Collision Tumor of Primary Laryngeal Mucosal Melanoma
and Invasive Squamous Cell Carcinoma with IL-17A and CD70
Sasis Sirikanjanapong•Biana Lanson•
Milan Amin•Frank Martiniuk•Hideko Kamino•
Beverly Y. Wang
Received: 21 April 2010/Accepted: 20 July 2010/Published online: 10 August 2010
? Humana 2010
larynx is the squamous cell carcinoma (SCC). The primary
malignant melanoma is quite rare in this location. Less than
60 cases of laryngeal melanomas have been reported to
date. To our knowledge, collision primary malignant mel-
anoma and invasive squamous cell carcinoma in the vocal
cords has not been reported. We report a 53-year-old male
patient who was diagnosed with a collision tumor of lar-
yngeal melanoma and invasive SCC. Multiple Th17 path-
way related genes including CTLA-4, IL-17A-F, PLZF,
FoxP3, RorcT, CD27, and CD70 were analyzed by reverse
transcriptase-polymerase chain reaction (Rt–PCR) in this
case. Both IL-17A and CD70 genes were detected in this
case of collision tumor. The results may define useful
biomarkers for early diagnosis of mucosal melanoma and
open an immunotherapeutic field for clinical management
with the potential benefit from the immunomodulators that
enhance both genes.
The most common primary malignancy of the
melanoma ? Squamous cell carcinoma ? IL-17A and CD70
Collision tumor ? Laryngeal mucosal
Approximately 40,000 new patients are diagnosed with
head and neck cancers each year in the United States, and
squamous cell carcinoma represents more than 90% of all
cases . Squamous cell carcinomas account for 95% of
primary laryngeal tumors. Most patients are in the fifth,
sixth, or seventh decades of life at the time of diagnosis,
with an average age of about 60 years. Smoking and
alcohol abuse are by far the principal risk factors .
Mucosal melanomas represent 1.3% of all cutaneous and
non-cutaneous melanomas . Among mucosal melano-
mas, 55% are located in the head and neck region ,
involving the nose and paranasal sinuses, followed by the
oral cavity . Rarely, primary melanomas arise in the
larynx. In fact, less than 60 cases of laryngeal melanomas
have been reported to date [4, 5].
The pathogenesis of mucosal melanoma is unknown.
Several speculated risk factors include exposure to sunlight,
human papilloma virus, chronic irritants, and carcinogenic
compounds . Recently, several studies reported that
malignant melanomas were related to an altered immune
system. Several TH17 related genes have been studied in
cutaneous melanoma, including CTLA-4, IL17, RORct, and
FoxP3, with very limited studies done in mucosal melano-
mas. These genes are of interest for immunomodulator
The term ‘‘collision tumor’’ indicates two coexistent but
independent tumors with distinct morphology. Collision
tumors affecting larynx are extremely rare. To the best of
Funded by grant number: NIH/NCRR 1UL1RR029893-01.
S. Sirikanjanapong ? B. Y. Wang (&)
Department of Pathology, New York University School
of Medicine, 550 First Avenue, TCH-461, New York,
NY 10016, USA
B. Lanson ? M. Amin ? B. Y. Wang
Department of Otolaryngology, New York University School
of Medicine, New York, USA
Department of Medicine, New York University School
of Medicine, New York, USA
Department of Dermatopathology, New York University School
of Medicine, New York, USA
Head and Neck Pathol (2010) 4:295–299
our knowledge, a laryngeal SCC coexisting with a primary
laryngeal mucosal melanoma has not previously been
We report a 53-year-old male patient who was diag-
nosed with collision tumor of laryngeal melanoma and
invasive SCC, with over-expression of IL-17A and CD70
genes, by reverse transcriptase-polymerase chain reaction
A 53-year-old man presented to the ear, nose and throat
(ENT) clinic complaining of a 6-month history of hoarse-
ness. He also had pain in his left throat and odynophagia.
He denied dysphagia or dyspnea. He reported a 25-pound
weight loss over the past 3 months. He had smoked half a
pack of cigarettes per day for 15 years. He is also a hep-
atitis C carrier. Physical examination did not demonstrate
any cutaneous pigmented lesion suspicious for malignant
melanoma. Computed tomography (CT) of head and neck
revealed a nodular enhancement along the anterior 2/3 of
the left true vocal cord and an enlarged left subclavicular
lymph node measuring 1.6 cm in the greatest dimension
(Fig. 1a). Laryngoscopic examination revealed a tan–blue
thin plaque on the left side of the larynx (Fig. 1b). The
lesion extended from the left false vocal cord to the true
vocal cord along the anterior 2/3 of the cord. The
remaining areas of larynx were clinically unremarkable.
The lesion was biopsied and the diagnosis was laryngeal
mucosal melanoma. Four weeks later the patient underwent
a total laryngectomy (Fig. 1c) and left selective neck
Postoperatively the patient recovered and was dis-
charged without any complications. Subsequently he
received high dose radiation therapy. He denied any
complication from the treatment. He has been followed up
at the clinic on a regular basis for 15 months. There is no
evidence of recurrence or distant metastases to date.
Microscopic examination of biopsied samples demon-
strated laryngeal squamous mucosa with proliferation of
pleomorphic heavily pigmented, rounded, and dendritic
melanocytes in the epithelial basal layer and in the upper
levels of the squamous epithelium (Fig. 2a). There were
melanocytes in the laminar propria admixed with heavily
enlarged nuclei, and some of them had basophilic nucleoli.
Confirmatory immunohistochemical studies by standard
ABC-Biotin immunoperoxidase (Dako, CA) were per-
formed. The tumor cells were positive for S-100 protein,
HMB-45, Melan-A, and PNL-2. The immunohistochemi-
cally positive melanocytes were distributed in the epithe-
lium and the lamina propria. Atypical squamous cells were
noted as well. A PAS stain demonstrated pseudophyphae of
Candida in the cornified layer. The biopsy was diagnosed a
laryngeal mucosal melanoma. Gross examination of the
total laryngectomy specimen revealed a 2.5-cm ulcerated,
black, nodular area located on the left laryngeal mucosa,
extending from the false vocal cord to the true vocal cord
(Fig. 1c). Microscopic examination revealed sheets of
heavily pigmented, round-to-plump melanocytes infiltrat-
ing in the submucosa, presented as single cells and in small
nests along and above the basal layer of the epithelium
(Fig. 2b–d). The melanocytes were highlighted by immu-
nopositivity for HMB-45 (Fig. 2e) and Melan-A. The depth
of melanoma was 2.8 mm. In addition, there was markedly
dysplastic squamous cells in the epithelium (Fig. 2b–c) and
submucosa (Fig. 2d), consistent with in situ and invasive
squamous cell carcinoma admixed with atypical pigmented
melanocytes (Fig. 2b–c). The final diagnosis was muco0
sal malignant melanoma and invasive SCC of the larynx.
The resection margins were uninvolved by tumor. No
Fig. 1 a Coronal section from non-contrast CT scan shows a 1.7-cm
mass present at the left vocal cords, filling the ventricular space
(arrow). b Endoscopic view shows a bluish and light brown mass on
the left larynx (arrow). c Total laryngectomy specimen shows a dark
pigmented mass involving the left vocal cords (arrow)
296 Head and Neck Pathol (2010) 4:295–299
angiolymphatic invasion was noted. No metastatic disease
was identified in six lymph nodes.
Formalin fixed paraffin tumor tissue was analyzed for the
genes CTLA-4, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E,
IL-17F, PLZF, FoxP3, RorcT, CD27, and CD70. RNA was
extracted with TRIzol after deparaffinization with xylene
and first strand cDNA synthesis was performed. PCR for
HGAPDH was used as an internal control to normalize
samples. Nested PCR for these genes was performed.
Agarose gel electrophoresis of the amplicons showed that
IL-17A and CD70 were detected (Fig. 3).
It is well known that more than 95% of primary laryngeal
malignancy is SCC. Most patients are in the fifth, sixth, or
seventh decades of life at the time of diagnosis, with an
average age of about 60 years . Smoking and alcoholic
factors are contributed to the development of laryngeal
Fig. 2 Low magnification from
the left vocal cord mass shows a
laryngeal mucosal tumor
composed of dark pigmented
cells (a). A closer view shows
the over-lined squamous
mucosa with high grade
dysplasia and pigmented
atypical melanocytes (b–c).
Invasive squamous cell
carcinoma infiltrating into
submucosa with mixed atypical
melanocytes (d). Immunostain
for HMB-45 highlights
melanocytes in squamous
epithelium and submucosal
Fig. 3 PCR amplicons. Appropriate sized bands were detected for
IL-17A and CD70. No bands were found for IL-17B, IL-17D, IL-17F,
PLZF, CD27, CD70, FoxP3, RorcT and negative control (no template
DNA). A 100 bp ladder is shown in the left lane. HGAPDH is used as
an internal control for reverse transcription
Head and Neck Pathol (2010) 4:295–299297
SCC. Other risk factors that have been proved, suggested,
or debated include prior radiation exposure, HPV infection,
genetic factors, air pollution, dietary deficiencies, chronic
gastrointestinal reflux, and occupational exposure to wood
or metal dusts and asbestos, etc. .
Mucosal malignant melanoma is infrequent. The head
and neck region accounts for more than half of all mucosal
melanoma cases, the rest are distributed in the anus and
rectum, the female genitalia, and the urinary tract [4, 7].
Mucosal melanoma of the head and neck area most com-
monly occur in the nasal cavity, followed by the oral cavity
. Primary laryngeal melanomas are rare, with less than
60 reported cases to date. Most primary laryngeal malig-
nant melanomas occur in elderly male patients, in their
sixth or seventh decades of life [5, 9, 10]. The majority of
the patients are white with only two reports of Asian
individuals [10, 11]. In contrast to laryngeal melanoma,
gender predilection is not observed in cutaneous melanoma
patients. And most cutaneous melanoma patients are
younger, in their fourth and fifth decades of life .
Patients can present with various symptoms including
sore throat and dysphagia, but the most common presenting
symptom is hoarseness. Tumors typically involve the
supraglottic larynx for primary laryngeal melanoma. The
second most common site is the glottis [5, 9, 10]. Risk
factors of laryngeal melanoma are unclear, but most studies
noted a strong association between a history of cigarette
smoking and laryngeal melanomas [5, 9]. Our patient was a
long-term smoker who also developed a concurrent lar-
yngeal invasive squamous cell carcinoma for which
smoking is a well-documented risk factor.
A collision tumor represents the coexistence of two
morphologically distinct tumors. A primary squamous cell
carcinoma of the larynx has been reported to occur simul-
taneously with other laryngeal neoplasms. Tomidokoro
et al. reported an isolated case of collision tumor of pri-
mary laryngeal SCC and leiomyosarcoma . The term
‘‘double tumor’’ has been suggested by Medina-Banegas
et al. for their case report of synchronous chondrosarcoma
and epidermoid carcinoma in situ of the larynx . In our
current case, it is important to distinguish between primary
laryngeal melanoma and metastatic malignant melanoma in
the larynx. Some authors suggest that the diagnosis of
primary laryngeal melanoma can not be made if lacking the
in situ melanoma component [9, 14, 15]. Although, it has
also been hypothesized that melanoma in the larynx may
arise from the submucosal melanocytes and melanocytes
from the mucoserous gland . We also believe that
primary laryngeal melanoma has to show a melanoma in
situ component. Additionally, the diagnosis should be
supported by the lack of melanoma elsewhere in the body.
Therefore, the diagnosis of primary laryngeal melanoma
may not be made solely by exclusion of other primary sites.
Morphologically, melanoma is a notorious mimicker,
which can appear to be epithelioid, spindled, and plasma-
cytoid. It can arrange in an organoid pattern or a solid
growth pattern. Immunohistochemical studies including
S-100, human melanoma black-45 (HMB-45), Melan-A,
microphthalmia transcription factor (MITF) and PNL-2 can
aid the confirmation of mucosal melanoma .
Recently, several studies reported that malignant mela-
nomas were related to an altered immune status. Several
genes of interest including IL-17, CTLA-4, CD70, and
CD27 have been extensively studied for immunotherapy
development [18–21]. IL-17E and IL-17A were found to be
higher in cell supernatants and blood serum of patients who
have oral epithelial squamous cell carcinoma when com-
pared to healthy people . In our study we were able to
identify two over-expressed genes including IL-17A and
CD70. It is impossible to determine whether these two
genes are associated with only SCC or only mucosal
melanoma, or both malignancies in this collision tumor.
Th17, a subset of CD4 that produces IL-17, has been
implicated in various autoimmune disorders including
rheumatoid arthritis and psoriasis. It has been shown to
have anti-tumor immunity in a number of studies . At
least six members of IL-17 have been identified. It has been
shown that IL-17A and IL-17C are able to induce mRNA
expression inflammatory cytokines including IL-1b, IL-16
and IL-23 . IL-23 recently has been demonstrated to be
a promising cancer vaccine for melanoma [23, 24]. Spec-
ulatively, this patient might carry a favorable prognosis
when compared to patients without an over-expressed
IL-17A. However, the role of Th17 on tumor has not yet
been clearly identified. Th17 has been shown to have dif-
ferent effects on tumor growth. Some studies demonstrated
that Th17 can promote the growth of tumors, while other
studies showed that it had an antitumor property. It has also
been shown that IL-17A can impair immune surveillance
by CD8 and promote neovascularization . Perhaps the
most important factor of antitumor immunity is the balance
of regulatory T cells (T regs) and Th17 in the tumor
microenvironment as illustrated in the work of Kryczek
et al. . It has been hypothesized that Th17 has a pro-
tective property in the early stage of the disease but
becomes permissive as the disease progresses . Whe-
ther Th17 has protective function for melanoma metastasis
is still controversial.
CD70, a tumor necrosis factor-related cell surface
ligand, has been extensively studied in the field of immu-
notherapy and it has been demonstrated to have anti-tumor
immunity. Transfection of tumor cells with CD70 enhances
antitumor immune response in murine tumor models .
Furthermore, Cormary et al. have been able to demonstrate
a stronger anti-tumor immune response elicited by CD40L
and CD70 expressing B16F10 tumor cells, based on the
298Head and Neck Pathol (2010) 4:295–299
fact that both CD40L and CD70 are costimulators needed
for full activation of T cells . They showed that the
tumor growth slowed down significantly when CD40L and
CD70 were coexpressed by the tumor [28, 29]. Therefore,
we speculate that our patient should elicit a strong immune
response against the tumor resulting in a favorable prog-
nosis based upon his overexpression of CD70 and IL-17A.
Like laryngeal SCC, the mainstay treatment for primary
mucosal melanoma in the head and neck region is radical
surgery with or without adjuvant therapy including radia-
tion and chemotherapy. Although radical surgery yields the
best local control, local recurrence occurs frequently . A
42% recurrence rate has been reported by Snow et al. .
Most recurrent cases occur in the nasal cavity . Shah
et al. reported a 64% recurrence rate. However, primary
laryngeal melanoma has a relatively low recurrence rate.
Yet the survival rate of laryngeal melanoma is still low.
The 5-year survival rate is less than 20% due to metastatic
disease [9, 16].
In summary, we report a 53-year-old male patient with a
collision primary laryngeal malignant melanoma and
invasive squamous cell carcinoma of the larynx with IL-
17A and CD70 over-expression. This gene expression may
open the door to the future where individual melanoma
patients will be treated with tailored immunotherapies. In
addition, this gene expression may serve as useful bio-
markers used for early diagnosis and predicting the prog-
nosis. However, more mucosal melanoma case studies with
long-term follow-up are necessary for better understanding
of the function of these genes in order to apply them in the
treatment for mucosal melanomas.
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