Human Pathology: Case Reports 24 (2021) 200497

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Human Pathology: Case Reports

journal homepage: www.elsevier.com/locate/ehpc

TUBA1A-GLI1 fusion in a soft tissue myoepithelial neoplasm

Yajuan J. Liu a, *, Michael J. Wagner b, c, Edward Y. Kim d, Eleanor Y. Chen a, *
a
Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195, United States
b
Division of Medical Oncology, University of Washington, Seattle, WA 98195, United States
c
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States
d
Department of Radiation Oncology, University of Washington, Seattle, WA 98195, United States

A R T I C L E I N F O A B S T R A C T

Keywords: Several types of benign and malignant neoplasms harboring GLI1 gene fusions with various partner genes,
GLI1 fusion including ACTB, MALAT1 and PTCH1, have been described. These neoplasms show a spectrum of morphologic
Soft tissue features and immunohistochemical profiles. However, the GLI1 gene fusion has not been described in soft tissue
Myoepithelial neoplasm
myoepithelial neoplasms previously. In this study, we reported a novel TUBA1A-GLI1 gene fusion in soft tissue
neoplasm occurring in the chest wall of a 35-year-old male. The neoplasm shows morphologic features and
immunophenotype of myoepithelial differentiation. FusionPlex analysis detected TUBA1A-GLI1 fusion in the
neoplasm. The fusion transcript is comprised of 3′ end of exon 1 of TUBA1A and 5′ end of exon 6 of GLI1,
retaining the FOXP coiled-coil domain and the DNA-binding zinc finger domains of GLI1. Promoter swapping
with the TUBA1A (tubulin alpha 1a) gene likely leads to deregulation of GLI1 expression and its downstream
targets. Despite the clinical presentation of multifocal disease and regional lymph node metastasis, the neoplasm
in our case study appeared to be stable in a 10-month follow-up, suggesting that this neoplasm likely pursues an
indolent clinical course. This study expands the morphologic and immunohistochemical spectrum of the neo­
plasms with GLI1 gene fusions and identifies a novel fusion in soft tissue myoepithelial neoplasms. As the
TUBA1A-GLI1 fusion event likely results in activated GLI1 expression, targeting the Hedgehog pathway is a
potential therapeutic option for treatment of patients with this neoplasm.

1. Introduction that harbors a novel TUBA1A-GLI1 fusion, thereby expanding the

The Gli (glioma associated oncogene) proteins, GLI1, GLI2 and GLI3,
are transcriptional effectors of the Hedgehog (Hh) signaling pathway,
which has important roles in embryonic development and oncogenesis
of various cancer types [1–3]. Gene fusions involving GLI1 have been
described in several benign and malignant neoplasms including peri­
cytoma with ACTB-GLI1 fusion [4,5], gastroblastoma and plexiform
fibromyxoma with MALAT1-GLI1 fusion [6,7] as well as “malignant
epithelioid neoplasms” with ACTB1/MALAT1/PTCH1-GLI1 fusion that
show frequent S100 expression and propensity for metastasis [8]. A
group of soft tissue neoplasms harboring GLI1 amplifications that show a
range of morphologic features and immunophenotypes has also been
described [9]. The GLI1 gene fusion or amplification in these tumors
leads to aberrant activation of GLI1 expression, which can serve as a
potential diagnostic biomarker and a therapeutic target.
In this study, we describe clinical, pathologic and molecular features
of a soft tissue tumor occurring in the chest wall of a 35-year-old male
morphologic and molecular spectrum of neoplasms with GLI1 gene
fusions.
2. Methods and materials
2.1. Case source
This project was approved by the Institutional Review Board at the
University of Washington. The case in this study was a consult case to
the Bone and Soft Tissue Subspecialty Service at the Department of
Pathology at the University of Washington.
2.2. Targeted RNA sequencing
Total nucleic acid (TNA) was extracted from the FFPE specimen
using AllPrep DNA/RNA FFPE kit according to the manufacturer’s rec­
ommended protocol (Qiagen, Valencia, CA, USA). The Fusionplex RNA-

* Corresponding authors at: Department of Laboratory Medicine and Pathology, 1959 NE Pacific Street, Seattle, WA 98195, United States.
E-mail addresses: yajuan@uw.edu (Y.J. Liu), eleanor2@uw.edu (E.Y. Chen).

https://doi.org/10.1016/j.ehpc.2021.200497
Received 13 January 2021; Received in revised form 4 March 2021; Accepted 8 March 2021
Available online 31 March 2021
2214-3300/© 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y.J. Liu et al.
sequencing assay was performed using a customized 115-gene panel
covering a wide spectrum of cancer genes known for their involvement
in gene fusions in solid tumors (ArcherDx, Inc. Boulder, CO). The
methods for Fusionplex RNA-sequencing analysis have been described
previously [10].
The data that support the findings of this study are available from
corresponding authors upon request.
3. Results
3.1. Clinical features
The case is a 35-year-old male presenting with a one-year history of
multiple palpable and painful small right axillary and chest wall nod­
ules. On Magnetic Resonance Imaging (MRI) and Computerized To­
mography (CT) studies (MRI in Fig. 1), there were multiple enhancing
masses seen extending circumferentially along the right chest wall.
These included a soft tissue mass anterior to the 5th costochondral
junction (4.6 × 1.6 cm), a posterior chest wall mass along the 4th
through 6th ribs (6.2 × 3.1 cm), a mass adjacent to the right fifth lateral
rib (1.6 × 1.5 × 1.3 cm) and right fifth anterior rib (2.9 × 2.3 × 1.6 cm)
(Fig. 1A). The right axillary nodule measured 1 cm in greatest dimension
(Fig. 1A). There is also bony destruction of the ribs. He underwent
excision of the symptomatic mass along the fifth rib and multiple right
axillary lymph nodes. His remaining chest wall tumors were not
considered amenable to curative resection or definitive radiotherapy. He
did not receive any treatment after surgery. At a 10-month clinical
follow-up, the remaining masses appeared stable in size with no addi­
tional disease on surveillance imaging studies.
3.2. Pathologic features
The excised chest wall mass and axillary lymph nodes measured up
to 2.1 cm and 1.0 cm in greatest dimension, respectively. Histologically,
the neoplasm was circumscribed and showed a nodular to plexiform
growth pattern (Fig. 2A). The neoplastic cells were ovoid to spindled in
shape with relatively uniform nuclei and arranged in nested, cord-like
and reticular patterns associated with myxoid and vascularized stroma
Fig. 1. Radiologic findings of the case study. (A) MRI imaging studies of the
case at presentation. Arrowheads indicate excised nodules.
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Human Pathology: Case Reports 24 (2021) 200497
(Fig. 2B, C). Only rare mitoses were identified. No necrosis was present.
Differential diagnosis based on the histologic findings includes soft tis­
sue myoepithelial neoplasm, vascular tumor (e.g. hemangioma or
hemangioendothelioma), pericytic neoplasm and soft tissue chordoma.
On immunohistochemistry, the neoplastic cells were positive for Keratin
AE1/AE3 (multifocal), S100, SOX10, GFAP (multifocal) and negative for
CD34, p63, SMA, Calponin, ERG and Brachyury (Fig. 3A-D). INI1
expression was retained. One of the masses appeared to involve a lymph
node, indicating regional metastasis (Fig. 2D).
Overall, based on the histologic findings and the immunoprofile, a
diagnosis of soft tissue neoplasm with myoepithelial differentiation was
rendered. Given the lack of definitive histologic classification, further
molecular analysis using FusionPlex was performed to assess for po­
tential presence of disease-specific gene fusion.
3.3. Molecular features
FusionPlex analysis using total nucleic acid (TNA) extracted from the
tumor mass of right chest wall detected TUBA1A → GLI1 fusion, and no
other fusions were detected in the rest of 114 genes sequenced by
FusionPlex analysis including EWSR1, FUS, PLAG1 and HMGA2 (Sup­
plemental Table 1). The fusion transcript identified in this case was
comprised of 3′ end of exon 1 (5′ UTR region) of TUBA1A
(NM_006009.3) and 5′ end of exon 6 of GLI1 (NM_005269.2), retaining
the FOXP coiled-coil domain and the DNA-binding zinc finger domains
of GLI1 (Table 1 and Fig. 4). This fusion gene was under the regulation of
TUBA1A promoter and predicted to initiate translation at GLI1 p.
Met185 (NM_005269.2) residue as this is the methionine closest to the
breakpoint. As the TNA extraction used contained a small portion of
DNA, this analysis also captured the fusion breakpoints at the DNA level
in intron 1 of TUBA1A and intron 5 of GLI1 (Table 1). The reciprocal
GLI1 → TUBA1A in-frame fusion transcript was also detected, but this
was expressed at a much lower level (1:13 ratio in comparison with the
TUBA1A → GLI1 fusion transcript) (Table 1 and Fig. 4).
4. Discussion
In this study, we identified a TUBA1A-GLI1 fusion in a soft tissue
neoplasm presented in the chest wall of a 35-year-old male. As TUBA1A
encodes an alpha tubulin subunit of microtubules, which form part of
the cytoskeleton in eukaryotic cells, and is expressed at high levels, the
resulting TUBA1A → GLI1 fusion product is predicted to activate GLI1
expression by promoter swapping with the highly expressed TUBA1A
(tubulin alpha 1a) gene, leading to deregulation of GLI1 expression and
its downstream targets. GLI1 has been described as a 3′ partner with
several 5′ partner genes in different tumors, such as ACTB/MALAT1/
PTCH1 in pericytoma and/or malignant epithelioid neoplasm [8,11].
The histologic features and immunophenotype (positive Keratin, S100,
SOX10 and GFAP expression) of the neoplasm in this study are most
consistent with a neoplasm with myoepithelial differentiation. The
neoplasm shows some features seen in soft tissue myoepithelioma, i.e.
nested or reticular growth of variably spindled or epithelioid cells with
myxoid stroma. Even though the patient presented with local regional
metastasis, the neoplasm appeared stable in the 10-month follow-up,
suggesting an indolent disease. In addition, the tumor lacks cytologic
atypia and increased mitotic activity seen in myoepithelial carcinoma.
In total, while the neoplasm likely belongs to the family of soft tissue
myoepithelial tumors, it might be best classified as a myoepithelial
neoplasm with uncertain biological potential until additional insight on
its biological behavior can be obtained from a larger series of these GLI1
gene fusion-positive tumors with myoepithelial differentiation. Addi­
tional diagnostic considerations such as vascular tumors (hemangioma
or hemangioendothelioma), soft tissue chordoma and pericytic tumor
were excluded by the lack of expression for ERG, Brachyury and SMA on
immunohistochemistry, respectively.
Approximately half of the myoepithelial tumors arising in soft tissue,
Y.J. Liu et al. Human Pathology: Case Reports 24 (2021) 200497

Fig. 2. Histologic features of the case study. (A) Hematoxylin and eosin image at 20× showing a neoplasm with nodular to plexiform growth pattern with cir­
cumscribed border. (B, C) Hematoxylin and eosin image of the neoplasm at 200X showing ovoid to spindled cells arranged in reticular, nested or cord-like pattern in a
myxoid and vascularized stroma. (D) Hematoxylin and eosin image of the neoplasm involving a lymph node at 40×.

Fig. 3. Immunohistochemical findings of the case study. Immunohistochemical stains demonstrate positive expression of (A) AE1/AE3, (B) S100, (C) SOX10 and (D)
GFAP in the neoplastic cells. Magnification: 200×.

bone or viscera harbor EWSR1 or FUS gene fusion with various partners
[12–14]. The myoepithelial tumors arising in the salivary glands or skin
frequently show PLAG1 or HMGA2 gene rearrangement [15–18]. The
neoplasm in this study was negative for EWSR1, FUS, PLAG1 or HMGA2
fusion in our FusionPlex analysis. It remains to be determined whether a
subset of the soft tissue myoepithelial tumors that are negative for
EWSR1, FUS, PLAG1 or HMGA2 gene rearrangement harbor other
recurrent gene fusions including GLI1.
Our reported neoplasm shows some morphologic similarities to the
previously described malignant epithelioid neoplasms with ACTB1/
MALAT1/PTCH1-GLI1 fusion [8], as characterized by monomorphic
cells in myxoid and vascularized stroma. However, in contrast to those
tumors which show nests of epithelioid cells with frequent S100
expression in most cases but are negative for SOX10 and rarely positive
for Keratin, the neoplasm in this study shows epithelioid to spindled
cells with an immunophenotype (positive expression for S100, GFAP,
SOX10 and Keratin AE1/AE3) consistent with myoepithelial
differentiation.
Pericytic tumors are perivascular myoid neoplasms that includes
myopericytoma, myofibroma and glomus tumor in a morphologic
spectrum. A subset of pericytic tumors has been demonstrated to harbor
the ACTB1-GLI1 fusion [4,5]. These tumors are characterized by sheets

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Y.J. Liu et al. Human Pathology: Case Reports 24 (2021) 200497

Fig. 4. TUBA1A-GLI1 fusion sequence-structure characterization. RNA sequencing reads and sequences including the breakpoints along with the gene structures and
their functional protein domains of TUBA1A (green boxes) (A), GLI1 (grey boxes) (B), reciprocal fusion transcripts of TUBA1A-GLI1 (C and D, Table 1). Untranslated
regions (5′ UTR and 3′ UTR) are shown as narrow bars. Exons are shown as boxes with numbers. The keys of protein domains are shown in the wider boxes behind
exons: N-terminal domain, intermediate domain, and C-terminal in TUBA1A; SNAG domain, interaction motif with SUFU, FOXP Coil-coil domain, and C2H2 Zn finger
domains in GLI1. The blue lines and arrows indicate the breakpoints and fusion points. Representative sequence reads of the TUBA1A-GLI1 fusions by paired-end RNA
sequencing (C and D), including exon mapping of chimeric transcripts over the breakpoints to the reference sequence at base resolution with amino acid translation in
frame. Total BAM coverages of the breakpoints (~1000 reads for TUBA1A → GLI1 fusion transcript and ~80 reads for GLI1 → TUBA1A fusion transcript) are shown
as grey bar scale. Example NGS read coverage of the breakpoints was shown in red bars (R1 reads) and blue bars (R2 reads). Sanger sequencing results that confirm
the breakpoints are shown at the bottom.

of SMA. Gastroblastoma is characterized by nested and lobular growth

Table 1
patterns as well as biphasic composition of an epithelioid component
Breakpoints in the fusions of TUBA1A and GLI1 detected in the case study.
and a spindle cell component, both of which are monomorphic and
Genes in fusions Genomic coordinates [GRCh37/hg19] bland. On immunohistochemistry, the epithelioid component is positive
5′ partner breakpoint 3′ partner breakpoint for keratin, and the spindle cell component is positive for SMA.
RNA: TUBA1A → GLI1 chr12:49582760, chr12:57859390, GLI1 Similar to the GLI1 gene fusions previously described in other tu­
(Exon 1::Exon 6) TUBA1A (Exon1) (Exon 6) mors, the GLI1 gene fusion in this case resulted in the retention of the
DNA: TUBA1A → GLI1 chr12:49580869, chr12:57859180, GLI1 zinc finger domain that is required for transcriptional activation of
(Intron 1::Intron 5) TUBA1A (Intron 1) (Intron 5) target genes. Up-regulated expression of GLI1 has been demonstrated in
RNA: GLI1 → TUBA1A chr12:57859038, GLI1 chr12: 49580616,
the previously described neoplasms with GLI1 gene fusion [4,7]. The
(Exon 5::Exon 2) (Exon 5) TUBA1A (Exon 2)
Reciprocal fusion TUBA1A-GLI1 fusion in the current study likely resulted in aberrant
activation of GLI1 gene expression as well, thereby making targeting the
Note: RefSeq numbers for TUBA1A, NM_006009.3 (encoded on negative strand)
Hedgehog pathway a potential therapeutic option for this tumor.
and for GLI1, NM_005269.2 (encoded on positive strand).
Taken together, neoplasms with GLI1 gene fusion can occur in a
range of anatomic locations and have a spectrum of morphologic fea­
of uniform ovoid to spindled cells arranged around delicate capillary
tures and variable immunohistochemical profiles. All of these neoplasms
vessels. The neoplastic cells are diffusely positive for smooth muscle
are characterized by monomorphic epithelioid and/or spindle cells and
actin. Electron microscopic examination demonstrates subcellular
are frequently associated with rich capillary-type vessels. The neoplasm
structures (e.g. external lamina, cytoplasmic myofilaments and
with TUBA1A-GLI1 fusion as described in the study shows morphologic
intermediate-type intercellular junction) that are consistent with a
features and immunophenotype of myoepithelial differentiation, war­
pericytic phenotype [4]. While the neoplasm in our study also demon­
ranting further investigation of EWSR1, FUS, PLAG1 or HMGA2 fusion-
strates ovoid-spindled cell morphology and close association with
negative myoepithelial tumors for other gene fusions including GLI1.
prominent capillary-type vessels, it lacks SMA expression on immuno­
Despite the initial presentation of multifocal disease and regional lymph
histochemistry to support pericytic differentiation.
node metastasis, the neoplasm in this study appeared to be stable in a 10-
Recurrent MALAT1-GLI1 fusion has been demonstrated in plexiform
month follow-up, suggesting an indolent clinical course.
fibromyxoma and gastroblastoma, which are two rare types of neoplasm
that occur in the stomach [6,7]. Plexiform fibromyxoma is morpholog­
5. Patient consent
ically characterized by plexiform or lobular growth of bland spindle cells
associated with myxoid stroma and delicate network of capillary vessels.
No patient consent was obtained as the case was de-identified, and
The neoplastic cells are myofibroblastic in type with positive expression
no patient-identifying information was included in the manuscript.

4
Y.J. Liu et al.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
CRediT authorship contribution statement
Yajuan Liu: Investigation, Methodology, Validation, Data curation,
Visualization, Writing - review & editing. Michael J. Wagner: Re­
sources, Visualization, Writing - review & editing. Edward Y. Kim:
Resources, Visualization, Writing - review & editing. Eleanor Y. Chen:
Conceptualization, Supervision, Investigation, Visualization, Writing -
original draft.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
Author contributions: The work was supported by the intra-
departmental funding support to EYC. This research did not receive
any specific grant from funding agencies in the public, commercial, or
not-for-profit sectors.
Ethics statement
This retrospective study was approved by the Institutional Review
Board at the University of Washington (protocol #9362). The study has
been performed in accordance with the Declaration of Helsinki.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ehpc.2021.200497.
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