Cardiac Angiosarcoma: Pathology & Cytogenetic Analysis

Original contribution
Cardiac angiosarcoma: histopathologic,
immunohistochemical, and cytogenetic
analysis of 10 cases
☆
Charles Leduc MD
a
, Sarah M. Jenkins MS
b
, William R. Sukov MD
a,b,c
,
Jeannette G. Rustin BS
c
,JosephJ.MaleszewskiMD
a,c,
⁎
a
Division of Anatomic Pathology, Mayo Clinic, Rochester, MN, 55905, USA
b
Division of Biomedical Statistics and Informatics, Rochester, MN, 55905, USA
c
Division of Laboratory Genetics, Mayo Clinic, Rochester, MN, 55905, USA
Received 20 August 2016; revised 20 September 2016; accepted 14 October 2016
Keywords:
Angiosarcoma;
Cardiac tumors;
Cytogenetics;
Molecular diagnostics;
Cardiac malignancy
Summary Angiosarcoma (AS) is the most common cardiac sarcoma with differentiation, and is poorly char-
acterized from a molecular genetic standpoint. Prognosis remains poor, owing to several factors including
aggressive tumor biology, poor response to adjuvant therapy, and lack of targeted therapy. The clinical,
pathologic and molecular cytogenetic features were studied in ten cardiac AS surgically resected at Mayo
Clinic (1994–2015) using a whole-genome, single-nucleotide polymorphism-based platform (OncoScan).
Mean patient age was 47.8 years, male/female ratio was 1:1.5, and overall median survival was 5.2 months.
The most common location was the right atrium (n = 7), with one case each occurring in the epicardium,
pericardium, and right ventricle. No patients had received thoracic irradiation. The most common morphol-
ogy was spindle cell (n = 8), with one case each of epithelioid and biphasic. ERG was the most sensitive
vascular marker, with diffuse immunoreactivity in all cases. Several recurrent (present in at least 3 cases)
aberrations were identified, including trisomies in chromosomes 4, 8, 11, 17, 20, as well as 1q+, and homo-
zygous deletion of CDKN2. Patients who received adjuvant therapy had longer overall survival than those
who did not (median 13.4 vs 3.2 months; P=.0283).Therewerenosignificant associations between tumor
location, histology, immunohistochemical findings, cytogenetic profile, and clinical outcome; however,
there was a trend towards improved overall survival in patients with tumors harboring 1q+ (median 31.8
vs 3.7 months, P= .06). This study confirms recurrent cytogenetic aberrations in cardiac AS, some of which
may have prognostic or predictive implications.
© 2016 Elsevier Inc. All rights reserved.
1. Introduction
Angiosarcoma (AS) is a rare malignant vascular neoplasm,
representing approximately 1% of all sarcomas [1]. It occurs
most commonly in the skin of the head and neck, breast, and
in deep soft tissues, and rarely in visceral locations such as
the liver and spleen [2]. While the heart is an uncommon site,
AS is somewhat overrepresented due to the relative rarity of
☆
Disclosures: The authors have no conflicts of interest or funding to
disclose.
⁎Corresponding author at: Division of Anatomic Pathology, Mayo Clinic,
200 First Street S.W. Rochester, MN, 55905, USA.
E-mail addresses: [e-mail] (C. Leduc),
[e-mail] (S. M. Jenkins), [e-mail]
(W. R. Sukov), [e-mail] (J. G. Rustin),
[e-mail] (J. J. Maleszewski).
www.elsevier.com/locate/humpath
http://dx.doi.org/10.1016/j.humpath.2016.10.014
0046-8177/© 2016 Elsevier Inc. All rights reserved.
Human Pathology (2017) 60,199–207

primary cardiac malignancies. As such, AS represents approx-
imately 40% of all cardiac sarcomas, and is thus the most com-
mon differentiated cardiac sarcoma [3,4]. Prognosis in cardiac
AS is poor, with median overall survival ranging from 5 to 13
months, owing to several factors including aggressive tu-
mor biology, the challenging nature of surgical resection,
poor response to adjuvant therapy, and the lack of targeted
therapy [4-6].
As a group, primary cardiac AS remains poorly character-
ized from an etiologic and molecular genetic standpoint.
Known risk factors for cutaneous and visceral AS, such as ion-
izing radiation and long-term exposure to synthetic material,
have each been documented in a single case of cardiac AS
[7,8]. However, most cases remain idiopathic. There appears
to be some basis for a genetic predisposition, based on the
clustering of cardiac AS within certain families [9,10].Inaddi-
tion, somatic point mutations in KRAS,TP53,andPLCG1
have been identified in some cases of cardiac AS, as have
large-scale genomic gains and losses, some of which appear
to be recurrent in a subset of cases [11,12]. However, owing
to the rarity of cardiac AS, reports to date have consisted of
case reports and small case series, which have limited the iden-
tification of recurring pathogenetically, and/or prognostically
significant, molecular aberrations.
This study aims to further characterize the molecular
genetic features of primary cardiac AS, and to correlate such
with histopathologic and clinical features. In particular, we
sought to identify recurrent cytogenetic aberrations that may
serve as prognostic markers as well as guide identification of
therapeutic targets.
2. Materials and methods
2.1. Case selection
Tissue registry archives at Mayo Clinic (Rochester, MN)
were queried for cases of cardiac AS (1995–2014). Cases with
sufficient tumor content (to reach target of 100 ng DNA) for
molecular genetic testing were retained for inclusion in the
study cohort. Patient demographics and clinical follow-up
and outcome were abstracted from the patient record. The
study was approved by the Mayo Clinic Institutional Review
Board and Biospecimens Subcommittee.
2.2. Gross and histopathologic features
Tumor location and size were abstracted from the patient
record (including radiology, surgery, and pathology reports).
Hematoxylin and eosin (H&E)–stained tissue sections of the
tumor were reviewed, and the following features were assessed:
cytomorphology (spindle cell, epithelioid, or biphasic); tumor
necrosis (present or absent); mitotic activity (number of mitotic
figures per 10 high-power fields [HPF, 400×] averaged over 30
HPFs).
2.3. Immunohistochemistry
Immunoperoxidase studies were carried out on formalin-fixed,
paraffin-embedded (FFPE) tissue sections (4-μm thick) with the
following antibodies: Broad-spectrum cytokeratin (Dako, Santa
Clara, CA; clones AE1 and AE3; 1:200); CD34 (Leica, Buffalo
Grove, IL, clone QBEnd/10, 1:50); CD31 (Dako, clone JC/70a,
1:350); ERG (Biocare, Concord, CA, clone 9FY, 1:25); and
FLI1 (Pharmingen, San Jose, CA, clone G146–254 BD, 1:50).
Tumor reactivity for immunohistochemical antibodies was scored
based on distribution (diffuse, N50% tumor cells; focal, b50%
tumor cells), and intensity (0, negative; 1+, weak reactivity;
2+, moderate reactivity; 3+, strong reactivity).
2.4. Molecular genetic analysis
DNA was extracted from 5-μm-thick scrolls prepared from
FFPE tissue block. DNA was extracted using QIAamp DNA
FFPE Tissue Kit (QIAGEN, Germantown, MD) following
the Affymetrix recommended protocol (Affymetrix P/N
703175 Rev. 2, Appendix E). Extracted DNA was quantified
using a Qubit 2.0 Fluorometer (Life Technologies/Thermo
Fisher Scientific, Waltham, MA) and the dsDNA BR assay
kit according to the manufacturer's protocol. This was done
in triplicate to provide each of the three test laboratories with
enough DNA to run the assays. The Affymetrix OncoScan as-
say was then performed per the manufacturer's protocol; this
whole-genome, single-nucleotide polymorphism–based micro-
array platform assesses copy number and loss of heterozygosi-
ty. The OncoScan assay utilizes molecular inversion probe
(MIP) technology, for the identification of copy number alter-
ations and loss of heterozygosity (LOH). MIP probes in the
OncoScan assay capture the alleles of over 220 000 SNPs at
carefully selected genomic locations, distributed across the ge-
nome with increased probe density within ~900 cancer genes.
Array fluorescence intensity data (CEL files), generated by
Affymetrix GeneChip Command Console (AGCC) Software ver-
sion 4.0, were processed using OncoScan Console software ver-
sion 1.1.034 to produce OSCHP files. After conversion of CEL
files by OncoScan Console, OSCHP files were loaded into the
Chromosome Analysis Suite (ChAS) software version 3.1 for
analysis of clinically relevant CN and LOH events. All regions
flagged by the ChAS software were investigated. Autosomal
probes were considered “copy number loss”if the genomic inter-
val deviating from normal contained a minimum of 25 probes.
Autosomal probes were considered “copy number gain”if the ge-
nomic interval deviating from normal contained a minimum of 50
probes. Absence of heterozygosity (AOH) calls were made when
a region of AOH was greater than 5 mb. Any regions not flagged
by the software but found to deviate from normal were investigat-
edandincludedinthelistoffinal calls as appropriate.
2.5. Statistical analysis
Overall survival was estimated with the Kaplan–Meier
method, and was summarized with the median survival and
200 C. Leduc et al.

95% confidence intervals (when estimable). Survival was
compared by patient groups via Cox proportional-hazards re-
gression using likelihood ratio tests. All analyses were per-
formed using SAS version 9.4 (SAS Institute Inc, Cary, NC),
and statistical figures were generated using R (http://www.R-
project.org/).
3. Results
3.1. Clinical features
Among 44 total primary cardiac sarcomas identified, fifteen
cases of primary cardiac AS were found, of which five had to
be excluded due to insufficient tissue for molecular testing. For
the ten cases retained for the study, the mean patient age was
47.8 ± 17.1 years (standard deviation) (range 23.4–78.0) with
a male/female ratio of 1:1.5 (Table 1). The most common tu-
mor location was the right atrium (n = 7, 70%), with one case
each occurring in the epicardium (n = 1), parietal pericardium
(n = 1), and right ventricle (n = 1). No patients had received
thoracic irradiation prior to the diagnosis of cardiac AS. All ex-
cept one patient were treated with attempted complete surgical
resection, with extensive surgery deemed inappropriate in the
remaining patient (Case 2) due to metastatic disease at presen-
tation. The majority (n = 7, 70%) received adjuvant chemo-
therapy (CT) or radiotherapy (RT) either alone (CT, n = 3;
RT, n = 1) or in combination (CT/RT, n = 3). Clinical
follow-up from time of diagnosis ranged from 1.2 to 61.2
months (median, 4.4 months), with half of the patients
experiencing metastasis, and the majority dying of dis-
ease (n =8, 80%). Of the patients alive, according to the most
recent available clinical documentation, one had residual
tumor within the right atrium 3 months following incomplete
resection (Case 8), while the other was disease free by
clinical and radiologic workup 61 months following diagnosis
(Case 7).
3.2. Gross and histopathologic features
Mean tumor size (maximum dimension) was 6.4 ± 2.8 cm
(range 2.5–9.8). Macroscopically, tumors ranged from dark
redtotan,withgenerallyinfiltrative lobulated borders that oc-
casionally merged imperceptibly with the endocardial surface
(Fig. 1 and Fig. 2). The most common cytomorphology was
spindle cell (n = 8, 80%), while one case exhibited pure epi-
thelioid morphology. The remaining case was biphasic, con-
taining both spindle cell and epithelioid morphology (Fig. 1).
Tumor necrosis was identified in all cases, and mean mitotic
was 13.2/10 HPF (5.7–23.7) (Table 2).
3.3. Immunohistochemical features
Immunoreactivity for broad spectrum cytokeratin was at
least focally present, in forty percent of cases (n = 4) total,
and was diffuse and strong in the one case with epithelioid
morphology. ERG was the most sensitive vascular marker,
with diffuse immunoreactivity in all cases, which was strong
(3+) in intensity in all but one case. Immunoreactivity for
FLI-1 was diffuse in the majority of cases (n = 6, 60%), and
at least focally present in all cases. Similarly, while the major-
ity of tumors showed diffuse immunoreactivity for CD31
(n = 7, 70%), two cases were only focally positive, and the
case with epithelioid morphology was completely non-
reactive. Finally, while the majority of cases were diffusely
immunoreactive for CD34, it had the lowest sensitivity com-
pared to the other vascular markers, as 2 cases were com-
pletely negative.
3.4. Molecular cytogenetic features
Whole-genome interrogation with Oncoscan identified
complex molecular profiles with aberrations identified in at
least 5 chromosomes in all cases (Fig. 2; Supplementary
Table 1. Several recurrent chromosomal abnormalities were
identified. The most common manifestation was single chro-
mosomal gain (trisomy), which was present in at least three
cases for the following chromosomes: 4, 8, 11, 17, and 20
(Table 3). Of these, trisomy 8 was the most common, being
identified in 80% (n = 8) of cases, followed by trisomy 17
(n = 6, 60%). All except 2 cases (Case 1, and Case 8) harbored
at least one of these aforementioned trisomies. Interestingly,
all cases with trisomy 11 also harbored trisomy 17 and trisomy
8. Other aberrations noted in at least 3 cases included gain of
the long arm of chromosome 1 (1q+) and homozygous dele-
tion of CDKN2. Finally, while there were no cases with specif-
ic amplification of MYC, small interstitial amplifications on
1q+ resulted in amplification of PIK3C2B/MDM4 in two
cases, and KIT was amplified in a single case (Supplementary
Table 1.
3.5. Survival analysis
The overall median survival across all patients was 5.2
months (95% CI: 1.2 months to 50.2 months), while the es-
timated 1-year survival was 35% (95% CI: 3.6% to 66.4%)
(Fig. 3). Patients who received CT/RT had significantly longer
overall survival than those who did not (median 13.4 vs 3.2
months; P= .0283). However, this may be a reflection of
patient age, as administration of CT/RT was significantly
associated with younger age (P= .0227), as the three pa-
tients who did not receive adjuvant therapy were ≥60 years
(mean 67.5), while all those who did were all b60 years of
age (mean 39.4). There was a trend towards improved overall
survival in patients with tumors harboring 1q+ compared to
those without (median 31.8 vs 3.7 months, P= .06) (Fig. 3).
There were no significant associations between tumor loca-
tion, histology, immunohistochemical findings, cytogenetic
profile, and clinical outcome.
201Molecular cytogenetics of cardiac angiosarcomas

4. Discussion
Toourknowledge,thisisthelargestcohortofcardiacASto
undergo cytogenetic analysis, and the first study to identify
prognostically significant recurrent molecular alterations.
The demographics of the patients in our cohort with a mean
patient age of 47.8 years are consistent with prior studies,
which demonstrate a wide age range and a peak incidence in
the fourth to fifth decade [3,13]. While a slight female predom-
inance was noted in the study group (1:1.5), it disappeared
with inclusion of the five cases excluded due to lack of tissue
for molecular characterization (8 men; 7 women). Thus, the
Mayo cohort also seems to be in keeping with prior reported
cohorts, showing a relatively equal sex distribution [3,5,13-15].
Tumor location, tumor size, and predominant histomorpho-
logic pattern in this study are in keeping with prior studies of
cardiac AS, which report a predilection for the right atrium,
an average tumor size of 6–7 cm (range 1.5–11), with solid
and vasoformative spindle cell tumor morphology
[4,5,15,16]. The epithelioid variant of angiosarcoma, while
unusual, is known to occur in the heart. It is a particularly chal-
lenging diagnosis given the unusual morphology and strong
cytokeratin expression, which requires a thorough clinical
and radiologic workup to rule out metastatic carcinoma. In
the one case included in this study, no primary mass was iden-
tified outside the heart, and, while the dominant morphology
was of solid sheets of epithelioid cells, there was also focal vascu-
lar formation and malignant cells with intracytoplasmic lumina
containing erythrocytes. While the lack of CD31 expression is un-
usual, at least a few studies have shown it to be less sensitive than
ERG in the diagnosis of cutaneous and hepatic angiosarcoma
[17,18]. While epithelioid sarcoma can exhibit similar mor-
phologic and antigenic characteristics, this diagnosis (to our
knowledge) has yet to be reported in this anatomic site.
Similar to our case, all previously reported cases of epithe-
lioid cardiac AS developed metastatic disease [19-22].Asin
soft tissue sites, the prognostic importance of epithelioid mor-
phology in cardiac AS remains unclear; however, most reports
suggest a worse prognosis compared to conventional cardiac
AS [20-22]. The factors contributing to somewhat prolonged
survival in our single case of AS (10.5 months) are unclear,
but may include relatively younger patient age and possible re-
sponse to adjuvant therapy. It is worth noting, in this context,
that dramatic response to chemotherapy has been reported in a
case of metastatic epithelioid AS, with complete remission of
disease up to 48 months on systemic therapy [19].
While AS is known to have variable histologic manifesta-
tions, well-defined biphasic histology, as seen in one of our
cases (Case 4, Fig. 1), is not a well-described phenomenon
[23]. The epicardial location causes consideration of epitheli-
oid hemangioendothelioma, but the vasoformative nature of
the tumor in association with prominent nuclear atypia and ne-
crosis were more in keeping with AS than epithelioid heman-
gioendothelioma [24]. The biphasic morphology in this case
raises the differential diagnosis of biphasic mesothelioma;
however, the immunoprofile was in keeping with a vascular neo-
plasm. Whether clear-cut biphasic histology has distinct prognos-
tic relevance remains unclear; however, knowledge of this
manifestation of AS highlights the challenging differential diagno-
sis of primary and metastatic tumors of the heart and serosal
membranes, and underlines the importance of a thorough immu-
nohistochemical workup including vascular markers.
The presence of immunoreactivity for cytokeratin in 40%
(n = 4) is somewhat higher than what is reported in the litera-
ture for malignant vascular tumors, which ranges from 0% to
35% [25-27]. However, if we exclude the case of epithelioid
AS, which are known to express cytokeratins [28], then the fre-
quency of immunoreactivity is 30% (n = 3). In addition, with
the exception of the epithelioid AS, all other cytokeratin-
reactive cases exhibited only focal immunoreactivity, which
may not have been considered “positive" in other studies. In-
terestingly, the one case with biphasic histology showed no
cytokeratin immunoreactivity in the epithelioid component
(Fig. 1); however, this is not entirely unexpected as even pure
epithelioid AS do not consistently express cytokeratins, with
immunoreactivity rates ranging from 50% to 85% across dif-
ferent sites [29-31].
ERG was diffusely reactive in all cases of cardiac AS, and
its superior sensitivity to CD31 and CD34 is consistent with
what has previously been reported [17]. Its utility as a marker
Table 1 Clinical characteristics and outcome in cardiac angiosarcoma
Case Age (y) Sex Site Size (cm) Metastasis Procedure Adjuvant CT/RT Follow-up (mo) Outcome
1 43 M Right ventricle 3.0 Bone Resection Yes/Yes 10.5 DOD
2 33 F Right atrium 2.5 Lungs, liver Biopsy No/Yes 5.2 DOD
3 57 F Right atrium 9.8 Lungs Resection Yes/No 13.4 DOD
4 23 F Epicardium N/A Nil Resection Yes/No 2.8 DOD
5 64 M Right atrium 9.6 Brain Resection No/No 3.7 DOD
6 42 M Right atrium 8.2 Bone Resection Yes/Yes 50.2 DOD
7 45 F Right atrium 7.3 Nil Resection Yes/Yes 61.2 NED
8 29 F Right atrium 5.5 Nil Resection Yes/No 3.2 AWD
9 77 M Pericardium N/A Nil Pericardiectomy No/No 1.2 DOD
10 60 F Right atrium 5.1 Nil Resection No/No 3.2 DOD
Abbreviations: N/A, not available; DOD, dead of disease; NED, No evidence of disease; AWD, Alive with disease.
202 C. Leduc et al.

of endothelial differentiation is of particular note in the one
case of epithelioid AS, which was uniformly non-reactive with
CD31. Given the occasional propensity of AS to originate on
serosal surfaces, ERG would also be preferred over CD34, as
the latter is known to be positive in other tumors at this site
such as solitary fibrous tumor, which, when malignant, could
potentially mimic areas of high-grade AS. Finally, FLI-1 did
not appear to offer any diagnostic advantage compared to
ERG, as the intensity and distribution were generally less,
which would limit its application in small biopsy specimens.
4.1. Molecular features
As highlighted by the most recent classification of tumors
of the heart by the World Health Organization, recurrent cyto-
genetic abnormalities were not thought to occur in cardiac AS
[32]. However, results from this study in concert with previ-
ously published reports clearly point to the contrary [11,12].
It is compelling that the three most common alterations in
the current study, namely trisomy 8 (n = 8), trisomy 17
(n = 6), and gain 1q (n = 4), were also the most common
Fig. 1 Gross, histologic, and immunohistochemical features of cardiac angiosarcoma. Corresponding macroscopy, microscopy and immunohis-
tochemistry of spindle cell (right column, Case 3), epithelioid (middle column, Case 1), and biphasic (right column, Case 4) AS. All photomicro-
graphs shown are on original magnification (×400).
203Molecular cytogenetics of cardiac angiosarcomas