Pediatric and Adult Hepatic EmbryonalSarcoma: A Comparative Ultrastructural

Study with Morphologic CorrelationsNarasimhan P. Agaram, MD,

Ann Baren, MA, and

Cristina R. Antonescu, MD

Department of Pathology,

Memorial Sloan-Kettering

Cancer Center, New York,

New York, USA

ABSTRACT Hepatic embryonal (undifferentiated) sarcoma (ES) is a rare

pediatric tumor occurring predominantly in the first decade of life, but a

few examples of adult ES have also been described. Isolated ultrastructural

reports describe contradictory lines of differentiation in these tumors. Four

pediatric and 3 adult ES cases were studied ultrastructurally and features

were correlated with morphology. Morphologically, tumors were composed

of mixture of plump spindle cells and bizarre giant cells, showing abundant

cytoplasmic eosinophilic globules. Ultrastructurally, the hallmark features in

all cases included dilated RERs and secondary lysosomes with dense preci-

pitates. Dilated mitochondria and mitochondrial–RER complexes were often

seen. Other features included intracytoplasmic fat droplets, scant actin

microfilaments, and focal glycogen pools. In summary, pediatric and adult

ES show similar morphologic and ultrastructural features. Ultrastructurally,

hepatic ES have distinctive findings, including dilated RER and electron-

dense lysosomal precipitates, which correlate with the eosinophilic hyaline

bodies seen microscopically. These findings suggest that ES are composed

of fibroblastic, fibrohistiocytic, and undifferentiated cells. Other lines of

differentiation were not identified.

KEYWORDS embryonal sarcoma, liver, ultrastructure, undifferentiated

Hepatic embryonal (undifferentiated) sarcoma (ES) is a rare tumor seen pre-

dominantly in the pediatric age group. It was initially reported as a distinct

entity by Stocker and Ishak in 1978 [18] when they reported 31 cases from

the AFIP files. In what has been the largest study to date, they categorized

‘‘undifferentiated sarcomas of the liver’’ as a distinct entity. The origin of

the tumor, although well accepted as mesenchymal, is still in debate, as it

does not show any particular line of differentiation. The immunohistochem-

ical staining pattern reported is not specific. Ultrastructurally, most hepatic

ES studied to date have been shown to demonstrate a fibroblastic=

fibrohistiocytic differentiation. Few isolated reports of myogenic [9] and

lipoblastic [6] differentiation have been described. We hereby report our

experience of hepatic ES with an emphasis on the ultrastructural features

Received 5 January 2006; accepted16 February 2006.

Address correspondence to Cristina R.Antonescu, MD, Department ofPathology, Memorial Sloan-KetteringCancer Center, 1275 York Avenue,New York, NY 10021, USA. E-mail:antonesc@mskcc.org

Ultrastructural Pathology, 30:403–408, 2006Copyright # Informa HealthcareISSN: 0191-3123 print=1521-0758 onlineDOI: 10.1080/01913120600854699

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and the morphologic correlation. We further com-

pare the pediatric tumors and the adult tumors to

look for any differentiating features.

MATERIALS AND METHODS

The Electron Microscopy Laboratory files of the

Department of Pathology of MSKCC were searched

for cases diagnosed as embryonal=undifferentiated

sarcoma of the liver. The available histologic and

immunohistochemical slides were reviewed by the

authors for confirmation of the diagnosis.

Patient demographics and clinical information,

such as age, sex, site, and size of the tumor, were

obtained from review of clinical charts and pathol-

ogy reports. The histopathologic findings recorded

were gross appearance, presence of a fibrous cap-

sule, cell type, type of stroma, presence of giant cells,

presence of eosinophilic globules, areas of increased

cellularity, presence of necrosis, inflammation and

entrapped bile ducts. The immunohistochemical

findings, if present, were recorded for each case.

For ultrastructural analysis, fresh tumor tissue was

immersed in a 3% formaldehyde–3% glutaraldehyde

fixative, postfixed in 1% osmium tetroxide, and

dehydrated in graded ethanol. The tissue was then

embedded in epoxy resin and stained with uranyl

acetate–lead citrate by using standard protocol.

Thick sections were cut and stained with toluidine

blue to select suitable areas for ultrastructural analy-

sis that was performed using a Philips-410 trans-

mission electron microscope.

The ultrastructural findings analyzed in each case

included the presence of secondary lysosomes and

dense precipitates, dilated rough endoplasmic reticu-

lum (RER), dilated mitochondria, intracytoplasmic fat

droplets, and cytoplasmic filaments.

Immunohistochemical studies were reviewed or

performed if material was available with antibodies

prediluted from Ventana Medical Systems (Tucson,

AZ), except where noted: vimentin, HHF-35, smooth

muscle actin (SMA), desmin (Dako, 1:50), myogenin,

Cam5.2, AE1:AE3, EMA, AAT, HepPar l, AFP, and

S-100 protein (1:500; Dako, Denmark).

RESULTS

Seven cases of embryonal (undifferentiated)

sarcoma (ES) were identified in the Electron

Microscopy files and diagnosis confirmed by review

of the histologic material available (Table 1). The 7

patients included were all females, 4 children and 3

adults. The pediatric patients were aged 3, 7, 8,

and 9 (mean: 6.7) years and the adults were aged

21, 33, and 50 (mean: 34.6) years. The tissue avail-

able for electron microscopy was from the primary

liver tumor in all except one case, where the lung

metastasis was studied instead.

The gross findings, available in 4=7 cases, showed

a well-circumscribed solitary mass with cystic areas,

hemorrhage, and necrosis (Figure 1). The solid areas

were tan and soft. These 4 tumors ranged in size

from 6.2 to 17.0 cm (mean: 13.1 cm).

Microscopically, all cases showed a cellular pro-

liferation of plump to spindle cells arranged in a

vague storiform pattern in a background of myxoid

stroma (Figure 2C). Scattered bizarre giant cells as

well as areas of necrosis were seen in all cases

(Figure 2A). A moderate amount of pale or vacuo-

lated cytoplasm was noted in most tumor cells. A

consistent and distinct finding was the presence of

cytoplasmic eosinophilic globules, of variable size

and number (Figure 2A). These cytoplasmic globules

were PAS positive and diastase resistant (Figure 2E).

A fibrous capsule was present in 4 of the 6 cases.

Areas of increased cellularity, resembling a small blue

round cell tumor, were identified in 4=6 cases

(Figure 2B). An inflammatory component was seen

in 4=6 cases and entrapped bile ducts were seen in

3 cases.

Immunohistochemical studies were available in

3 cases. Positivity for vimentin (diffuse) and desmin

(patchy, focal) (Figure 2D) was seen in all 3 tumors

FIGURE 1 Left partial hepatectomy resection for a large

(17 cm), solitary, cystic, and hemorrhagic mass in a 50-year-old

woman (case 7).

N. P. Agaram et al. 404

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tested. Two cases showed positivity for AAT

(a-1-antitrypsin) (Figure 2F) and focal positivity for

muscle-specific actin (HHF 35). Low and high mol-

ecular weight cytokeratins, S-100 protein, Hep Parl,

AFP, and EMA were performed on 2 cases and were

negative. Myogenin was negative in all the 3 cases.

Electron microscopically, the tumors were com-

posed of spindle cells in a loose myxoid stroma

(Table 2). The ultrastructural hallmark of this tumor,

as seen in all cases studied, was the presence of

dilated RER and electron-dense, membrane-bound

secondary lysosomes (Figure 3B–D). The cisternal

dilatation of RER (Figure 3B) correlated with the

vacuolated appearance of the cytoplasm histologi-

cally, while the electron-dense precipitates (Figure

3D) corresponded to the eosinophilic globules seen

morphologically. Dilated mitochondria were seen

in 6=7 cases. Three cases showed the presence of

the characteristic mitochondria–RER complexes, with

the dilated RER wrapping tightly around the

mitochondria (Figure 3E). Rare giant cells with

irregular nuclei were identified (Figure 3A). In 4=7

cases, an undifferentiated component of rounded

cells with minimal cytoplasm and scarce cytoplasmic

organelles was identified. These areas correlated

with the primitive cellular component noted on his-

tology. Intracytoplasmic fat droplets were seen in

varying amounts in 5=7 cases. Scant subplasmalem-

mal actin microfilaments were identified in 3=7 cases

and focal glycogen pools were seen in 2=7 cases.

Follow-up information was available in 6=7 cases

and ranged from 6 months to 21 years. One of the

patients, a 21-year-old female, died of disease within

12 months of the diagnosis. The remaining 5 cases

showed no evidence of disease at 6 months, 8

months, 9 months, 8 years, and 21 years (Table 1).

FIGURE 3 Ultrastructural findings: (A) low magnification of a

bizarre tumor cell with multilobated nucleus and dilated, branch-

ing RER cisternae (case 1, 3 6600); (B) cystically dilated RER

adjacent to electrondense, membrane-bound lysosomes (case 7,

3 7040); (C) complex and heterogeneously electrondense cyto-

plasmic inclusion (case 3, 3 10,650); (D) electrondense membrane

bound secondary lysosome (case 4, 3 20,400); (E) cytoplasmic

detail showing characteristic mitochondria–RER complexes,

with dilated RER wrapping tightly around mitochondria (case 7, 3

16,640).

FIGURE 2 Histologic appearance and immunohistochemical

profile of ES: (A) Bizarre cells with abundant cytoplasmic eosino-

philic globules (case 7, 3 200); (B) small cell undifferentiated

component (Case 1, 3 200); (C) pleomorphic spindle cells in a

myxoid stroma (case 6, 3 200); (D) desmin reactivity mainly seen

in large pleomorphic cells (case 6, 3 200); (E) PAS-diastase

resistant positivity in cytoplasmic inclusions (case 6, 3 400);

(F) a-1-antitrypsin immunoreactivity highlight the hyaline glo-

bules (case 1, 3 400).

405 Hepatic and Embryonal Sarcoma

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DISCUSSION

Embryonal (undifferentiated) sarcoma (ES) of the

liver is a histologically distinctive neoplasm with a

peak incidence in the age range of 6–10 years. It is

considered the fourth [18] in frequency of primary

pediatric hepatic tumors, after hepatoblastoma and

hepatocellular carcinoma. By definition, ES shows

no specific line of differentiation, such as smooth

or striated muscle, or lipoblasts by routine micro-

scopic exam, while ultrastructurally, the small

number of cases examined indicated inconclusive

results with regard to histogenesis.

In the present study, similar ultrastructural find-

ings in both the pediatric and adult ES tumors were

found, including dilated RER cisternae and promi-

nent electron-dense bodies. These findings suggest

a fibroblastic or fibrohistiocytic lineage, while no fea-

tures supporting other lines of differentiation, which

have been reported previously, were identified in

this study.

Most ultrastructural reports of ES make note of

the prominent RER in these tumors, specifically of

cisternal dilatation, a finding commonly seen in

fibroblastic proliferations [1, 4, 6, 9]. Another consist-

ent electron microscopic feature is the presence of

abundant secondary lysosomes composed of the

dense precipitates [1, 4, 6, 9]. The number and elec-

tron density of these precipitates varies, with some

being larger or denser than others. These electron-

dense complexes distinctly correlated with the hya-

line globules seen histologically, which have been

reported to be composed of a-1-antitrypsin (AAT)

[1], and were thought to be indicative of a fibrohistio-

cytic differentiation. The presence of lipid droplets is

variable, but is seen in most cases studied [4, 6].

Exceedingly rare, the presence of abundant lipid

deposits ultrastructurally can lead to an erroneous

diagnosis of liposarcoma [5]. Subplasmalemmal con-

densation of actin microfilaments can be found in a

minority of the cells, but these findings are considered

insufficient for a smooth muscle differentiation.

The fibrohistiocytic features of ES were initially

highlighted by Keating and Taylor [10], when they

reported the ultrastructural findings of 2 cases and

TABLE 1 Demographic and Clinicopathologic Data in 7 Patients with Embryonal Sarcoma

Case Age (years) Sex Site Size (cm) Gross FU interval Survival

1 3 F Liver 6.2 Pink tan, soft, areas of hemorrhage and necrosis 9 months NED

2 7 F Liver NA NA 21 years NED

3 8 F Liver NA NA NA NA

4 9 F Liver 15.0 Well-circumscribed, encapsulated, tan, soft

friable with hemorrhage and necrosis

8 years NED

5 21 F Lung NA NA 6 months DOD

6 33 F Liver 14.5 Well-circumscribed, myxoid, tan variegated

cut surface, areas of hemorrhage and necrosis

6 months NED

7 50 F Liver 17.0 Cystic mass with hemorrhage and necrosis 8 months NED

Note. FU, Follow-up; NA, not available; NED, no evidence of disease; DOD, dead of disease.

TABLE 2 Ultrastructural Features of Pediatric and Adult Embryonal Sarcoma

Case Age (years) Site Cell type

Lysosomes with

dense precipitates

Dilated

RER

Fat

droplets

Dilated

mitochondria

MIT–RER

complex Actin

1 3 Liver Plump=spindle þ þ þ þ þ =� þ � þ2 7 Liver Plump oval þ þ þ þ � � � þ3 8 Liver Plump oval þ þ þ þ þ þ � �4 9 Liver Spindle þ þ þ þ þ þ þ þ �5 21 Lung Spindle þ þ þ þ þ þ � �6 33 Liver Spindle þ þ þ þ þ þ þ þ �7 50 Liver Plump=spindle þ þ þ þ þ þ þ þ þ þ

Note. RER, rough endoplasmic reticulum; MIT–RER, mitochondria–rough endoplasmic reticulum complex.

N. P. Agaram et al. 406

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reviewed the published literature. The presence of

prominent lysosomal and phagolysosomal com-

plexes with dense inclusions was found to be very

similar to the ultrastructural findings of malignant

fibrous histiocytoma. Our study is very much in

keeping with this interpretation, since the cell consti-

tuents identified in both pediatric and adult ES

included fibroblastic, fibrohistiocytic, myofibroblas-

tic, and undifferentiated cells, similar to the ones

seen in the so-called malignant fibrous histiocytoma.

Interestingly, mitochondrial–RER complexes were

found in 3 of our cases. These structures are not cell-

type or tumor-type specific and have been described

in other tumors, including chordomas, malignant pleu-

ral mesotheliomas, parathyroid adenoma, and malig-

nant fibrous histiocytoma [8]. Although the function

of these complexes is unknown, it is speculated that

they formwhen freshRER is being synthesizedand that

the mitochondria provide energy for this process.

Our findings do not support the hypothesis put

forth by Parham et al. [15] that hepatic ES form an

ultrastructural and immunohistochemical continuum

with rhabdomyosarcoma. Their comparative study of

13 hepatic undifferentiated sarcomas along with two

rhabdomyosarcomas found overlapping features

and suggested a common histogenesis, probably

from a multipotential mesenchymal stem cell. In

our opinion, rhabdomyosarcoma has distinct immu-

nohistochemical (desmin and myogenin reactivity)

and electron microscopic (thick myosin filaments,

Z-bands, myosin-ribosome complexes) features,

which can be distinguished from the undifferentiated

phenotype of ES.

Miettinen et al. [13] described epithelial features in

2 ES tumors, supported by cytokeratin positivity, as

well as tonofilaments and desmosome-like ultra-

structurally. Most, but not all, other studies have

shown that cytokeratin is not a consistent finding in

these tumors [10, 17]. None of our cases showed

cytokeratin positivity, tonofilaments, or well-formed

junctional complexes. The presence of epithelial ele-

ments in the tumor was addressed by Stocker and

Ishak [18] in their paper. They inferred these epi-

thelial elements to be entrapped bile ducts and not

differentiated tumor components, as they are located

mainly along the periphery of the tumor in transition

with the adjacent liver and also due to the fact that

they have distinct basement membranes around

them. In our study, we found similar entrapped bile

duct components along the periphery of the tumor

nodules in half the cases.

The hypothesis of ES arising from a mesenchymal

hamartoma of the liver was first suggested by de

Chadarevian et al. [7]. In their case report, a 12-

year-old girl was diagnosed with an undifferentiated

sarcoma of the liver arising in conjunction with a

mesenchymal hamartoma. Lauwers et al. [12]

reported subsequently a case of 15-year-old girl with

an undifferentiated sarcoma of the liver arising

within a mesenchymal hamartoma. DNA ploidy

studies showed that the mesenchymal hamartoma-

like areas within the tumor were diploid as opposed

to the aneuploid tumor. Cytogenetic studies of their

tumor showed alterations of chromosome 19, which

have also been reported in mesenchymal hamar-

toma. Since then, there has been only one other case

report, by O’Sullivan et al. [14]. None of the 6 cases in

our study showed any evidence of an associated

mesenchymal hamartoma. Additional studies are

required to further confirm this association.

An undifferentiated component, with increased

cellularity and high N=C ratio, resembling a small,

blue round cell tumor was noted in 4 cases in our

study. Such foci, if present alone in a small biopsy

material, can cause diagnosis problems with other

primary hepatic tumors in the pediatric age group,

such as hepatoblastoma and rhabdomyosarcoma.

Other clinical parameters, including age and levels

of serum AFP, will be helpful in differentiating hepa-

toblastomas. Especially difficult might be the distinc-

tion from rhabdomyosarcoma, since both tumors

stain with desmin. Additional immunohistochemical

markers for myogenin or myoD1 will be helpful in

differentiating these tumors. Myogenin stain was

negative in 3 of our cases that were desmin positive.

Cytogenetic analysis of ES has revealed complex

chromosomal abnormalities. Sowery et al. [16], in a

study of 6 cases of ES by Comparative Genomic

Hybridization, showed that the pattern of chromo-

somal changes includes gains of chromosomes 1q,

5q, 6q, 8q, and 12q, and losses of chromosomes

9q, 11p, and 14.

The usual mode of spread of these tumors is either

local extension or hematogenous metastasis. The most

common sites of matastasis include lung parenchyma

and the pleural and peritoneal fluids. Local extension

into the diaphragm, abdominal organs, and lung

has been reported. Hepatectomy, followed by

407 Hepatic and Embryonal Sarcoma

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chemotherapy, is currently the most common thera-

peutic approach, with most reported studies using

the rhabdomyosarcoma chemotherapy protocol.

Since most of the reports on hepatic ES are

case reports, few studies have looked at the survival

data of patients with these tumors. In the study by Lack

et al. [11] in 1991, 13=16 patients died of disease with a

median survival of 12 months. Aoyama et al. [2] studied

8 cases and 6=8 of these cases died between 2 and 16

months after diagnosis. In a recent study by Bisogno et

al. [3], 10=17 cases were alive in follow-up ranging

from 2.4 to 20 years. In that study modern multimodal

therapy and supportive therapy has improved the sur-

vival of these tumors and the prognosis should no

longer be considered poor. In our study, 5 cases

showed no evidence of disease at follow-up duration

ranging from 6 months to 21 years.

In conclusion, hepatic ES have distinctive ultra-

structural findings, including dilated RER and dense

lysosomal precipitates, which correlate with the

eosinophilic hyaline bodies seen microscopically.

These findings suggest that ES are composed of

fibroblastic, fibrohistiocytic, and undifferentiated

cells. Both the pediatric and adult ES show similar

morphologic and ultrastructural features. With recent

multimodality therapy, these tumors seem to have a

better prognosis.

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