2021, Volume 37, Number 3, Page(s) 226-232
What Would Next Generation Sequencing Bring to the Diagnosis and Treatment of Sarcomas? A Series of 20 Cases, a Single Institution’s Experience
Ýbrahim KULAÇ, Pýnar BULUTAY, Çisel AYDIN MERÝÇÖZ
Department of Pathology, Koç University, School of Medicine, ISTANBUL, TURKEY
Keywords: Sarcoma, Soft tissue tumors, Next generation sequencing, Molecular pathology
Soft tissue tumors comprise a small proportion of a pathologist’s routine practice. Although morphology and immunohistochemistry
are quite helpful for diagnosing these tumors, many require molecular tests. Fluorescence in-situ hybridization has been the most commonly
used method for the detection of specific genomic alteration, but next generation sequencing (NGS) could be more informative in many ways.
Here we present our targeted NGS experience on soft tissue tumors with a series of 20 cases.
Material and Method: The Laboratory Information System (LIS) was screened for soft tissue tumors that had been sequenced by NGS (between
January 2018 - February 2021). 20 consecutive cases were included in the study. All cases were sequenced using a commercial targeted sequencing
panel designed for soft tissue tumors.
Results: We were able to run a reliable sequencing study for 16 (80%) of the cases but 4 (20%) of them failed in quality tests. We have found
pathogenic alterations in 12 (60%) of the cases. The most common alterations were EWSR1 fusions, FLI1 being the most common partner. NGS
results drastically changed the initial diagnosis, and thus the treatment modalities, in 3 cases (15%): the case with ETV6-NTRK3 fusion, the case
with FUS-TFCP2 fusion, and the case of rhabdomyosarcoma (RMS) that was favored to be of the alveolar subtype and turned out to lack FOXO1
Conclusion: A targeted NGS panel is robust and very informative. It not only allows pathologists to further specify and/or confirm their diagnosis
but it could also play an important role in predicting the outcome.
Soft tissue sarcomas may occur at any age and at any
localization. American Cancer Society estimates that more
than 12 000 people will be diagnosed soft tissue sarcoma in
. Although sarcomas seem to comprise only a small
part of a pathologist’s daily practice, the diagnosis can
often be challenging, time consuming and laborious. Many
reports with inconclusive diagnosis that require molecular
tests are commonly signed out inevitably.
Classification of these tumors has become more
complicated and detailed after each WHO Classification
of Tumors of Soft Tissue and Bones book edition 2.
Hematoxylin & eosin (H&E) sections and a panel of
immunohistochemistry with relevant antibodies with the
guidance of a detailed radiological & clinical evaluation
can be quite helpful for an accurate diagnosis. However,
many entities have close resemblance at the H&E level
and even their immunoexpressional profile is very similar.
Molecular tests for a significant number of cases are usually
needed and can be definitive. The key genetic event for soft tissue tumors is gene fusions and demonstrating those
genetic alterations is not only diagnostic but may also have
predictive value. There are a number of methods available
today and most institutions prefer fluorescence in-situ
hybridization (FISH) or reverse transcriptase PCR (RTPCR).
FISH has been one of the most widely used techniques
worldwide to detect gene re-arrangements 3,4.
Traditionally, dual color and split signal FISH probes
are used and allow investigations to detect the break of
that gene, which is suggestive for a rearrangement 5.
Although this approach is highly valuable, it usually lacks
the information of the partner gene. Knowing the partner
gene can be invaluable for the diagnostic dilemma such as
seen with desmoplastic small round cell tumor (DSRCT)
versus Ewing sarcoma since both might have fusions
involving EWSR1 but with a different partner gene. There
are other types of FISH probes that target each gene, and
a fusion signal is suggestive for a rearrangement of the
two. This approach is highly useful, although it provides no information on the exons involved. Another method is
RT-PCR which aims to amplify the target cDNA converted
from the RNA extracted from the tumor tissue 3,6. This
approach is fast, cheap and can help to identify the specific
exonal regions of the fusion. However, when the differential
diagnosis includes numerous entities, all with different
genomic alterations, it is substantially cumbersome to test
all the specific alterations by FISH or RT-PCR for each one.
Massive parallel sequencing (next generation sequencing
/ NGS) has recently become a method of choice in many
institutions for sequencing studies. It allows to sequence
multiple genes at once with reasonable speed. cDNA
synthesized from RNA or genomic DNA obtained from
paraffin block can be used as the starting material. This
technology has been used for years for cancer research
and has more recently been introduced into clinical
practice. Detection of cancer associated germline or
somatic alterations has never been this practical. Although
it still has a high price point and there are issues about
the regulations and reimbursements, NGS is providing
invaluable information about tumors.
Here we report a series of 20 soft tissue tumors that were
evaluated by NGS for further genomic characterization
using a commercial targeted NGS panel.
The Koç University Hospital, Department of Pathology’s
Laboratory Information System (LIS) was screened for
all soft tissue tumors that had been sequenced by NGS
(between January 2018-February 2021). Among the 600
various NGS studies, it was found that 20 cases with a
(proposed) diagnosis of soft tissue tumor were sequenced
using the ArcherDx Sarcoma Panel. The ArcherDx sarcoma
panel covers the most commonly altered genes in sarcomas
(ALK, BCOR, BRAF, CAMTA1, CIC, CSF1, EGFR, EPC1,
ERG, ESR1, EWSR1, FGFR1, FGFR2, FGFR3, FOS, FOSB,
FOXO1, FUS, GLI1, HMGA2, JAZF1, MDM2, MEAF6,
MET, MGEA5, MKL2, NCOA1, NCOA2, NR4A3, NTRK1,
NTRK2, NTRK3, NUTM1, PAX3, PDGFB, PHF1, PLAG1,
PRKCA, PRKCB, PRKCD, RET, ROS1, SS18, STAT6,
TAF15, TCF12, TFE3, TFG, USP6, YAP1, YWHAE). Eleven
of the cases were consults and 9 were from our department.
Qiagen AllPrep DNA/RNA FFPE Kit was used for RNA
extraction following the manufacturer’s protocol. RNA
quantity was measured with the Qubit fluorometric
quantification system (Life Technologies). Before library preparation, cDNA was synthesized from all RNA samples
and a control PCR for quality assessment was performed.
Although the library preparation kit requires a Ct value
of <27, we had to include samples with Ct values of 27-30
because the cases did not have any other tissue samples.
After the QC PCR run, cDNA library was prepared with the
Archer FusionPlex Sarcoma kit (ArcherDX, Boulder, CO)
following the manufacturer’s protocol. Libraries were run
on the Illumina NextSeq 500 with compatible flow cells.
All the analyses were done by using the ArcherDx Analysis
software (version 6.2.7) and variants were confirmed with
publicly available somatic variant databases.
|Demographics and Basic Information About Cases/
The median age of the patients was 20 (1-76) years. The
female to male ratio was 9/11. We were unable to perform
the study for four (20%) of the cases that had a Ct value of
>30 at the QC PCR study. All 16 cases had acceptable read
numbers and coverage. 11 (55%) of the cases were consults
and 9 (45%) of them were from our department. 18 (90%)
of the samples were paraffin blocks, 1 of the remaining two
cases was a fresh frozen tissue stored in a -80°C freezer for
a month after the surgery, and the other sample was a cell
block from a mediastinal fine needle aspiration.
We have found pathogenic alterations in 12 (60%) of the
cases (Table I). The most common alteration was EWSR1-
FLI1 fusion (three cases) and fusions involving FOXO1
(two cases). All the cases with EWSR1-FLI1 fusion were
referred to us for confirmation of the proposed Ewing
Click Here to Zoom
|Table I: All the cases included to the study with their initial and final diagnosis along with genomic alterations detected by targeted
NGS results drastically changed the initial diagnosis, and
thus the treatment modalities, in 3 cases (15%): the case
with ETV6-NTRK3 fusion, the case with FUS-TFCP2
fusion, and the case of rhabdomyosarcoma (RMS) that was
favored to be of the alveolar subtype and turned out to lack
Two (10%) of the cases had no specific diagnosis other
than high-grade malignant mesenchymal tumor and
undifferentiated sarcoma, and we were unable to detect any
specific alterations in these two tumors.
Although soft tissue tumors comprise only a small part
of a pathologist’s routine practice, cases can sometimes
be diagnostically challenging. In adults, liposarcoma and leiomyosarcoma are among the most common sarcoma
types and most of the time do not require any further studies
other than H&E evaluation and immunohistochemistry.
However, other relatively common tumors such as
rhabdomyosarcoma and Ewing’s sarcoma/PNET often
need the identification of the pathognomonic translocation
because they have overlapping features with several entities
and immunohistochemistry could be helpful only at a
certain level. In daily practice we also encounter some
rare soft tissue tumors that have less known but specific
genetic alterations. These alterations can be investigated
by techniques such as FISH, RT-PCR and NGS. They
all have advantages and disadvantages. FISH and RTPCR
are tests that require testing for each gene with a
separate reaction. They can be very helpful for some cases.
However, especially when the morphology is vague and the
immunohistochemistry does not direct towards a specific diagnosis, it would be hard to test the tumor tissue for every
possible alteration with FISH and RT-PCR. In that case an
NGS with a targeted panel would help substantially. There
are two major approaches to hunt gene fusions by NGS
that mainly differ by the starting material; the genomic
DNA-based approach and the RNA-based approach. A
RNA-based application enables the detection of fusions
and even rare genomic abnormalities with high confidence
if the isolated nucleic acid’s quality is high enough. There
are several in-house and commercial targeted NGS panels
that are designed for different sequencing platforms; one
we have been using and reported in this study is RNA
based and has a good coverage for many genes related to
soft tissue tumors.
There are two major aspects of this study, and we would
like to discuss them separately:
Common Fusions That Require the Identification of
Ewing sarcoma can overlap with many similar entities, especially
with desmoplastic small round cell tumor (DSRCT),
morphologically, clinically, and immunohistochemically;
both are tumors of children and young adults, and although
DSRCT is commonly located intraabdominally it can be
encountered anywhere, as can Ewing sarcoma 7. For an
experienced soft tissue pathologist, this differential may not
be an issue for most of the cases using relevant morphological
clues and some immunohistochemistry studies.
Although WT1 is commonly used to differentiate these two
and considered to be highly reliable 8 it may not be that
helpful for all cases. A FISH study can be performed but a
break-apart probe would only tell whether there is a rearrangement
involving EWSR1. Using fusion-specific probes
(for all possible genes) will increase the cost and time. A
NGS panel that covers the most common genes rearranged
in round cell sarcomas would be more efficient as regards
cost and time. Moreover, undifferentiated round cell
tumors of the bone and soft tissues have recently become
more diverse. Genetically and clinically different round
cell sarcomas were recently identified: round cell sarcoma
with EWSR1-non ETS (NFATC2 and PATZ1 being the
most common ones), CIC-rearranged sarcomas and sarcomas
with BCOR genetic alteration. These tumors can have
morphological features resembling Ewing sarcoma 9-11.
Although these tumors share morphological similarities,
they are distinct entities with different clinical and prognostic
features. The differentiation is almost solely possible by
a NGS panel that covers all the relevant genes. In our series,
we had (non-RMS) 5 round cell tumors that were directed
to molecular pathology for the detection of relevant alterations
and all had EWSR1-FLI1 fusions. Moreover, it was
shown that different fusion transcripts may have various
degrees of clinical impact for many neoplasms including
Ewing sarcoma 12,13.
The detection of characteristic fusion is becoming a
requirement for rhabdomyosarcomas as well. RMSs
are histologically classified as alveolar, embryonal,
pleomorphic, spindle cell/sclerosing with alveolar and
embryonal RMSs being the most common types 2. In
the past, the differentiation was made morphologically
(with the help of immunohistochemistry) but for at least
two decades the diagnosis is being supported by a FISH
study using a FOXO1 break-apart probe. The Children’s
Oncology Group recently published a report that FOXO1
fusion status was the most important prognosticator after
metastatic status 14. Although it is very much possible to say that the tumor most likely to have this mutation by
morphology, a FISH or a NGS study would be more helpful
Rare Fusions in Rare Tumors Either for Diagnosis or for
In our series we had three interesting and rare cases
that deserve more attention. One of the cases was a
molecular consult that was directed to our department
for sequencing. The patient was a 33-year-old female, and
her tumor was localized in the maxilla. The tumor was
composed of epithelioid and spindle areas with extensive
necrosis, and immunohistochemistry showed positivity
with pancytokeratin, Desmin and MyoD1, which were
all noted in an external pathology report. The differential
included entities such as rhabdomyosarcoma and
sarcomatoid carcinoma along with others and the case was
reported as “sarcoma with epithelioid morphology” with
a comment that said, “malignant tumor, favor EWSR1-
PATZ1 fusion sarcoma (EPS)” with a note: “sequencing
was recommended”. Only one representative block was
sent to our department for sequencing. A hematoxylin &
eosin section of the block revealed an epithelioid/rhabdoid
looking malignant tumor (Figure 1A). A RNA based
approach was used as described in detail in the material
– methods section and the study revealed the FUS-TFCP2
fusion. The case was diagnosed as “epithelioid and spindle
cell rhabdomyosarcoma with FUS-TFCP2 fusion” in
accordance with the morphological, immunohistochemical
and molecular findings 15,16.
Another patient was a 7-year-old male with a tumor at the
pleura. The tumor looked spindly, cellular, and relatively
monotonous (Figure 1B). The initial report favored
angiomatoid fibrous histiocytoma and the patient reached
our department for sequencing. Our sequencing study
revealed an ETV6-NTRK3 fusion. We have finalized the
report by saying “infantile fibrosarcoma and inflammatory
myofibroblastic tumor can be considered in the differential
diagnosis with the morphological, immunohistochemical
and molecular findings 17-19”. Apart from the final
diagnosis, this patient could be a candidate for targeted
therapy because of this NTRK3 fusion 20.
Click Here to Zoom
|Figure 1: Hematoxylin & eosin sections of two selected cases. One (A) was initially called a malignant tumor with epithelioid
morphology and considered an EWSR1 non-ETV fusion sarcoma. An NGS study revealed a TFCP2-FUS fusion; the case was eventually
diagnosed epithelioid and spindle rhabdomyosarcoma with TFCP2-FUS fusion. The other case (B) was initially called angiomatoid
fibrous histiocytoma and NGS study revealed ETV6-NTRK3 fusion. It was eventually diagnosed as inflammatory myofibroblastic tumor
/ infantile fibrosarcoma. (Fusions of the cases are demonstrated below H&E images for each case)
The last case was a 12-year-old boy with a mass in right thigh.
The tumor was removed, and the specimen was evaluated
at an outside pathology laboratory. The case was considered
sclerosing epithelioid fibrosarcoma (SEF) although it was
negative for MUC4, immunohistochemically. The patient
was referred to our department for NGS studies. We have
detected a fusion between KMT2A and YAP1 genes and this was supportive of the initial SEF diagnosis. SEFs are
rare tumors harboring EWSR1 fusions commonly with
CREB3L1 gene. Recent studies suggested that there is a
group of tumors with KMT2A-YAP1 fusion that have
almost identical morphological features with SEF but
without the MUC4 positivity and EWSR1 fusion. So far,
researchers have called these tumors MUC4-negative
SEF 21,22. The data is limited on these tumors, and it
is not well documented whether these genomic alterations
have different impact on prognosis. We think that, this
clarification will most likely be possible when a sufficient
number of cases are reported with their detailed clinical
and morpho-molecular workup.
Detailed morphological evaluation and extensive
immunohistochemical studies were performed on all three
tumors discussed above. Today’s knowledge on soft tissue
tumors makes us more aware of rare entities with specific
genetic alterations, which entails us to perform molecular
studies. Although molecular findings would not add any
prognostic or predictive information for some tumors,
it would have a huge impact in some. There are several
publications on the utility and the benefits of a targeted
NGS for soft tissue sarcomas 23-25.
NGS may seem like a highly advantageous technique
especially in the context of gene fusions, but it is rather long and still costs a lot. Although we think that NGS
will overtake many techniques in the future, we cannot
deny the fact that the use of techniques such as RT-PCR
and FISH is currently more practical for many patients.
Finally, we would like to emphasize that NGS is only an
ancillary technique like FISH or RT-PCR. They all need to
be used in conjunction with microscopic evaluation and
immunohistochemistry and need to be interpreted with
caution by a pathologist. Although not within the scope
of this paper, we would like to indicate that preanalytical
variables are extremely important and all the samples
should be fixed and stored with care. A last word should
be said on the interpretation of NGS results, as they should
be interpreted according to bioinformatic metrics and one
should take coverage, number of reads and other quality
parameters into consideration.
In conclusion, a RNA based NGS approach is highly
valuable for the diagnosis of soft tissue tumors especially
while dealing with rare cases with less known genomic
alterations. This technique allows pathologists to further
specify and/or confirm their diagnosis while providing
predictive outcomes in some cases. Results of an NGS
study should carefully be evaluated with clinical,
histomorphological and immunohistochemical findings by
the pathologist to finalize the case, in the best way possible.
CONFLICT of INTEREST
The authors declare that they have no conflict of interest.
Concept: IK, Design: IK, CAM, Data collection or
processing: IK, PB, CAM, Analysis or Interpretation: IK,
PB, CAM, Literature search: IK, CAM, Writing: IK, CAM,
Approval: IK, PB, CAM.
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