autopsy.
The most important factor for successful management of
MM is early diagnosis. Patients with metastatic melanoma
have limited treatment opportunities. In cases of localized
metastasis, surgical resection can enable in some patients a
prolonged interval of recurrence-free survival.
Precise pathological diagnosis of mediastinal lymphadenopathy
in cases of MM is crucial for effective treatment.
Open thoracic surgery and mediastinoscopy are standard
methods for hilar and mediastinal lymph node staging.
However, they are costly, invasive, require general
anesthesia, and can lead to complications. Endobronchial
ultrasound-guided transbronchial needle aspiration
(EBUS-TBNA) cytology is a minimally invasive, safe and
suitable procedure that can be used for diagnosing hilar and
mediastinal lymphadenopathy. However, there is limited
experience with this procedure. Thus, there is a case report
5 and a series of nine cases (seven metastatic) 6 in the
literature using this technique to detect metastatic MM to
the mediastinal lymph nodes.
In this study, we investigated the feasibility of EBUS-TBNA
for evaluating hilar and mediastinal lymphadenopathy in
MM. Hilar and/or mediastinal lymphadenopathy presented
as a recurrence or a primary diagnosis in a series of eleven
patients diagnosed with MM.
The methods used are similar to those performed in
a previous EBUS-TBNA study in clear cell renal cell
. Between January 2011 and March 2018,
we performed 1,500 EBUS-TBNA studies of hilar or
mediastinal lymph nodes for a variety of clinical indications
including malignancy. All the cases were identified inhouse.
The lymph nodes sampled were enlarged (short axis
> 1cm) according to computed tomography (CT) scans,
and they were associated in some cases with nodular lesions
in the lung.
Tumor staging was established according to the 7th edition
of the AJCC Cancer Staging Manual 8.
EBUS-TBNA was performed under local anesthesia
and midazolam and fentanyl sedation as an outpatient
procedure, using a flexible bronchoscope Olympus BFUC160F-
OL8 (Olympus, Tokyo, Japan) and an ultrasound image processor Olympus EUS Exera EU-c60 (Olympus,
Tokio, Japan). Specimens were obtained with a 22-gauge
needle. The average number of needle passes from each
location was 3 (range 1-6).
An on-site evaluation was performed in all the cases and
the specimen was assumed adequate. Each case had aspirate
smears that were stained with Diff-Quick and Papanicolaou
method. In all the cases we had cell block preparations.
Sections of the cytoblocks were stained with hematoxylin and
eosin. Considering the tissue limitation, we did not use the
Fontana-Masson silver method for melanin. This pigment
was evaluated in the sections stained with hematoxylin
and eosin. Immunohistochemical study was carried out
on formalin-fixed 4-μm-thick paraffin-embedded tissue
sections using the EnVision FLEX Visualization System
(Dako, Agilent Technologies, SL, Las Rozas, Madrid,
Spain). Antibodies used in the immunohistochemical
study are detailed in Table I. The immunohistochemical
reactions were performed using appropriate tissue controls.
Automatic staining was performed on a Dako Omnis
autostainer (Agilent Technologies, SL).
PDL1 immunohistochemistry and BRAF mutation analysis
were performed in four and seven cases respectively.
In the remaining cases, there was not enough material
in the cytoblock. PDL1 protein expression was carried
out on paraffin-embedded tissue cut into 3 μm sections
using two different antibody clones, Dako 28-8 and Dako
22C3. Formalin-fixed, paraffin-embedded agar cell pellets
prepared from the PD-L1-positive and PD-L1-negative cell
line and tonsil tissues were used as controls. Assessment of
PD-L1 staining was performed by a pathologist previously
trained on the 28-8 and 22C3 Dako pharmDx assays. The
percentage of tumor cells with linear membranous staining at any intensity was reported following 28-8 and 22C3 Dako
pharmDx assays interpretation guides.
In seven cases, DNA extraction was carried out to study
BRAF mutation analysis. After hematoxylin-eosin-stained
slide review and tumor tissue selection, genomic DNA was
extracted from 5 to 10 (5-μm-thick) sequential tissue sections
for each specimen using the QIAGEN Deparaffinization
Solution and the QIAamp DNA FFPE Tissue Kit (Qiagen,
Germany) according to manufacturer’s guidelines. The
concentrations were evaluated by spectrophotometry
(NanoDrop 1000 Spectrophotometers, Thermo Scientific
Inc., MA, USA). Pyrosequencing of BRAF mutation
regions (codon 464-469 and codon 600) was performed
with the Therascreen BRAF Pyro Kit (Qiagen, Germany)
on a PyroMark Q24 System (Qiagen, Germany) according
to the manufacturer’s handbook.
Because of limited material and variation of the staining
panel over the years, not all tumors were stained with the
same series of antibodies.
This study was approved by the Ethics Committee of
IDIVAL Research Institute (CI: 2018.053) and confirmed to
the provisions of the Declaration of Helsinki.
Over the seven-year and four-month study period, we
analyzed the data from eleven patients who underwent
EBUS-TBNA with cytoblock. The patients underwent
this procedure because of suspected hilar or mediastinal
metastasis according to CT. Eight patients had been
diagnosed with MM in the past (mean 54.4 months, range
18 to 115 months) while in three this tumor was primarily
diagnosed in the staging phase by means of EBUS-TBNA
). There were 6 male and 5 female patients (M:F,
6:5) and the mean age was 60.3 years (range 42 to 88 years).
Click Here to Zoom
|Table II: Clinical details of patients with mediastinal metastases caused by melanoma
CT scans with contrast enhancement in the eight patients
showed well-defined, large, heterogeneously-enhancing
solid masses in the hilar zone or anterior mediastinum
(Figure 1A-C). The mean hilar or mediastinal lymph node
size detected with CT was 3.0 cm (range 1.1 to 8.1 cm).
Eight (72.7%) cases had metastases to the lung associated
with metastases in the mediastinal lymph nodes; and in
four (50%) of these cases, the lung metastasis was solitary.
Three (27.3%) cases had metastases in the mediastinal
lymph nodes in absence of lung metastases (Table II).
Endobronchial ultrasound study of the lymph nodes
included increased size, irregularity, non-homogeneity,
hypervascularization and increased eco-quality (Figure
Click Here to Zoom
|Figure 1: Axial thoracic CT scans and endobronchial ultrasound. A) CT scan (Case 1). The red arrow shows a rounded pathological
lymph node with low attenuation values located in the subcarinal region (level 7). B) CT scan (case 5). The arrow indicates a rounded,
homogeneous hypodense nodular image at level 10R, corresponding to a pathological lymph node by size and location. C) CT scan (case
8) The arrows indicate a large, hypodense heterogeneous lymphadenopathic conglomerate. It has been located in the left paratracheal
region (4L) that extends to the aortopulmonary window and the ipsilateral hilum. It produces the stenosis of the left main pulmonary
and contacts more than 50% of the aortic circumference. D) Endobronchial ultrasound (case 8). A needle is observed within a large,
heterogeneous lymph node conglomerate in the left paratracheal region.
Cytological smears revealed a lymphoid and hematic
background on which there were atypical cells arranged in
small clusters (37.5%) or scattered as discohesive groups or
isolated elements (62.5%). Cellularity was moderate (27.3%)
to high (72.7%). The cytological type varied between
epithelioid (54.5%), spindle cell (27.3%) and epithelioid
and spindle cell (18.2%). Epithelioid or round cells formed
groups of large, disaggregated, atypical cells or isolated
elements occasionally binucleated, or multinucleated. The
nuclei were frequently in an eccentric position (Figure 2).
These nuclei usually had a regular outline, and the nuclear
chromatin was evenly distributed. Nucleoli were prominent.
Nuclear pseudoinclusions (intranuclear cytoplasmic
invaginations) were seen in isolated cells in most cases
(Figure 3A). Presence of macronuclei was noticed. Multiple
small cytoplasmic vacuoles were observed in smears stained
with Diff-Quick (Figure 3B). Singly atypical dispersed cells
showed melanin pigment in five (45.4%) of cases (Figure
3C). Smears containing a mixture of epithelial-type and
spindle cells or only spindle cells usually formed cohesive
Click Here to Zoom
|Figure 2: Cytological smear showing a hemorrhagic background
on which there are epithelioid malignant cells with lack of
cohesiveness. Eccentric nuclei are often seen. There is a binucleate
tumor cell. Cytoplasmic melanin pigment is not apparent (case 5)
(Diff-Quick stain; x400).
Click Here to Zoom
|Figure 3: Cytological smear. A) A binucleate cell in the center
of the image shows a pseudonuclear inclusion (intranuclear
cytoplasmic invagination) (case 1) (Papanicolaou stain; x400).
B) Two cells showing multiple small cytoplasmic vacuoles
measuring <1 μm in diameter can be seen (case 3) (Diff-Quick
stain; x400). C) Tumor cells with dusty melanin pigment stained
in blue with Diff-Quick stain (case 2) (Diff-Quick stain; x400).
All cases were categorized as positive for metastatic
The cell blocks showed groups or masses of round to
oval tumor cells with moderate amount of cytoplasm and
moderately pleomorphic nuclei with coarse chromatin
(Figure 4A). Nucleoli were prominent. Three main cytologic
types were recognized: epithelioid (54.5%) (Figure 4B),
spindle cell (27.3%) (Figure 4C) and mixed epithelioid and
spindle cells (18.2%). Multinucleated cells were prominent
in one case (Figure 4D). Melanin pigment was present in
some cells in seven (63.6%) cases. Immunohistochemistry
revealed positive reactivity for HMB45 (8/11, 72.7%)
(Figure 5A), Melan A (9/11, 81.8%) (Figure 5B), SOX10
(10/11, 90.9%) (Figure 5C), and S100 protein (10/11,
90.9%) (Figure 5D) in tumor cells (Table II). Cytokeratin
(CK) AE1/AE3 (0/6, 0%), CK7 (0/2, 0%), CD 56 (0/2,
0%), synaptophysin (0/2, 0%), p40 (0/2, 0%) leukocyte
common antigen (CD45, 0/2, 0%) and TTF1 (0/8, 0%)
were no reactive. In the immunohistochemical study of
the melanoma markers (HMB45, Melan A, SOX10, S100
protein) it was observed that four markers were positive in
7 (63.6%) cases, three markers in 1 (9.1%) case, and two
markers in 3 (27.3%) cases.
Click Here to Zoom
|Figure 4: Routine hematoxylin-eosin stain of the cell blocks. A) Presence of tissue fragments of melanoma in a background of red cells
(case 5) (H&E; x200). B) Epithelioid cells (case 1) (H&E; x400). C) Spindle cells (case 6) (H&E; x400). D) Multinucleated giant cells (case
7) (H&E; x400).
Click Here to Zoom
|Figure 5: Immunohistochemistry. Neoplastic cells show reactivity for A) HMB45 (case 7) (IHC; x400). B) Melan A (case 5) (IHC; x400).
C) SOX10 (case 5) (IHC; x400). D) S100 protein (case 5) (IHC; x200).
The histological study of the cytoblocks plus immunohistochemistry
was considered confirmatory of the diagnosis. PDL1 biomarker was negative for all cases. The study of the
BRAF mutations showed positivity in two (28.6%) of seven
cases studied (Table II). Mutations were detected in exon
15, codon 600 of the BRAF gene.
In this study, there were no inadequate cytological samples.
No clinical complications were observed in patients due to
the use of the procedure.
MM is an aggressive neoplasm that can metastasize to
all organs of the human body and its manifestations are multifaceted. The metastatic pattern was analyzed by
autopsy of 216 patients by Patel et al 9
. The most common
organs involved were the lymph nodes (73.6%), lungs
(71.3%), liver (58.3%), brain (49.1%), bone (48.6%), heart
(47.2%), adrenal glands (46.8%), and gastrointestinal tract
(43.5%). Single organ metastases were very uncommon in
cutaneous MM. On the other hand, Webb in a radiologic
and pathologic study of 65 patients with intrathoracic
metastases from MM observed that 35 (54%) out of them
had hilar or mediastinal lymph node metastases. In 80% of
the cases, these lymph nodes were enlarged 10
In patients with tumors arising below the diaphragm, the
spread to intrathoracic nodes occurs via the thoracic duct,
most likely by reflux of tumor cells. This duct, as a rule,
drains lymph from the pelvis and abdomen. In cases with
tumors arising above the diaphragm, the lymphatic spread
to mediastinum nodes occurs by means of lymphatics
connecting axillary, cervical, and mediastinal chains. In
addition, in patients with metastatic pulmonary nodules
(indicative of hematogenous spread to the lungs), secondary
spread of tumor cells to hilar and mediastinal lymph nodes
may occur 10.
The thorax should be the main focus for the screening of
initial systemic MM dissemination 11. Mediastinoscopy
with a sensitivity of 80% to 85% and a specificity of about
100% is considered the standard method for diagnosis of
mediastinal lymphadenopathy with tissue confirmation.
However, this method has limited access to nodal stations
2 and 4 (paratracheal), and 7 (subcarinal), and the access to
hilar nodes can be difficult and may require thoracoscopy. In
addition, mediastinoscopy is associated with a considerable
rate of morbidity. Thus, this technique is associated with a
neck scar and a 2% risk of morbidity and 0.08% of mortality. Furthermore, the method cannot be repeatedly conducted
on the same patient 12-14.
It is usually accepted that a normal lymph node has a
maximum short-axis diameter of 10 mm or less 15.
Metastasized lymph nodes tend to be larger than normal
or benign lymph nodes. Computed tomography (CT) scan
is usually used in the staging of metastatic melanoma and
for control of high-risk patients after loco-regional surgical
treatment. Thus, CT scanning is the most reliable radiographic
method for evaluating intrathoracic metastases including
mediastinal and hilar lymphadenopathy 16,17. However,
CT scanning is insensitive to the presence of small nodal
metastases and can show false-positive results evaluating
large nodes 18. On the other hand, echofeatures alone are
not reliable in determining the underlying etiology of hilar
and mediastinal lymphadenopathy, including sarcoidosis, tuberculosis, lymphoma or metastatic recurrence 19. In
addition, it should be taken into account that there is a wellknown
association between non-Hodgkin lymphoma and
melanoma 20. Furthermore, up to 20% of patients with
melanoma may develop synchronously or metachronously
other malignancies such as carcinomas, lymphomas, or
EBUS-TBNA has emerged as a minimally invasive, and
highly precise technique for sampling intrathoracic lymph
nodes. EBUS-TBNA combines endoscopic visualization
with high-frequency ultrasound imaging which warrants
obtain cytological and histological samples. Nevertheless,
definitive and accurate cytologic diagnosis is challenging
owing to the varied morphologic appearances of melanoma
in cytologic preparations 21. However, the precise
cytological diagnosis of metastatic melanoma starts with clinical history. Cytodiagnosis clues include poorly cohesive
cells, blatant malignant features, epithelioid and spindled
shapes, eccentric placement of nuclei, prominent nucleoli,
nuclear pseudoinclusions, macronuclei, bi- or multinucleation,
and small intracytoplasmic vacuoles. These small
vacuoles measuring <1 μm in diameter are characteristic of
melanoma cells 22. The melanin production, although
diagnostic, is only observed in ≤50% of cases 22,23. In
this setting, melanin pigment can be difficult to distinguish
from hemosiderin or anthracotic pigment. Melanin appears
as fine dust-like intracytoplasmic pigment in atypical cells.
Melanophages can also be distinguished. Fontana-Masson
silver method can recognize the melanin pigment in
difficult cases. From the practical point of view, the presence
of tumor cells with dark pigment in a smear should include
melanoma in the differential diagnosis.
The study of the cytoblock and the help of immunohistochemistry
is especially useful in the evaluation of
intrathoracic lymph nodes for suspected metastases.
Immunohistochemical staining is crucial in differentiating
metastatic melanoma from other tumors and imitators.
Thus, a sensitivity of 94% to 95% and a specificity of 100%
of the EBUS-TBNA method has been reported 12,24. In
this study, we observed immunohistochemical positivity at
least for two melanoma markers. On the other hand, the
procedure can provide sufficient tissue for biomarkers and
It is important to assess BRAF mutation status before
treatment of metastatic melanoma. The frequency of BRAF
mutation in primary melanoma ranges from 36 to 45%,
and 42-55% in metastatic melanoma 25. The presence of
a BRAF mutation in early melanoma shows no association with disease-free interval or overall survival. However,
the presence of a BRAF mutation in metastatic melanoma
is associated with a poorer survival 25. Furthermore,
the determination of the BRAF gene mutation allows the
treatment with the appropriate BRAF kinase inhibitor
vemurafenib (PLX4032) 26,27.
The complication rate for EBUS-TBNA is low and varies
from 1.23% 25 to 1.44% 26. They include device breakage,
hemorrhage, pneumothorax, infections (mediastinitis,
pneumonia, pericarditis, sepsis), and death (large cerebral
infarction reported in one case) 28,29.
In conclusion, EBUS-TBNA is an alternative minimally
invasive technique for surgical mediastinal staging
of MM. It can also be used to repeat procedures for
additional required testing including biomarkers and
molecular studies. The study of the cytoblock and the
help of immunohistochemistry is especially useful in the
evaluation of intrathoracic lymph nodes for suspected
melanoma metastases. The procedure complications are
CONFLICT OF INTEREST
The authors declare no conflict of interest.
No external funding for this work.
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