Keywords
CTNNB1
- Wnt - pathogenetics - sinonasal teratocarcinosarcoma - teratocarcinosarcoma - β-catenin
Introduction
Teratocarcinosarcomas are aggressive tumors arising primarily in the sinonasal region
and anterior cranial base. They are extremely rare, with less than 100 cases ever
reported in the literature.[1] These tumors are heterogeneous, and comprised epithelial, mesenchymal, and neural
tissues.[2] Sinonasal teratocarcinosarcomas are highly difficult to treat, in part due to their
location in the anterior skull base (with difficulty to achieve adequate margins)
and in part due to their inherent aggressiveness. Current standard of care includes
aggressive surgical resection followed by radiation therapy.[3] There are no established surgical guidelines, but surgical options historically
reported include open anterior craniofacial resection, maxillectomy, and lateral rhinotomy.[1] Currently open, endoscopic, or a combination thereof have been used to approach
sinonasal teratocarcinosarcomas to achieve complete resection.
Unfortunately, disease recurrence is frequent (31.4%), with a high rate for metastasis
(17.6%) and intracranial extension.[1] Most frequently, the lungs are the site of distant metastasis. Overall survival
historically was dismal, and although improving (56% overall survival at 45 months
after surgery and radiation),[1] patients still have a guarded prognosis.
Little is known biologically or genetically about teratocarcinosarcomas. Although
previous immunohistochemical staining has confirmed the presence of epithelial, mesenchymal,
and neural markers,[2]
[4] there has been no insight into the pathogenesis of these tumors. The genetic pathways
governing teratocarcinosarcomas have not been studied, and no reported disruptive
genomic events driving these tumors have been described. Identification of the underlying
genetic drivers of sinonasal teratocarcinosarcomas could provide potentially successful
adjuvant or alternative precision medicine treatment options for these aggressive
tumors.[5]
[6]
[7]
[8] Here, we describe a case of a patient with a sinonasal teratocarcinosarcoma and
perform targeted next-generation sequencing to identify critical genomic drivers of
the disease.
Methods
Patient Data
Patients were identified from the University of Michigan Cranial Base Program. Institutional
Review Board (IRB) approval was obtained from the University of Michigan IRB. Patient
data and images were collected from clinical records retrospectively.
Targeted Exomic Sequencing
Using targeted, amplicon-based sequencing with the Oncomine Cancer Panel, we assessed
the mutation status of common oncologic therapeutic targets as described.[9]
[10] Briefly, amplicon-based DNA sequencing was performed using 40 ng of DNA isolated
with a RNA/DNA formalin-fixed paraffin-embedded isolation kit (Qiagen) on the Ion
Torrent Personal Genome Machine, utilizing the AmpliSeq Oncomine Cancer Panel.[11] Nucleotide variants and indels were identified using the Torrent Variant Caller
plugin, annotated using Annovar.[12] Sequencing the tumor library on a single 318 chip, we generated 5,254,571 mapped
reads with a mean depth of 315x and 96% of all targeted bases covered by at least 20 reads. Alignment and variant
calling were performed using standard plugins in the Torrent Server Suite (v3.6).
We identified 1,209 variants, which were then filtered through a standardized pipeline
to remove low confidence calls, sequencing errors, germline variants and common polymorphisms,
and results were compared with known mutations in Catalogue of Somatic Mutations in
Cancer (COSMIC), the Oncomine Powertools Database (containing pan-cancer The Cancer
Genome Atlas [TCGA] data), and our internal database of cancer sequencing results.
Sanger Sequencing
Confirmatory Sanger sequencing of the index tumor, and sequencing of additional index
patient tissue was performed to confirm the mutation was a somatic variant. Forward
and reverse primers were designed to amplify the region of the c.134C>T mutation ([Supplementary Table 1]). Standard polymerized chain reaction conditions were used to amplify the DNA product.
Sanger sequencing was performed by the University of Michigan Sequencing Core. Data
analysis was performed with Sequence Scanner Software 1.0 from Applied Biosystems,
Inc..
Immunohistochemical Staining of β-catenin
Immunohistochemical staining for β-catenin overexpression was performed on patient
tumors. Briefly, the formalin-fixed paraffin-embedded sections from the primary tumor
were heated, and underwent peroxidase blocking. A β-catenin mouse monoclonal antibody
(224M; Cell Marque) was applied at a 1:50 dilution. A Mouse EnVision + HRP Kit (Dako)
was used for staining. The slides were then counterstained with Harris's hematoxylin.
Analysis of Data from the Cancer Genome Atlas and COSMIC
The COSMIC is an online database cataloguing mutations in a wide array of cancers.[13] TCGA is an online repository of mutational findings collected from next-generation
sequencing on a variety of cancers.[14] Mutational reports from both datasets are publicly available and were accessed to
identify mutations in β-catenin in other tumors as previously described.[15]
[16]
Results
Patient Clinical Courses
The index patient was a 48-year-old man who initially presented with a left-sided
nasal mass. His symptoms included a 2-month history of left-sided nasal obstruction,
hyposmia, and recurrent left-sided epistaxis. On clinical exam, he had a large friable
left-sided nasal mass filling the nasal cavity and extending into the nasopharynx.
Biopsy in clinic demonstrated poorly differentiated sarcomatoid adenocarcinoma. Imaging
demonstrated a T2 hyperintense, T1 hypointense left nasal cavity mass extending to
the left ethmoid sinuses and nasopharynx. It also involved the lamina papyracea, abutting
the left orbit and extending up to the cribriform plate ([Fig. 1A]). Positron emission tomography imaging did not reveal any metastatic disease, and
he was staged as T2N0M0.
Fig. 1 (A) Preoperative and (B) 24-month postoperative axial T1-weighted MRI with contrast of the index teratocarcinosarcoma.
Intraoperative appearance of sinonasal teratocarcinosarcoma (C) before and (D) after resection. MRI, magnetic resonance imaging.
The patient underwent a margin-negative endoscopic endonasal approach for resection
of his nasal mass, including cribriform resection and nasoseptal vascularized pedicled
flap reconstruction ([Fig. 1C, D]). Final pathology was confirmed as a sinonasal teratocarcinosarcoma, with negative
margins. He underwent 60 gray of postoperative radiation to the left neck and bilateral
retropharyngeal nodal basins. He recovered well postoperatively, and is currently
43 months status posttreatment with no evidence of disease recurrence ([Fig. 1B]).
The second patient was a 50-year-old man who had slowly progressive headaches, nausea,
and emesis for 2 to 3 years. He had worsening headaches, nausea, and emesis, along
with behavioral changes, dysosmias, a recurrent right-sided nasal obstruction and
epistaxis. Magnetic resonance imaging of the brain revealed a large mass in the right
inferior frontal lobe ([Fig. 2A, B]). Biopsies were consistent with sinonasal teratocarcinosarcoma. The tumor was not
surgically resectable; thus, the patient underwent chemoradiation. While he initially
tolerated chemoradiation, his neurologic status deteriorated. Subsequent imaging revealed
progressive disease and hemorrhagic infarctions ([Fig. 2C, D]). Given his worsening clinical status, he was made comfort care and died ∼1 month
after presentation.
Fig. 2 (A, B) Initial and (C, D) follow-up axial T1-weighted MRI of the second teratocarcinosarcoma. The follow-up
MRI showed new hemorrhagic infarcts over the course of chemoradiation treatment. MRI,
magnetic resonance imaging.
Targeted Exome Sequencing of the Sinonasal Teratocarcinosarcoma
Given the limited understanding of the biology behind sinonasal teratocarcinosarcomas,
we screened for commonly mutated cancer drivers, as determined by the Oncomine Cancer
Panel. This analysis pipeline prioritized a high confidence mutation in the CTNNB1 (β-catenin) gene, resulting in a p.S45F codon change. To confirm our finding of the
p.S45F mutation in β-catenin, we performed Sanger sequencing. We confirmed the c.134C>T
point mutation in one copy of β-catenin ([Fig. 3A]), resulting in the p.S45F activating mutation. Sequencing of surrounding tissue
did not reveal this mutation ([Fig. 3B]), verifying this is a somatic, not germline, mutation.
Fig. 3 (A) Chromatogram of c.134C>T mutation in β-catenin, resulting in p.S45F. Arrow pointing
to the mutation. (B) No mutation seen in surrounding tissue, confirming this is a somatic mutation.
Immunohistochemical Staining of β-catenin
We performed immunohistochemical staining to identify β-catenin protein expression
patterns in our index tumor and the second tumor. In the tumors, the carcinoma component
was adenocarcinoma. The teratoma component contained benign respiratory, benign squamous,
and primitive mesenchymal components. Both tumors demonstrated significant overexpression
of β-catenin throughout the tumor, when compared with surrounding nontumor tissue
([Fig. 4]). Specifically, β-catenin staining was predominantly nuclear in the mesenchymal
component and in a subset of the epithelial component. The majority of the epithelial
component demonstrated membranous β-catenin staining.
Fig. 4 (A, C) Immunohistochemistry demonstrating overexpression of β-catenin in index and second
teratocarcinosarcoma tumor tissues, with surrounding normal tissue with no overexpression
of β-catenin. (B, D) Corresponding hematoxylin and eosin stains were captured. ×20 magnification.
β-catenin S45F Mutations Identified in Mutation Databases
We next searched for p.S45F (c.134C>T) β-catenin mutations in publically available
cancer databases using Oncomine. Overall, 454 mutations in β-catenin were identified
in 10,194 tumor samples (4.5% of all tumors). Of the 454 mutations, 44 were missense
mutations in S45 (9.7% of all β-catenin mutations), and 15 were specifically p.S45F
mutations (3.3% of all β-catenin mutations). The tumors containing this specific mutation
were most frequently hepatocellular carcinomas ([Table 1]).
Table 1
Known p.S45F β-catenin mutations in other tumors
|
Database
|
% Tumors with β-catenin mutations
|
% β-catenin mutations p.S45F
|
p.S45F tumor types
|
|
COSMIC
|
10.1% (4,974/49,121)
|
10.4% (518/4,974)
|
65% Desmoid fibromatosis (337/518)
12% Colorectal (61/518)
11% Hepatic (59/518)
4% Wilm's tumor (21/518)
3% Endometrial (13/518)
2% Melanoma (8/518)
1% Biliary (5/518)
< 1% Adrenal (3/518)
< 1% Ovarian (3/518)
< 1% CNS (2/518)
< 1% Cervical (1/518)
< 1% Gastric (1/518)
< 1% Lung (1/518)
< 1% Lymphoma (1/518)
< 1% Pancreatic (1/518)
< 1% Urethral (1/518)
|
|
TCGA
|
4.5% (454/10,194)
|
3.3% (15/454)
|
60% Hepatic (9/15)
13% Endometrial (2/15)
7% Adrenal (1/15)
7% Lung (1/15)
7% Colorectal (1/15)
7% Melanoma (1/15)
|
Abbreviations: CNS, central nervous system; COSMIC, Catalogue of Somatic Mutations
in Cancer; TCGA, The Cancer Genome Atlas.
We then queried the p.S45F (c.134C>T) mutation in the COSMIC database. The p.S45F
mutation was identified in more than 500 combined soft tissue tumors (including adrenal,
biliary tract, central nervous system, endometrial, kidney, and liver systems, [Table 1]). The most common tumor harboring the mutation is desmoid fibromatosis.
Discussion
The Wnt//β-catenin pathway is a well-studied cell proliferation pathway. Dysregulation
in Wnt//β-catenin signaling has been implicated in several cancers.[17]
[18]
[19] Using next-generation sequencing techniques, we have identified an actionable p.S45F
mutation in β-catenin in a sinonasal teratocarcinosarcoma. This mutation is of great
interest as it leads to decreased degradation of β-catenin, resulting in increased
signaling for cell growth and division.[20]
[21] We have confirmed that this mutation results in an overexpression and nuclear translocation
of β-catenin based on immunohistochemical stains. Given the resulting constitutive
Wnt/β-catenin signaling, we postulate this mutation is a potential genetic driver
mutation and an alluring prospect for treatment using inhibitors of the Wnt/β-catenin
pathway.[22]
Interestingly, the p.S45F mutation is identified in tumors with a significant mesenchymal
component (particularly desmoid fibromatosis of soft tissue). This is consistent with
the histology in this sinonasal teratocarcinosarcoma, which contains a significant
amount of mesenchymal tissue. Notably, this mutation has been demonstrated to predict
increased recurrence and poor response to meloxicam therapy in desmoid tumors.[23]
[24] The presence of the p.S45F mutation in multiple soft tissue tumors, and identification
of a worse prognosis in certain tumors with this mutation, suggests a potential beneficial
role for inhibitors targeting β-catenin.
Precision medicine trials are increasingly being employed to identify and apply targeted
therapy options in cancers refractory to current treatment paradigms.[25]
[26] Currently, multiple Wnt/β-catenin pathway agents are in development, including agents
targeted specifically against β-catenin.[27]
[28] Future trials using these inhibitors in patients with p.S45F mutations will be informative
in their applicability. In patients with this specific mutation, targeted regulators
of the Wnt pathway upstream of β-catenin may prove to be ineffective given the likely
dominant overexpression of the β-catenin protein. Rather, specific inhibitors of β-catenin
and downstream targets may prove to be more successful.
Given the current limitations in care and poor prognosis of patients with sinonasal
teratocarcinosarcoma, new treatment paradigms need to be identified. Next-generation
sequencing techniques provide valuable data for potential targeted therapy options.
Specifically in this instance, we have identified a p.S45F mutation in β-catenin in
a patient with a sinonasal teratocarcinosarcoma, providing a targetable treatment
option if this index patient were to suffer disease recurrence. This is the first
study to identify a targetable genetic mutation and possible tumor driver in a sinonasal
teratocarcinosarcoma. In the future, it will be important to screen for p.S45F mutations
and other mutations/aberrations in β-catenin in other teratocarcinosarcoma specimens
to identify if this is a common driver mutation in these tumors. If so, application
of Wnt/β-catenin pathway inhibitors may provide a successful adjuvant or alternative
treatment option in a historically difficult-to-treat cancer. Further sequencing of
teratocarcinosarcomas and other rare skull base tumors will be useful to increase
our knowledge of potential genetic drivers of disease.
