MolecularVariability of TLS-CHOP Structure Shows NoSigni¢cant Impact on the Level of Adipogenesis:AComparative Ultrastructural and RT-PCRAnalysis of14 Cases of Myxoid/Round Cell Liposarcomas

Hsuan-Ying Huang, MD

Department of Pathology, Chang Gung Memorial Hospital,Kaoshiung Medical Center, Kaoshiung, Taiwan; andDepartment of Pathology, Memorial Sloan-Kettering CancerCenter, New York, New York, USA

Cristina R. Antonescu, MD

Department of Pathology, Memorial Sloan-Kettering CancerCenter, New York, New York, USA.

A specific TLS-CHOP fusion gene derived from the t(12;16) ispresent in at least 95% of myxoid/round cell liposarcomas (MLS).Rare cases of MLS show a variant t(12;22) translocation, resulting inEWS-CHOP fusion gene. The CHOP gene encodes a leucine-zippertranscription factor, which is implicated in both oncogenictransformation and inhibition of adipogenesis. To examine whetherthe molecular variability of TLS-CHOP or EWS-CHOP fusiontranscript structure is associated with the degree of inhibition ofadipogenesis, a comparative ultrastructural and RT-PCR-basedanalysis of 14 MLS was performed. The specimens consisted of 9primary, 2 locally recurrent tumors, and one representative sampleeach from 3 patients with multifocal soft tissue metastases.Histologically, there were 8 high-grade and 6 low-grade MLS using5% round cell (RC) component as the cutoff point. By RT-PCR assaythere were 13 cases with TLS-CHOP fusion transcripts: 7 cases oftype 5-2 (known as type II), 4 cases of type 7-2 (known as type I),1 case of type 8-2 (known as type III), and 1 unique case of type6-2. The remaining 1 case showed an EWS-CHOP fusion transcript.Ultrastructural examination revealed that tumor cells were composedof a moderate-to-predominant proportion of well-formed lipoblasts in4 cases, while in 6 cases such lipoblasts were very scant.The remaining 4 tumors were arrested in the stage of transitional cells.The heterogeneity of TLS-CHOP fusion transcript showed noapparent impact on adipogenesis, since both TLS-CHOP type I and IIcases could randomly display various levels of lipoblasticdifferentiation. Furthermore, the 4 cases without definite lipoblastsshowed no preference for any specific fusion variants and consistedof one each of TLS-CHOP subtypes. In addition, the fusion transcriptvariants did not correlate with other ultrastructural features, such asthe presence and amount of glycogen, mitochondria, roughendoplasmic reticulum, vimentin-like intermediate filaments, andexternal lamina. However, there appeared to have a trend suggestingthe predilections of glycogen particles and vimentin-like intermediatefilaments in primitive mesenchymal cells and/or transitional cells.These findings cannot substantiate the hypothesis that the molecularvariability of fusion transcripts has a biological impact onadipogenesis of MLS, and other factors might be implicated in theirlevel of differentiation.

Keywords adipogenesis, EWS-CHOP, glycogen, RT-PCR, TLS-CHOP,vimentin

Myxoid round cell liposarcoma (MLS) is the mostcommon subtype of malignant adipose-tissue tumors,typically presenting as a deeply located mass in theextremities of young adults. The tumor is composedhistologically of various cell types having differentdegrees of maturation in adipogenic process, rangingultrastructurally from undifferentiated mesenchymalcells through transitional cells with scant lipid dropletsto mono-vacuolated signet ring-like lipoblasts set

Received18 November 2002; accepted 24 January 2003.

This study was performed while Dr. Hsuan-Y|ng Huang worked as a visiting

research fellow at Memorial Sloan-Kettering Cancer Center.

We thank Ann Baren and Elizabeth Weiss for technical assistance with ultra-

structural analysis.

Address correspondence to Dr. Cristina R. Antonescu, MD, Department

of Pathology,Memorial Sloan-Kettering Cancer Center,1275,York Avenue,New

York,NY10021,USA.E-mail: antonesc@mskcc.org

Ultrastructural Pathology, 27:217–226, 2003

Copyright # Taylor & Francis Inc.

ISSN: 0191-3123 print/1521-0758 online

DOI: 10.1080/01913120390225426

217

Ultr

astr

uct P

atho

l Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ritis

h C

olum

bia

on 1

0/29

/14

For

pers

onal

use

onl

y.

within a myxoid matrix. A proportion of MLS casesshow histological progression to round cell (RC)histology, associated with a significantly poor prog-nosis [1, 2]. The molecular events associated witht(12;16) result in the chimeric fusion of the CHOP geneat 12q13 and FUS/TLS gene at 16p11 and the gen-eration of TLS � CHOP hybrid gene, which encodes anaberrant transcriptional regulator. Similar to otherrearrangements producing specific gene fusions, thegenomic breakpoints of the t(12:16) are widely dis-persed in specific introns of the TLS and CHOP genesand vary from one tumor to another [2^6]. To date, 9variants of TLS-CHOP chimeric transcripts have beenidentified in the MLS [7], most of which were gen-erated through the fusion of exons 7, 5, or 8,respectively, of TLS with exon 2 of CHOP [5^7]. Inaddition, a handful of MLS exhibit the variant trans-location involving 12q13 and 22q11-12, leading to thevariant EWS-CHOP gene fusion [8, 9]. In a recentstudy, we found that the molecular variability of fusiontranscripts in MLS appeared not to have significantprognostic impact, as opposed to some othertranslocation-associated sarcomas [2].

The CHOP gene encodes a leucine-zipper trans-cription factor belonging to the CCAAT/enha-ncer-binding protein (C/EBP) family implicated in theadipogenesis and growth arrest [3, 5, 10, 11]. Kurodaet al. has reported that TLS/FUS-CHOP type IIchimeric protein played critical roles in both

oncogenic transformation and inhibition of adipocyticconversion of preadipocytes in murine cell line [5].To our knowledge, no systematic study hasaddressed whether the different structure of thefusion partners of CHOP gene in MLS has anyimpact on the degree of adipogenesis. In the currentstudy, we sought to investigate this issue bycorrelating the ultrastructural findings and moleculargenetic variants of 14 cases of MLS, with specialemphasis on adipocytic cell maturation.

MATERIALS AND METHODS

Study Group and Case MaterialAmong the cases of MLS operated at Memorial

Sloan-Kettering Cancer Center that were previouslyconfirmed to harbor CHOP gene rearrangement bySouthern blotting or t(12;16) by conventionalcytogenetics [2, 3], we identified 14 cases withavailable tumor tissue for molecular analysis and fixedin glutaraldehyde for electronmicroscopic examination.Clinical parameters, including gender, age, location,and size, were obtained from medical chart records.In all 14 cases hematoxylin^eosin stained slides fromthe corresponding en bloc resection specimen wereavailable for pathologic review, and on average 1block/cm3 of tumor was submitted for histological

FIG. 1 Ultrastructural appearance of a primitive undifferentiated mesenchymal cell (case 4, type 5-2 fusion)showing a moderate amount of glycogen particles and mitochondria in the cytoplasm with discontinuousexternal lamina (arrows). Fat droplets were absent in such cells. Original magnification, �3,175.

218 H.-Y. Huang and C. R. Antonescu

Ultr

astr

uct P

atho

l Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ritis

h C

olum

bia

on 1

0/29

/14

For

pers

onal

use

onl

y.

examination in each case. The tumors were scored forthe percentage of round cell (RC) component, whichwas estimated by scanning all individual sections, byusing the entire tumor volume as denominator [2].

Ultrastructural ExaminationFor ultrastructural analysis, fresh tumor tissue was

immersed in 2% glutaraldehyde, postfixed in 1%osmium tetroxide, dehydrated in graded ethanol,embedded in epoxy resin, and stained with uranylacetate^lead citrate using standard protocol. Thicksections were cut and stained with toluidine blue ineach case to select suitable areas for ultrastructuralevaluation under a Philips 410 transmission electronmicroscope. To interpret the ultrastructural findingsobjectively, we prospectively scoped these 14 cases inblind fashion without prior knowledge of fusiontranscript types and histological features. Since therehas been a lack of standardization among variousultrastructural studies in terminology and criteria usedfor the morphologic definition of lipogenic cells, weadopted Lagace’s method to classify the constituentcells of MLS into 3 categories: the undifferentiatedmesenchymal cells,the transitional or intermediate cells,and definite lipoblasts [12]. The undifferentiatedmesenchymal cells were characterized byround-to-oval nuclei, high nuclear^cytoplasmic ratio,and minimal cytoplasmic differentiation (Figure 1). Thekey feature of definite lipoblasts was the cytoplasmicaccumulation of many multivesicular lipid droplets orlarge confluent fat vacuoles that indented or pushedaside the nuclei (Figure 2). The transitional or inter-

mediate tumor cells had features between the former2 with only scant inconspicuous lipid vacuoles(Figure 3). The following ultrastructural features wereanalyzed for various cell types: the stages of lipogenicdifferentiation, their relative proportion in each tumorexamined, the amount and distribution of glycogen,vimentin-like intermediate filaments (IFs), and roughendoplasmic reticulum (RER) as well as the presenceor absence of external lamina and pinocytotic vesicles.At least 100 tumor cells were examined in each case,and the aforementioned variables were determinedsemiquantitatively by using the following classifyingscheme:7 , absent; þ, scant or inconspicuous;þþ ,moderate;þþþ , many or predominant.

Reverse Transcriptase^Polymerase ChainReaction (RT-PCR) for TLS-CHOP andEWS-CHOP

Tumor samples for molecular assay weresnap-frozen in liquid nitrogen and stored at770�C.Analysis by RT-PCR for TLS-CHOP and if indicated forEWS-CHOP transcripts was performed as previouslydescribed [2, 3]. In brief, extraction of total RNA wasbased on the guanidinium isothiocyanate^phenolchloroform method using the RNA Wiz reagent(Ambion, Austin, TX). The adequacy of extracted RNAwas assessed by RT-PCR, using primers forphosphoglycerate kinase transcripts [2]. Negativecontrols that lacked either tumor RNA or reversedtranscriptase were used in parallel. Three micrograms oftotal RNA were subjected to RT-PCR with QiagenOne-Step RT-PCR kit (Qiagen, Valencia, CA), and the

FIG. 2 Ultrastructural appearance of well-formed lipoblasts (case 10, type 7-2) showing numerous microvesiculardroplets indenting (left) or pushing aside (bottom) the nuclei. A moderate amount of mitochondria wasfound in the cytoplasm, whereas vimentin-like filaments and glycogen particles were lacking or scant.External lamina was seen focally surrounding the cell surfaces (arrows). Original magnification, �1,400.

TLS-CHOP Structure and Adipogenesis 219

Ultr

astr

uct P

atho

l Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ritis

h C

olum

bia

on 1

0/29

/14

For

pers

onal

use

onl

y.

FIG. 3 The transitional stage cells were distinguished from the undifferentiated mesenchymal cells by the presenceof scant small lipid droplets (A) Low-power view (case 12, type 5-2 fusion) showing a cluster oftransitional cells loaded with bundles of vimentin-like filaments and a moderate amount of mitochondria.No well-formed lipoblasts were seen elsewhere in this case, which was ‘‘arrested’’ in the process oflipogenesis. Original magnification, �1,741. (B) High-power view of a transitional cell (case 14, type7-2) displaying a moderate amount of vimentin filaments and scant glycogen. Original magnification�4,133. (C) A transitional cell of another case (case 7, type 5-2) showing discontinuous external lamina(arrowheads) and pinocytotic vesicles (arrows). Original magnification, �4,125.

220 H.-Y. Huang and C. R. Antonescu

Ultr

astr

uct P

atho

l Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ritis

h C

olum

bia

on 1

0/29

/14

For

pers

onal

use

onl

y.

following primer sets: exon 5 of TLS forward (5’-CAGCCA GCA GCC TAG CTA TG-‘3) or exon 7 of EWSforward (5’-CTG GAT CCT ACA GCC AAG CTC CAAG-‘3) and exon 3 of CHOP reverse (5’-TGT CCC GAAGGA GAA AGG CAA TG-3’). The Qiagen 1 StepRT-PCR conditions were carried out at 50�C for 30min, 95�C for 15 min, 35 cycles of 95�C for 45 s, 64�Cfor 45 s, 72�C for 1 min, and final extension at 72�C for7 min. The RT-PCR products were identified byagarose gel electrophoresis. The expected sizes were250 bp for type 5-2, 526 bp for type 7-2, 625 bp fortype 8-2 TLS-CHOP, and 179 bp for EWS-CHOP type7-2 fusion transcripts. All non-type 5-2 transcriptswere verified by automatic direct sequencing.

RESULTS

Clinical FindingsSalient clinical features of the 14 cases are

summarized in Table 1. There were 11 males and 3females. The age at the first presentation ranged from13 to 68 years (mean, 42.1 years; median, 36 years).Seven patients (50%) were younger or equal to 35years when first seen. In 9 cases the tissue available forboth ultrastructural examination and molecular assaywas obtained from primary tumor specimens, all ofwhich arose from the lower extremity or buttock. Of theremaining 5 cases, one each was selected from 2patients with locally recurrent tumors occurring in thethigh and retroperitoneum, respectively, and from 3with either synchronous or metachronous multifocalsoft tissue metastases at presentation (Table 1). Themean and median sizes of tumors examined were 16.1and 15 cm, respectively (range, 5^30 cm). Except for 1case with synchronous multicentric lesions (case 5),the other 13 patients included in this study did notundergo chemotherapy prior to the surgical resection oftumors.

The Correlations Between FusionTranscript Variants and MorphologicalFeatures

Table 2 summarizes the RT-PCR results, the per-centage of RC component, and electron microscopicfindings of 14 cases of MLS.

RT-PCR ResultsAlthough the case number was quite limited in the

current study, the distribution of fusion variants ofTLS-CHOP chimeric transcripts was generally inkeeping with our recent large-scale study, whichsubstantiated that the current study group did notconsist of a skewed population. The most commonTLS-CHOP fusion type was 5-2, identified in 7 cases(50%), followed by 7-2 in 4 cases (28.6%) (Figure 4).In addition, TLS-CHOP fusion type 8-2, TLS-CHOPtype 6-2, and EWS-CHOP type 7-2 were found in 1case each, respectively. Direct sequencing of theTLS-CHOP type 6-2 case revealed a unique fusiontranscript, joining the 5’ portion of exons of TLS of exon2 of CHOP. The TLS break was located within exon 6(position 733), as previously reported (2). TheEWS-CHOP type 7-2 case showed a 179-bp productby direct sequencing.

Microscopic Findings and the Correlation withFusion Transcript Variants

All 14 tumors revealed the characteristic appearanceof MLS with tumor cells at different stages oflipogenesis as well as delicate plexiform capillarynetworks set within myxoid stroma, Eight cases,including 4 cases showing TLS-CHOP type 5-2, 3showing TLS-CHOP type 7-2, and 1 with EWS-CHOPtype 7-2, were classified as high grade, using the cutoffpoint of 5% of RC component, which had beenvalidated as a strong prognostic predictor in our prior

TABLE 1 Clinical Features of 14 Myxoid/Round Cell Liposarcomas

Case no. Sex Age at first presentationLocation of tumor for EMexamination T|ssue sample Size (cm)

1 M 29 Right thigh Primary 62 M 33 Right thigh Metastasesa 143 M 35 Thigh 1st recurrence 124 M 33 Retroperitoneum 1st recurrence 235 F 61 Right thigh Metastasesa,b 126 M 31 Left buttock Primary 237 M 37 Left thigh Primary 158 F 32 Intraabdominal Metastasesa 309 F 63 Right thigh Primary 1510 M 42 Right thigh Primary 1911 M 68 Right thigh Primary 10.212 M 13 Left thigh Primary 513 M 65 Right calf Primary 2014 M 47 Left calf Primary 21

aMulticentric somatic soft tissuemetastases.bStatus post preoperative chemotherapy.

TLS-CHOP Structure and Adipogenesis 221

Ultr

astr

uct P

atho

l Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ritis

h C

olum

bia

on 1

0/29

/14

For

pers

onal

use

onl

y.

TABLE

2Histo

logical

Fea

ture

s,Ultra

stru

ctura

lFindings,

and

Fusion

Tra

nsc

riptSubty

pes

of14

Myx

oid/R

ound

CellLiposa

rcomas

Case

no.

Rou

ndcell

compon

ent

(%)

Fusion

tran

scrip

tsubtype

Prim

itive

cells

Interm

ediate

cells

Definite

lipob

lasts

Am

IFGly

Mt

RER

ELAm

IFGly

Mt

RER

ELAm

IFGly

Mt

RER

ELAdd

ition

alfeatures

10

8-2

3þ

2þ

1þ

2þ

2þ

71þ

1þ

1þ

2þ

1þ

77

77

77

7Den

segranules

220

7-2

3þ

1þ

1þ

2þ

2þ

1þ

2þ

71þ

2þ

2þ

71þ

77

1þ

1þ

7Celljunctions

30

6-2

3þ

72þ

2þ

2þ

71þ

71þ

2þ

2þ

77

77

77

74

05-2

2þ

2þ

2þ

2þ

1þ

1þ

1þ

77

2þ

77

2þ

77

1þ

1þ

75

25

5-2

2þ

2þ

2þ

2þ

1þ

72þ

1þ

1þ

1þ

1þ

71þ

77

1þ

77

Onionskin-likevessels

610

7-2

3þ

2þ

2þ

2þ

2þ

1þ

1þ

2þ

2þ

1þ

1þ

77

77

77

77

05-2

2þ

2þ

1þ

2þ

2þ

1þ

2þ

2þ

2þ

2þ

1þ

71þ

1þ

72þ

1þ

1þ

Pinocy

tosis

830

EWS-C

HOP

2þ

71þ

1þ

1þ

1þ

2þ

71þ

2þ

1þ

72þ

77

2þ

1þ

7Celljunctions

935

5-2

1þ

71þ

77

71þ

1þ

1þ

2þ

1þ

1þ

3þ

77

2þ

1þ

2þ

10

<5

7-2

1þ

71þ

77

71þ

1þ

1þ

2þ

1þ

72þ

71þ

2þ

1þ

1þ

11

40

5-2

1þ

2þ

1þ

2þ

1þ

73þ

2þ

2þ

2þ

1þ

1þ

1þ

71þ

2þ

1þ

712

05-2

2þ

2þ

2þ

1þ

1þ

72þ

2þ

2þ

2þ

1þ

77

77

77

713

10

5-2

2þ

71þ

2þ

1þ

72þ

2þ

1þ

1þ

1þ

71þ

71þ

2þ

71þ

Pinocy

tosis

14

60

7-2

2þ

2þ

77

77

2þ

2þ

1þ

2þ

77

1þ

77

77

7

Note.Am,relativeam

ount

ofind

ividua

lcelltyp

e;IF,vim

entin

-likeinterm

ediatefilam

ents;G

ly,glyco

genco

nten

t;Mt,mito

chon

dria;R

ER,rou

ghen

doplasmicreticulum

,EL,externa

llam

ina;

7,ab

sent;þ,

scan

torinc

onspicuou

s;þþ,m

ode

rate;þþþ,m

anyorp

redo

minan

t.

222

Ultr

astr

uct P

atho

l Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ritis

h C

olum

bia

on 1

0/29

/14

For

pers

onal

use

onl

y.

study [2]. The percentage of RC component in these 8high-grade cases ranged from 10 to 40%, and genuinelipoblasts were rarely found in the high-grade RCcomponent. The RT-PCR results of the remaining 6low-grade cases were 3 TLS-CHOP type 5-2transcripts and 1 each of TLS-CHOP type 7-2, type 6-2,and type 8-2 transcripts. The amount and maturation oflipoblasts varied in both low-grade MLS and non-RCareas of high-grade cases, ranging from a pre-dominance of banal plump spindly mesenchymal cellswith inconspicuous adipogenesis to fully blownsignet-ring lipoblasts. No histological features over-lapping with either well-differentiated or pleomorphictypes of liposarcomas were observed in any case,including 1 case occurring primarily in the retro-peritoneum (Table 1).

Ultrastructural Findings and the Correlations withFusion Transcript Variants

At the ultrastructural level, all 14 cases showedvarying amounts of primitive mesenchymal cells(Figure 1) and transitional lipogenic cells withinconspicuous lipid droplets (Figure 3), while therewere 4 cases exhibiting an arrest of adipogenesis at thetransitional cell stage without any definite lipoblastsafter exhaustive search (Figure 3A). In the remaining10 cases having definite lipoblasts, 4 contained amoderate-to-predominant proportion of such lipogeniccells characterized by multivesicular lipid droplets orlarge confluent signet ring-like fat vacuoles (Figure 2),and another 6 cases revealed only an inconspicuous

percentage of well-formed lipoblasts. From theperspective of fusion transcript structures, the mol-ecular heterogeneity among various subtypes appearedto have no apparent impact on the maturation oflipoblasts. Except for a single EWS-CHOP caseshowing a moderate amount of definite lipoblasts,those tumors with only scant definite lipoblasts orarrested in the intermediate stage of adipogenesis couldoccur in all TLS-CHOP fusion transcript subtypes.Furthermore, the cases belonging to the 2 major fusiontranscript variants, TLS-CHOP type 5-2 and type 7-2,had a rather random distribution in regard to the extentof lipogenesis. For instance, a moderate-to-predominant proportion of definite lipoblasts wasobserved in 2 out of 7 TLS-CHOP type 5-2 cases, scantdefinite lipoblasts were found in four, and no definitelipoblast was seen in one. Likewise, regarding therelatively amounts of definite lipoblasts in the 4TLS-CHOP type 7-2 cases, one of them was moderate,two displayed a scant proportion, and another one wascompletely absent.

Similarly, no apparent correlations were observedbetween the subtypes of fusion transcript variants andother ultrastructural features, including the presenceand amount of glycogen, mitochondria, RER,vimentin-like IFs, and external lamina. Nevertheless,there was a trend toward an association between thelevel of lipoblastic differentiation and the relativelyamount of some cytoplasmic contents, such as thevimentin-like IFs and glycogen. Irrespective of fusiontranscript structures, vimentin-like IFs (Figure 3) andglycogen particles (Figures 1, 3B, C) were frequentlyencountered within the cytoplasm of primitivemesenchymal cells or transitional cells or both in themajority of all 14 cases. By contrast, among 10 tumorsdifferentiating into the stage of definite lipoblasts, therewas only 1 case (TLS-CHOP type 5-2) showing a scantamount of intermediate filaments and 3 cases(TLS-CHOP type 5-2, n¼2;TLS-CHOP type 7-2,n¼1) having glycogen particles appearing in thedefinite lipoblasts. With one exception (case 14), themitochondria (Figures 1, 2, 3A, B) and RER werenearly consistently present in each case and could berandomly appearing in any stage of lipoblasticdifferentiation. Although external laminae could beobserved in 9 out of 14 cases (64.3%), they weregenerally focal, discontinuous, and related to neitherthe fusion variants nor the level of adipocytic differ-entiation (Figure 1, 2, 3C). Other miscellaneousultrastructural findings were only occasionallyobserved, including micropinocytotic activitiesappearing in 2 TLS-CHOP type 5-2 cases, occasionalrudimentary cellular junctions present in 1 each ofTLS-CHOP type 5-2, TLS-CHOP type 7-2, andEWS-CHOP type 7-2 cases, and intracytoplasmicelectron-dense granules in 1 TLS-CHOP type 8-2 case.The capillaries observed in the majority of MLS wereultrastructurally unremarkable. However, one tumorundergoing preoperative chemotherapy displayedpericapillary proliferation of spindle cells with onionskin-appearing concentric array (Figure 5).

FIG. 4 Detection of TLS-CHOP transcript byRT-PCR. M1, size marker (PhiX174RFDNA/HaeIII); II, type TLS-CHOP 5-2 fusion(type II, 250 bp, case 4); I, type TLS-CHOP7-2 fusion (type I, 526 bp, case 2); III, typeTLS-CHOP 8-2 fusion (type III, 625 bp, case1). þ /7 , with/ without reversetranscriptase.

TLS-CHOP Structure and Adipogenesis 223

Ultr

astr

uct P

atho

l Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ritis

h C

olum

bia

on 1

0/29

/14

For

pers

onal

use

onl

y.

DISCUSSIONThe consistency and specificity of TLS-CHOP

rearrangement as a genetic hallmark of MLS, as well asthe notion that cases with a RC component belong tothe MLS category, have been well documented. Theextensive homology between TLS and EWS suggestedthat the 2 genes are closely related and may haveoriginated from a common ancestor gene. Therefore, itis not surprising that in rare cases of MLS, the EWSgene is an alternative translocation partner of CHOP [2,3, 5^9, 13].

Prior investigations documented that in MLS thejuxtaposition of an effector domain from TLS or EWSRNA-binding proteins to the targeting domain ofCHOP leads to the creation of a potent oncogene [4^7,9]. This oncogenic effect is mediated not only byaberrantly strong transcriptional activity but also by thedysregulation of normal CHOP function to inducegrowth arrest at G1/S checkpoint [2, 3, 14]. Althoughat least 9 variants of TLS-CHOP chimeric transcriptshave been reported to date in the MLS [7], most ofthem are generated through the fusion of exons 7,5 or8, respectively, of TLS with exon 2 of CHOP.Nevertheless, in our recent large series study, themolecular variability of fusion transcripts in MLS didnot carry a significant impact on either histologic gradeor disease-free survival, even though an associationwas found between the P53 status and type 5-2 fusion[2].

Adipogenesis is a process in which undifferentiatedmesenchymal cells are able to mature into postmitotic,fat-laden adipocytes. This differentiation process

results in dramatic changes in gene expression and aspectacular alteration in cell morphology. The earlyphase of adipocytic differentiation is accompanied bythe induction of transcriptional factors that promoteactivation or inhibition of downstream cell-specificgenes [10, 11]. Interestingly, the CHOP(C/EBPhomologous protein) gene is one of the severaldownstream negative regulators of adipogenesis [11,15]. Previous in vitro studies also indicated thatTLS-CHOP oncoprotein, homologous toCCAAT/enhancer binding (C/EBP) protein family, wasextremely effective at preventing adipocyte conversionfrom preadipocytes by directly interfering with C/EBPb-driven lipogenesis [5,10]. In the current study, wesought to determine whether there are different levelsof adipocytic differentiation among MLS with varioustypes of fusion or translocation variants. We thereforeultrastructurally analyzed a series of 14 MLS withregard to the relative proportions of primitivemesenchymal cells, transitional cells, and definitelipoblasts of each case in a semiquantitative manner.However, several lines of evidence argue against anassociation between the molecular heterogeneity andthe degree of adipogenesis. Firstly, theMLS included inthis study comprised a representative population offusion subtypes, with TLS-CHOP type 5-2 (50%) andtype 7-2(28.6%) being the 2 most prevalent variants, afinding in concordance with data from our previouslarge-scale molecular analysis of genetic heterogeneityof TLS-CHOP fusion transcripts. Secondly, the casesbelonging to the 2 major fusion transcript variants,TLS-CHOP type 5-2 and type 7-2, displayed a rather

FIG. 5 The only case receiving preoperative chemotherapy (case 5, type 5-2) showing vascular reactioncharacterized by concentric array of proliferative spindle mesenchymal cells surrounding a capillary.Original magnification, �4,125.

224 H.-Y. Huang and C. R. Antonescu

Ultr

astr

uct P

atho

l Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ritis

h C

olum

bia

on 1

0/29

/14

For

pers

onal

use

onl

y.

random distribution in regard to the extent oflipogenesis, ranging from absent to having a scantpercentage, to having a moderate or conspicuousamount of definite lipoblasts. Thirdly, it was possiblefor all TLS-CHOP fusion subtypes to occur in thetumors with only scant definite lipoblasts or arrested inthe intermediate stage of adipogenesis. These findingswould suggest that within TLS-CHOP, TLS exons 6-8do not play a critical role from the perspective oflipogenic function.

Vimentin filaments provide an intracellular functionthat influences the formation and maintenance of lipiddroplets in adipocytic conversion [16^18]. Franke et al.have shown in murine 3T3-L1 cells that during thisprocess, the extended fibrillar organization of vimentinIFs in undifferentiated cell was altered and thesefilaments were reorganized to surround the nascentlipid droplets, forming a complex cage with amonolayer of regularly spaced vimentin IFs [16]. Inaddition, using both anti-filament antibodies andtransfection of mutant vimentin cDNA in these cells,Lieber and Evans also found that perturbation of theorganization of vimentin IFs appeared to significantlyinhibit the formation of lipid droplets [18]. Ourultrastructural observations of vimentin IFs in MLSwere conceptually in keeping with the results ofabove-stated studies since we found a trend indicatinga preponderance of the linear fibrillar vimentin IFsappearing in either primitive or transitional cells.Conversely, the definite lipoblasts with larger fatvacuoles might encompass the vimentin IFs mostlyarranged in the form of complex cages, which werehardly amenable to being seen clearly without suf-ficient magnification [16, 17]. However, the types ofTLS-CHOP fusion transcripts appeared to have nosignificant association with the vimentin IFs in ourstudy. This suggested that the molecular heterogeneityof fusion genes in MLS did not play a crucial role in thespatial organizing dynamics of vimentin IFs implicatedin the lipid droplets accumulation.

There seems to be a close relationship between thepresence of glycogen and the production of lipid in thedeveloping lipoblasts [19^21]. However, the questionof whether the amount of glycogen is significantlyassociated with the level of differentiation of adipocyticcells remained controversial. Using tissue culture toinvestigate the growth behavior of liposarcoma, Tardioet al. found that the cytoplasm of tumor cells becameprogressively loaded with glycogen and followed byaccumulation of lipid droplets, which was generallyseen in all types of liposarcoma with minimal variation[19]. Their findings were in accordance with ourultrastructural observation of MLS displaying apreference of glycogen particles in primitive as well astransitional cells, although Fu et al. demonstratedabundant glycogen in mid-stage lipoblasts but foundvery little in precursor cells and transitional cells [21].In the current study, there is no apparent correlationbetween the molecular heterogeneity and the presenceand amount of glycogen, indicating that even ifglycogen serving as an indispensable factor during

adipogenesis, its metabolism and activation of per-tinent enzymes are not directly influenced byTLS-CHOP structure.

The other ultrastructural features, includingmitochondria, RER, and external lamina, were related toneither the types of chimeric fusion transcripts nor thelevel of adipocytic cell maturation. Only two MLS inthis series showed evidence of increased pinocyticactivity, which was low in incidence as compared toprevious studies [12, 20, 22]. In summary, we havereported a series of 14 MLS and correlated theultrastructural findings with RT-PCR results.Nevertheless, neither the level of adipogenesis nor thedistribution and amount of cytoplasmic contents wasinfluenced by the genetic heterogeneity of chimericfusion transcripts.

REFERENCES1. Mentzel T, Fletcher CD. Lipomatous tumours of soft tissues: an

update. Virchows Arch. 1995;427:353^363.2. Antonescu CR, Tschernyavsky SJ, Decuseara R, et al. Prognostic

impact of P53 status, TLS-CHOP fusion transcript structure, andhistological grade in myxoid liposarcoma: a molecular andclinicopathologic study of 82 cases. Clin Cancer Res.2001;7:3977^3987.

3. Antonescu CR, Elahi A, Humphrey M, et al. Specificity ofTLS-CHOP rearrangement for classic myxoid/round cellliposarcoma: absence in predominantly myxoidwell-differentiated liposarcomas. J Mol Diagn. 2000;2:132^138.

4. Knight JC, Renwick PJ, Cin PD, Van den Berghe H, Fletcher CD.Translocation t(12;16)(q13;p11) in myxoid liposarcoma andround cell liposarcoma: molecular and cytogenetic analysis.Cancer Res. 1995;55:24^27.

5. Kuroda M, Ishida T, Horiuchi H, et al. Chimeric TLS/FUS-CHOPgene expression and the heterogeneity of its junction in humanmyxoid and round cell liposarcoma. Am J Pathol.1995;147:1221^1227.

6. Panagopoulos I, Mandahl N, Ron D, et al. Characterization of theCHOP breakpoints and fusion transcripts in myxoid liposarcomaswith the 12;16 translocation. Cancer Res. 1994;54:6500^6503.

7. Panagopoulos I, Mertens F, Isaksson M, Mandahl N. A novelFUS/CHOP chimera in myxoid liposarcoma. Biochem BiophysRes Commun. 2000;279:838^845.

8. Dal Cin P, Sciot R, Panagopoulos I, et al. Additional evidence of avariant translocation t(12;22) with EWS/CHOP fusion in myxoidliposarcoma: clinicopathological features. J Pathol.1997;182:437^441.

9. Panagopoulos I, HoglundM,Mertens F, mandahl N, Mitelman F,Aman P. Fusion of the EWS and CHOP genes in myxoidliposarcoma. Oncogene. 1996;12:489^494.

10. Adelmant G, Gilbert JD, Freytag SO. Human translocationliposarcoma-CCAAT/enhancer binding protein (C/EBP)homologous protein (TLS-CHOP) oncoprotein preventsadipocyte differentiation by directly interfering with C/EBPbetafunction. J Biol Chem. 1998;273:15,574^15,581.

11. Tang QQ, Lane MD. Role of C/EBP homologous protein(CHOP-10) in the programmed activation ofCCAAT/enhancer-binding protein-beta during adipogenesis.Proc Natl Acad Sci USA. 2000;97:12,446^12,450.

12. Lagace R, Jacob S, Seemayer TA. Myxoid liposarcoma. Anelectronmicroscopic study: biological and histogeneticconsiderations. Virchows Arch A Pathol Anat Histol.1979;384:159^172.

13. Antonescu CR, Elahi A, Healey JH, et al. Monoclonality ofmultifocal myxoid liposarcoma: confirmation by analysis ofTLS-CHOP or EWS-CHOP rearrangements. Clin Cancer Res.2000;6:2788^2793.

TLS-CHOP Structure and Adipogenesis 225

Ultr

astr

uct P

atho

l Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ritis

h C

olum

bia

on 1

0/29

/14

For

pers

onal

use

onl

y.

14. Kuroda M, Ishida T, Takanashi M, Satoh M, Machinami R,Watanable T. Oncogenic transformation and inhibition ofadipocytic conversion of preadipocytes by TLS/FUS-CHOP typeII chimeric protein. Am J Pathol. 1997;151:735^744.

15. Batchvarova N, Wang XZ, Ron D. Inhibition of adipogenesis bythe stress-induced protein CHOP (Gadd 153). EMBO J.1995;14:4654^4661.

16. Franke WW, Hergt M, Grund C. Rearrangement of the vimentincytoskeleton during adipose conversion: formation of anintermediate filament cage around lipid globules.Cell.1987;49:131^141.

17. Wang SM, Fong TH, Hsu SY, Chien CL, Wu JC. Reorganizationof a novel vimentin-associated protein in 3T3-L1 cells duringadipose conversion. J Cell Biochem. 1997;67:84^91.

18. Lieber JG, Evans RM. Disruption of the vimentin intermediatefilament system during adipose conversion of 3T3-L1 cellsinhibits lipid droplet accumulation. J Cell Sci.1996;109:3047^3058.

19. Tardio J, Escalona-Zapata J, Diez-Nau DD. The behaviour ofliposarcoma in tissue culture: a specific growth pattern. J Pathod.1993;170:451^456.

20. Rossouw DJ, Cinti S, Dickersin GR. Liposarcoma. Anultrastructural study of 15 cases. Am J Clin Pathol.1986;85:649^667.

21. Fu YS, parker FG, Kaye GI, Lattes R. Ultrastructure of benign andmalignant adipose tissue tumors. Pathol Annu. 1980;15:67^89.

22. Vuzevski VD, van der Heul RO. Comparative ultrastructure ofsoft-tissue myxoid tumors. Ultrastruct Pathol. 1988;12:87^105.

226 H.-Y. Huang and C. R. Antonescu

Ultr

astr

uct P

atho

l Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f B

ritis

h C

olum

bia

on 1

0/29

/14

For

pers

onal

use

onl

y.