Virchows Archiv

https://doi.org/10.1007/s00428-020-02963-w

ORIGINAL ARTICLE

Clinicopathologic and molecular features of six cases of phosphaturic

mesenchymal tumor
Lulu Sun 1 & Carina Dehner 1 & Jason Kenney 1 & Samantha M. McNulty 1 & Xiaopei Zhu 1 & John D. Pfeifer 1 &
Horacio M. Maluf 1 & John S. A. Chrisinger 1

Received: 6 July 2020 / Revised: 19 October 2020 / Accepted: 30 October 2020

# Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Phosphaturic mesenchymal tumors (PMT) are rare neoplasms characterized by secretion of FGF23, resulting in renal phosphate
wasting and osteomalacia. This tumor-induced osteomalacia (TIO) is cured by complete resection; thus, diagnosis is important,
particularly on biopsy. Although PMT have a classic histologic appearance of bland spindled cells with conspicuous vascular
network and characteristic smudgy basophilic matrix, there is a broad histologic spectrum and variant histologic patterns can
make recognition difficult. Recent studies have demonstrated FN1-FGFR1 and FN1-FGF1 gene fusions in PMT; however,
approximately 50% of cases are negative for these fusions. We sought to characterize 6 cases of PMT in-depth, compare fusion
detection methods, and determine whether alternative fusions could be uncovered by targeted RNA sequencing. Of the 6 cases of
PMT in our institutional archive, 3 were not given diagnoses of PMT at the time of initial pathologic examination. We
characterized the immunoprofile (SMA, D2-40, CD56, S100 protein, desmin, SATB2, and ERG) and gene fusion status (FN1
and FGFR1 rearrangements by fluorescent in situ hybridization (FISH) and two targeted RNA sequencing approaches) in these
cases. Tumors were consistently positive for SATB2 and negative for desmin, with 5/6 cases expressing ERG and CD56. One
specimen was acid-decalcified and failed FISH and RNA sequencing. We found FN1 gene rearrangements by FISH in 2/5 cases,
and a FN1-FGFR1 fusion by targeted RNA sequencing. No alternative gene fusions were identified by RNA sequencing. Our
findings suggest that IHC and molecular analysis can aid in the diagnosis of PMT, guiding excision of the tumor and resolution of
osteomalacia.

Keywords Phosphaturic mesenchymal tumor . RNA sequencing . Fluorescence in situ hybridization . FN1-FGFR1 . FN1-FGF1 .
Tumor-induced osteomalacia

Introduction
Phosphaturic mesenchymal tumors (PMT) are rare neoplasms
characterized by secretion of fibroblast-like growth factor 23
(FGF23), resulting in renal phosphate wasting,
hypophosphatemia, bone pain, and fractures [1–4]. This
tumor-induced osteomalacia (TIO) is cured by complete re-
section. Most cases occur in middle age; however, cases are
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/s00428-020-
02963-w.
* John S. A. Chrisinger
jschrisi@wustl.edu
1 to epithelioid cells [17].
Department of Pathology and Immunology, Washington University
School of Medicine, 425 S. Euclid Ave., Campus Box 8118, St.
Louis, MO 63110, USA
reported from 9 months to over 80 years [5, 6]. Most PMT
occur in the soft tissue or bone and are solitary and benign;
however, rare multifocal and malignant cases occur [1, 7–17].
Exceptional tumors are extra-musculoskeletal [17–19].
PMT are generally recognized based on history of osteo-
malacia, elevated serum FGF23, and histomorphology.
Histologically, PMT are characterized by bland spindled to
stellate cells, prominent vascular network, and distinctive
smudgy basophilic matrix which can undergo “grungy” or
flocculent calcification [1, 7]. Mitotic activity is usually low.
Additional features include the presence of adipose tissue,
myxoid changes, osteoid-like or chondroid-like matrix,
microcysts, osseous metaplasia, and osteoclast-like giant cells
[1, 7, 13, 20]. Interestingly, some cases show a somewhat
endocrine-like morphology with trabecular growth of round
While lesions with typical histologic features in the setting
of TIO are unlikely to cause confusion, some tumors are

apparently “non-phosphaturic” due to occult osteomalacia,
early complete resection, or inherent biologic characteristics
of the tumor or patient [1, 6, 13, 20–23]. In addition, tumors
may be histologically heterogeneous and variant patterns may
be encountered, including cases resembling angiomyolipoma,
glomangiopericytoma, osteoblastoma, osteosarcoma,
chondromyxoid fibroma, solitary fibrous tumor, giant cell tu-
mor of the bone, glomus tumor, and high-grade spindle cell or
spindle cell and pleomorphic sarcoma [13, 17, 24]. Thus, rec-
ognition may be difficult, particularly on a small biopsy and in
patients without known TIO.
In such cases, ancillary techniques including immunohis-
tochemical (IHC) studies and fluorescent in situ hybridization
(FISH) can be helpful. IHC studies for FGF23, FGFR1, D2-
40, SSTR2A, SATB2, CD56, and ERG have been reported to
be positive in PMT, and focal or patchy positivity for SMA
and DOG1 is also seen [1, 13, 20, 25–28]. S100 protein,
keratins, CD34, and desmin are typically negative. Further,
recent studies have demonstrated FN1-FGFR1 and FN1-
FGF1 gene fusions in 42% and 6% of PMT, respectively
[29, 30]. These findings suggest a role of FGFR signaling in
the tumorigenesis of PMT and in the FGF23 hypersecretion
phenotype. The genetic basis of tumors without FN1-FGFR1
and FN1-FGF1 fusions is less clear, though one recent study
demonstrated overexpression of α-klotho or less commonly
β-klotho in fusion-negative cases [31].
We sought to address multiple aims: first, to describe the
clinical and histologic features of additional cases of this rare
tumor; second, to validate previously reported IHC findings;
third, to investigate methods for fusion detection, and, finally,
to test the hypothesis that alternative fusions, including in-
volving other genes in the FGFR family, could be found in
PMT by RNA sequencing.
Methods
Case selection
This study was approved by the Institutional Review Board.
All potential PMT cases from December 1990 to October
2017 were retrieved from the pathology departmental archive
of Washington University School of Medicine. We searched
for cases that met at least one of the following criteria: (i)
histologic diagnosis of PMT, (ii) clinical history of TIO or
elevated FGF23 levels and/or, and (iii) resolution of osteoma-
lacia after tumor removal. Six cases were found. Formalin-
fixed, paraffin-embedded (FFPE) blocks and slides were re-
trieved. Slides were examined by two pathologists who con-
firmed the diagnosis of PMT and histologic features were
noted. Clinical characteristics were obtained from pathology
reports and the electronic medical record.
Virchows Arch
Immunohistochemistry
We performed a panel of IHC studies: smooth muscle actin
(SMA, clone 1A4, Cell Marque, Rocklin, CA), SATB2 (clone
EP281, Cell Marque, Rocklin, CA), podoplanin (clone D2-40,
Cell Marque, Rocklin, CA), CD56 (clone 123C3, Ventana-
Roche, Basel, Switzerland), S100 protein (clone 4C4.9,
Ventana-Roche, Basel, Switzerland), desmin (clone DE-R-
11, Ventana-Roche, Basel, Switzerland), and ERG (clone
EPR3864, Ventana-Roche, Basel, Switzerland). Unstained
slides were cut from each case and staining was run on a
Ventana IHC autostaining platform. Five cases had a subset
of these stains performed during the routine clinical diagnostic
work-up, and these stains were not repeated. Immunostaining
was scored as diffuse, patchy (scattered positive areas), focal
(few positive cells), and negative.
FISH
Interphase FISH was performed on 5-μm-thick FFPE tissue
sections. The FN1 Break Apart (BA) Probe Kit was synthe-
sized by Empire Genomics (Buffalo, NY, USA), with the 5′
and 3′ regions of the FN1 gene labeled with CytoGold and
CytoRed, respectively. The FN1-FGFR1 Tri-color Fusion/
Translocation FISH Probe Kit, synthesized by CytoTest
(Rockville, MD, USA), labels the 5′ region of the FN1 gene,
the 5′ region of the FGFR1 gene, and the 3′ region of the
FGFR1 gene with CytoAqua, CytoGreen, and CytoOrange
fluorophores, respectively.
Processing is outlined here, with detailed protocols includ-
ed in the supporting information. In brief, FFPE specimen
slides were baked, deparaffinized, and dehydrated. Then,
slides were pretreated, washed, protease digested, washed,
and dehydrated again. After applying the probe, slides were
coverslipped, then codenatured at 80 °C for 5 min and hybrid-
ized at 37 °C overnight. After washing, nuclear
counterstaining was performed and slides were coverslipped.
Slides were stored at − 20 °C until analysis. Images were
captured on an Olympus BX61 Fluorescent scope using an
E-Cite SERIES 120PC Q light source using CytoVision
Version 7.5 Build 72 software.
Normal fat and muscle sections from non-cancer amputa-
tions were used to establish baseline performance characteris-
tics for both probe sets. All available tissue was scanned and
no abnormal patterns were found in the controls. A tricolor
filter was used in conjunction with single band filters to eval-
uate the FN1-FGFR1 Tri-color probe set. The FN1-BA probe
was evaluated using single band filters. We scanned all avail-
able tissue for abnormal (split-signal) patterns, allowing for up
to 1 abnormal pattern per field of view at × 100. For both
probe sets, we required split signals to be separated by more
than 2 signal diameters to be considered a positive rearrange-
ment in a given cell.
Virchows Arch

Medically managed, never resected

TruSight RNA Fusion

normalization of phosphorus
Normalization of phosphorus
Complete resolution of TIO

Complete resolution of TIO

Complete resolution of TIO

Resection after biopsy with

Areas containing tumor were circled on H&E-stained slides,
Follow-up Status at last follow-up

and 1 mm punches were taken from the corresponding areas

on the FFPE tissue blocks. Deparaffinization and RNA extrac-
tion were performed using RecoverALL Total Nucleic Acid
Isolation Kit for FFPE (Thermo Fisher, Waltham, MA). RNA

levels

levels
quality and concentration were assessed using an Agilent
Bioanalyzer (Agilent, Santa Clara, CA). Quality of RNA
was determined using DV200 values, which represent the per-
duration

13 yrs

centage of RNA fragments that are greater than 200 nucleo-

2 mo
2 mo
4 yrs

3 yrs
2 yrs

tides in length, a measurement useful for FFPE samples,

consistent with TIO

which have RNA fragmentation. A DV200 of < 30% is not
Benign angiomatous

Periosseous fibroma
Procedure Original histologic

recommended by the manufacturer for sequencing. Based on

lesion, NOS
Glomus tumor

the DV200 values, 100 ng of RNA was used for library prep-
aration per TruSight RNA Fusion Panel kit (Illumina, San
diagnosis

Diego, CA) instructions.

PMT

PMT
PMT

A targeted RNA sequencing panel specific for fusion anal-

ysis was performed. This TruSight RNA Fusion Panel cap-
Resection

Resection

Resection
Resection
Biopsy

Biopsy

tures 507 fusion-associated genes (compiled from data from

COSMIC and other databases) and their fusion partners.
Library preparation was performed per protocol using the
Left 4th rib
Left thigh

Left thigh
pleura
Thoracic
proxi-
verte-

TruSight RNA Fusion Panel kit (Illumina, San Diego, CA).

F female, M male, mo months, PMT phosphaturic mesenchymal tumor, TIO tumor-induced osteomalacia, yrs years
Tumor

tibia
brae

mal
Right
T12

Blunt-ended double-stranded cDNA was synthesized from

site

extracted RNA. 3′ ends were adenylated, and multiple

Phosphorus
(mg/dL)**

indexing adapters ligated. After PCR amplification, the library

was quantified, and targeted probes were hybridized and cap-
tured. After washing, a second hybridization and capture was
0.6
1.4

2.3
1.5
--

--

performed, followed by purification and a second PCR ampli-

fication. The amplified and enriched library was cleaned up by
FGF23

mL)*
(RU/

AMPure XP beads (Beckman Coulter, Pasadena, CA). The

238
890

396
166
--

--

concentration and quality of the library were measured by

Bioanalyzer. Sequencing was performed on the Illumina
before diagnosis (years)
Duration of symptoms

NextSeq 550 system (Illumina, San Diego, CA), at a read

length of 2 × 76 bp. Total reads were down-sampled to ap-
proximately 3 million reads per sample, according to kit rec-
*Reference range ≤ 180RU/mL, **reference range 2.5–4.5 mg/dL
Chronic pain, multiple fractures At least 15

At least 8

ommendations (to decrease false positives). Reads were

aligned to the human reference assembly (GRCh37) using
Clinical and pathologic characteristics of PMT

STAR aligner. Samtools and bedtools were used to tally the

2

Bone pain, multiple fractures, 3

number of reads that were aligned to the genome and targeted

loss, hip fracture, weakness

Bone pain, multiple fractures,

Bone pain, multiple fractures

Back and thigh pain, weight

regions. Fusions were assessed using the STAR-Fusion suite

[32]. To minimize false-positive results, each fusion call had
to be supported by at least one spanning read and at least one
junction read, with a minimum of 5 supporting reads in one
weakness

weakness
Symptoms

category.
Unknown

FN1 FusionPlex
F (years)

Fusion partners for FN1 were also assayed at the University of

M/ Age

71
53

23

24
63
79

Washington Laboratory for Precision Diagnostics. Total

PMT06 M
PMT01 M

PMT04 F
PMT02 F

PMT03 F

PMT05 F

nucleic acid was extracted from unstained FFPE slides and

Table 1

cDNA synthesis was performed. Pre-sequencing QC was per-

Case

formed by diluting the first-strand cDNA with a PreSeq QC


Fig. 1 Histologic features of phosphaturic mesenchymal tumors (PMT).
A. Classic appearance of PMT with bland spindle cells, prominent small
vascular network and hemangiopericytomatous vessels, and
characteristic grungy, calcified matrix (upper right) (PMT06, H&E,
100x magnification). B. Prominent adipocytic component with nests of
small epithelioid cells and large muscular vessels (PMT02, H&E, 200x
magnification). C. Striking dilated vascular spaces with minor adipocytic
and bony components (PMT03, H&E, 40x magnification). Scattered cells
primer and quantifying the cDNA. A pre-sequencing QC cy-
cle number of 29 was used as the quality control cutoff. A
targeted and enriched DNA library was constructed using
FusionPlex BBI Solid Tumor panel and reagent kit (Archer,
Virchows Arch
with clear cytoplasmic vacuoles with nuclear scalloping were also present
(inset, H&E, 600x magnification). D. Infiltrative growth with entrapment
of lamellar bone (PMT04, H&E, 100x magnification). E. Trabecular
growth of epithelioid cells set in myxoid stroma (PMT02, H&E, 200x
magnification) with only very focal smudgy basophilic matrix (inset,
H&E, 400x magnification). F. Somewhat glomoid proliferation of
spindle to ovoid cells (PMT01, H&E, 200x magnification)
Boulder, CO). The library was quantified by quantitative
PCR. Paired-end sequencing was performed on a NextSeq
sequencer (Illumina, San Diego, CA). Archer Analysis soft-
ware was used to process FASTQ output files with a
Virchows Arch

Table 2 Immunohistochemical staining patterns of PMT symptoms before identification of PMT ranged from 2 years
Case Desmin SATB2 SMA S100 D2-40 CD56 ERG to more than 15 years. Symptoms included multiple bone
fractures, bone pain, and weakness. Of the cases with docu-
PMT01 Neg Diffuse Focal Neg Diffuse Patchy Diffuse mented FGF23 levels, 3/4 were elevated. Phosphorus levels
PMT02 Neg Diffuse Focal Neg Neg Patchy Diffuse were low in 4/4 cases with lab values available. In 4 cases,
PMT03 Neg Diffuse Focal Neg Neg Neg Neg phosphorus levels normalized after resection, without supple-
PMT04 Neg Diffuse Focal Neg Patchy Patchy Patchy mental therapy, and in one case, a significant increase in bone
PMT05 Neg Diffuse Neg Neg Neg Diffuse Diffuse density was observed after tumor removal. One tumor was
PMT06 Neg Diffuse Diffuse Focal Focal Focal Diffuse unable to be resected due to its location and patient
comorbidities.
Three tumors were initially diagnosed as PMT, and one
tumor was diagnosed as periosseous fibroma, consistent with
minimum of 4.5 million paired-end reads. This software was TIO. Of the two remaining cases, one (PMT01) was diag-
used to annotate gene fusions. nosed as glomus tumor, while the other (PMT02) was diag-
nosed as a benign angiomatous lesion (Table 1). All cases
were composed of bland spindled, stellate, ovoid, round and/
Results or small epithelioid cells with conspicuous vasculature and
infiltrative growth (Fig. 1). Smudgy basophilic matrix was
Six cases that met inclusion criteria were found (Table 1). identified in all but one case, though it was very focal in three
Three cases arose in the bone (vertebrae, tibia and rib), two cases including the cases diagnosed as glomus tumor and
cases in the soft tissue (both from left thigh), and one from the benign angiomatous lesion. Mitotic activity was low (0–1/10
thoracic pleura. Patient age ranged from 23 to 79 years, with hpf) and all cases showed an adipocytic component, though of
two cases from men and four from women. Duration of variable extent. Hemosiderin deposition and giant cells were

Fig. 2 Immunohistochemical (IHC) features of phosphaturic mesenchymal tumor. Representative IHC images showing that tumor cells are diffusely
positive for SATB2 (A) and ERG (B), while negative for desmin (C) and S100 protein (D) (200x magnification)
 Virchows Arch

Table 3 Specimen characteristics

and quality control metrics Case Age of specimen Type of RNA quality TruSight % on- FusionPlex pre-
(years) decalcification (DV200*) target reads seq QC

PMT01 9 None 43 82 Passed

PMT02 12 None 46 91 Failed
PMT03 3 Acid Undetectable N/A N/A
PMT04 2 EDTA 74 92 Passed
PMT05 2 None 68 93 Failed
PMT06 22 None 43 91 Passed

*DV200: percentage of RNA fragments that are > 200 nucleotides in length

frequently present and microcysts were noted in a subset. One
case showed scattered bland multivacuolated cells with nucle-
ar scalloping.
Six of 6 cases of PMT were negative for desmin, while
diffusely positive for SATB2 (Table 2 and Fig. 2). ERG was
frequently expressed and was diffuse in 4 cases. Staining with
CD56 was also frequently present albeit variable in extent.
Focal SMA staining was present in 4 cases. Tumors were
negative for S100 protein, except one case which showed
focal staining. D2-40 showed variable staining.
FN1 break-apart FISH and FN1-FGFR1 Tri-color fu-
sion FISH was successful in 5/6 cases. In PMT3, which
lacked detectable probe hybridization, the specimen was
acid decalcified (Table 3). FN1 break-apart FISH showed
FN1 gene rearrangements in 2 of the 5 successfully hy-
bridized cases (Table 4 and Fig. 3). Both cases showed
greater than 40% tumor cells with split signals. FN1-
FGFR1 Tri-color fusion FISH was negative in all cases
(Table 4 and Fig. 3).
To verify FISH findings and further characterize FN1 fu-
sions, the 5 cases that had successful FISH performance were
assayed for FN1 fusions only using FusionPlex technology.
PMT02 and PMT05 failed pre-sequencing quality control
(QC) metrics (Table 3). PMT06 was found to have a FN1-
FGFR1 fusion that was in frame, between the 5′ end of exon
11 of FN1 and the 3′ end of exon 7 of FGFR1. No FN1 fusions
were detected in PMT01 and PMT04 (Table 4).
A more extensive targeted RNA sequencing approach was
employed to investigate possible alternative fusions. The
TruSight targeted RNA fusion panel contains 507 cancer-
associated genes, including FGFR1-4, but not FN1. In 5 cases,
RNA extracted from the FFPE blocks met quantity and quality
control metrics for TruSight RNA sequencing, including an
EDTA decalcified specimen and specimens ranging from 2 to
22 years of age (Tables 3 and 4). Library preparation and
sequencing were successful in these 5 cases, with a high per-
centage of on-target reads after alignment (Table 3). The case
with insufficient RNA was the same specimen that failed
FISH hybridization. No FGFR1 fusions were detected in the
5 cases sequenced. No fusions in all other cancer panel genes
were found in the 5 cases (Table 4).
Discussion
PMT are rare tumors that cause TIO. Diagnosis is critical
as complete resection of the tumor is curative. However,
the diagnosis can be fraught with difficulties on multiple
levels as broadly evidenced by the fact that in our study the
duration of time from onset of symptoms to histologic di-
agnosis ranged from 2 to more than 15 years. One reason is
that even when TIO is clinically suspected the tumor may
evade detection for years, even with advanced imaging
techniques [33–35]. In addition, at the time of initial

Table 4 Gene rearrangement

results by FISH and targeted Case FN1 Break- FN1-FGFR1 FusionPlex targeted FN1 RNA TruSight Targeted
RNA sequencing apart FISH Fusion FISH sequencing RNA
sequencing

PMT01 - - No fusions detected No fusions detected

PMT02 + - Failed QC No fusions detected
PMT03 Inconclusive Inconclusive Not performed Insufficient/poor
quality RNA
PMT04 - - No fusions detected No fusions detected
PMT05 - - Failed QC No fusions detected
PMT06 + - FN1-FGFR1 fusion No fusions detected
Virchows Arch
Fig. 3 FISH analysis demonstrates FN1 gene rearrangement in 2 cases.
The top panels show representative images from FN1 break-apart FISH
for two cases, PMT02 and PMT06, with the 5’ and 3’ regions of the FN1
gene labeled with gold (artificially colored green for visualization) and
red, respectively. A combined signal and separate gold and red signals,
representing a non-rearranged allele and a rearranged allele, respectively,
can be seen in 2 of the 3 nuclei depicted in each case. The bottom panels
pathologic examination, 3 of our 6 cases (PMT01, PMT02,
and PMT06) were not given specific diagnoses of PMT. In
the first two cases, misclassification was presumably due
to lack of known TIO. The third case (PMT06) was diag-
nosed as “periosseous fibroma, consistent with tumorigen-
ic osteomalacia.” This interpretation was rendered in 1997,
about 10 years after the groundbreaking description by
Weidner and Cruz [7] but 7 years before the seminal work
of Folpe et al. [1] providing further support for the lack of
acceptance of PMT as the main cause of TIO at that time.
Further, the unusual basophilic matrix was only very fo-
cally present in 3 of our cases and absent in one. These
findings highlight some of the historical and persistent dif-
ficulties in identifying the source of TIO and accurately
diagnosing PMT, which suggests the need for ancillary
studies in at least some cases.
We confirmed that PMT generally show an unusual IHC
profile characterized by positivity for SATB2, CD56, and
ERG, consistent with prior studies [1, 13, 20, 25–28]. This
pattern of immunostaining can provide some support for a
diagnosis of PMT; however, it lacks a high degree of speci-
ficity thus limiting its utility.
We found 2 cases with FN1 gene rearrangements by FISH,
one of which was positive for the FN1-FGFR1 gene fusion by
FusionPlex technology. Notably, the fusion in this case was
not found by FN1-FGFR1 Tri-color fusion FISH, nor by the
TruSight RNA fusion panel, which contains FGFR1 as a
show images from FN1-FGFR1 Tri-Color fusion FISH, with the 5’ re-
gion of the FN1 gene in aqua, the 5’ region of the FGFR1 gene labeled
green, and the 3’ region of the FGFR1 gene labeled orange (artificially
colored red for visualization). All complete nuclei show two combined
green-orange signals, representing two intact FGFR1 alleles, and two
separate aqua signals, representing non-translocated FN1 genes
target. However, the FusionPlex result is likely to be correct,
given that it is a reported fusion in PMT and FISH was pos-
itive for FN1 rearrangement. There could be multiple potential
reasons for this discrepancy. First, the probe sets for the FISH
assays are different, and so precise locations of the
breakpoints and the target regions of the probes may lead to
different hybridization patterns. Second, there are differences
in technology and treatment between the Illumina TruSight
RNA fusion panel and the Archer FusionPlex panel. This is
further illustrated by the fact that 2 cases that failed QC on the
FusionPlex panel passed QC metrics on the TruSight protocol,
with successful on-target reads. This is a high percentage of
cases that did not pass QC; however, it does not appear to
correlate with the age of the specimen, as the oldest specimen
(22 years old) had a successfully discovered fusion. Of note,
FN1 rearrangements are not entirely specific for PMT. They
have also been reported in synovial chondromatosis and soft
tissue chondromas [36], so if FISH for FN1 rearrangements is
used for routine diagnosis, the results should be integrated
with the pathologic and clinical findings.
In addition to evaluating the IHC and cytogenetic fea-
tures, we sought to detect novel fusions which could po-
tentially have diagnostic utility and provide additional in-
sight into the pathogenesis of cases lacking FN1-FGFR1
and FN1-FGF1 fusions. We chose the TruSight targeted
RNA sequencing panel designed to look for gene fusions,
with good performance in FFPE specimens. The

advantages of such a targeted RNA sequencing panel in-
clude the following: (i) the targeted nature enables high
coverage at target sites without prohibitive cost, which
could aid in the detection of fusions expressed at low
levels, or found in few cells; (ii) the hybrid capture ap-
proach permits detection of novel fusion partners to the
already extensive 507 genes included; and (iii) the assay
is designed for degraded samples, including FFPE
specimens.
The panel included all 4 FGFR family genes (FGFR1-4)
that we hypothesized could be rearranged to promote down-
stream FGF23 expression. However, we did not detect gene
fusions in any FGFR genes by targeted RNA sequencing in
any of the 5 sequenced cases, nor were there fusions in any
other genes assayed. The TruSight Fusion Panel did not in-
clude FN1 or FGF1, so would not have detected that specific
rearrangement in our 2 cases with positive FN1 rearrangement
by FISH. Further, our approach was limited in that it was not a
whole-transcriptome analysis. Alternatively, non-fusion ge-
netic changes could underlie PMT without FN1-FGFR1/
FGF1 fusions, including upregulation of α-klotho, β-klotho,
or other FGFR signaling regulatory factors [31].
In conclusion, our study provides additional clinico-
pathologic description and illustrates that a distinctive
IHC profile and FISH for FN1 rearrangements can aid in
interpretation, especially in cases with unusual histology
and lack of known TIO. We demonstrated that different
results for fusion detection may be obtained with various
modalities, cautioning against over-reliance on an isolated
or unexpected result. We did not find additional gene
fusions that could be utilized in diagnosis or provide ad-
ditional insight into the molecular basis of cases without
FN1 rearrangements. These cases require additional ex-
ploration to discover their genetic underpinnings.
Authors’ contributions Lulu Sun: Project conception and planning, FISH
and RNA sequencing, histology examination, data collection, data anal-
ysis, manuscript writing
Carina Dehner: FISH, data collection, data analysis, manuscript
writing
Jason Kenney: RNA sequencing, data collection, data analysis, man-
uscript writing
Samantha McNulty: Project planning, RNA sequencing, data collec-
tion, data analysis, manuscript writing
Xiaopei Zhu: FISH, data collection, data analysis, manuscript writing
John Pfeifer: Project planning, FISH and RNA sequencing, data anal-
ysis, manuscript writing
Horacio Maluf: Project conception and planning, histology examina-
tion, data collection, data analysis, manuscript writing
John Chrisinger: Project conception and planning, histology examina-
tion, data collection, data analysis, manuscript writing
Funding Funding for this research was provided by the AMP
Translational Research Project Fund at Washington University School
of Medicine, Department of Pathology. Illumina provided reagents only
(TruSight RNA Fusion Panel kits).
Virchows Arch
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts of
interest.
Ethics approval This study was approved by the Washington University
School of Medicine institutional review board.
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