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From the Departments of Pathology
*
and Surgery,
Memorial Sloan-Kettering Cancer Center, New York; and Ludwig Institute for Cancer Research,
New York, New York
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unlike melanomas, most CCS are characterized cytogenetically by a recurrent chromosomal translocation, t(12;22)(q13;q12), resulting in fusion of the EWS gene on 22q12 with the ATF1 gene on 12q13.12 In the resulting chimeric protein, the C-terminal of EWS, which contains an RNA binding domain, is replaced by a functional bZIP DNA-binding and dimerization domain of ATF1, a transcription factor which is normally regulated by cAMP.13, 14 EWS-ATF1 binds to ATF sites in cAMP-responsive promoters through the bZIP domain derived from ATF1 and its function as a constitutive transcriptional activator is dependent on an activation domain within EWS containing repetitive elements.15 EWS-ATF1 functions as a potent constitutive activator of several cAMP-inducible promoters when assayed by transfection in cells lacking the EWS-ATF1 fusion.13, 14 However, the in vivo targets of EWS-ATF1 in CCS cells still remain unclear.16 Nonetheless, it appears that the viability of CCS cells is dependent on the activity of EWS-ATF1, based on a study using intracellular anti-ATF1 antibody.17 Recent data suggest that EWS-ATF1 may operate at least partly by interfering with P53 function, through competition with the latter for the transcriptional coactivator CBP.18
Since most of the genetically confirmed CCS have been studied by conventional karyotyping and published as isolated case reports in the cytogenetic or molecular diagnostic literature,19, 20, 21, 22, 23, 24, 25 the exact prevalence of t(12;22)-positive cases as confirmed by molecular techniques among cases histologically diagnosed as CCS remains uncertain. In this study, we assessed the prevalence of EWS-ATF1 fusion as detected by reverse transcriptase polymerase chain reaction (RT-PCR) in a group of 12 pathologically confirmed cases of CCS. In addition, we present for the first time an RT-PCR assay for the detection of EWS-ATF1 transcripts applicable to tumor RNA extracted from formalin-fixed paraffin-embedded material and we show that CCS expresses the melanocyte-specific form of the MITF transcription factor, further supporting its melanocytic differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Criteria for the microscopic diagnosis of CCS included the presence of polygonal cells with abundant clear to pale eosinophilic cytoplasm, vesicular nuclei with prominent nucleoli, and arrangement in a nested pattern. The cases were assessed for the presence of melanin pigment, multinucleated giant cells, and also for the presence of an association with tendon or aponeurotic structures.
Immunohistochemical studies for S-100 protein [Biogenex, San Ramon, CA; 1:50,000; citrate buffer] and HMB45 [DAKO, Carpinteria, CA; 1:500; citrate buffer] were done in all cases. Immunostaining with D5 (Labvision, Fremont, CA), a monoclonal antibody generated against human MITF, was also performed in all 12 cases. The D5 antibody binds a portion of MITF encoded by both melanocyte-specific (MITF-M) and non-melanocyte-specific isoforms of MITF transcripts.11, 26
Ultrastructural examination was performed on seven tumors. Representative fresh tumor tissue was fixed in 2% glutaraldehyde, postfixed in 1% osmium tetroxide, and embedded in epoxy resin using standard procedures.
Molecular Analysis
Frozen tissue, collected under an Institutional Review
Board-approved protocol, was available in 4 of the 12 cases (CCS
1, 8–10), and represented primary (CCS 8 and 10) or metastatic (CCS 1
and 9) tumor samples. Tumors were snap-frozen and stored at -70°C.
Total RNA was extracted using 1 ml of Trizol reagent (Gibco BRL Inc.,
Gaithersburg, MD). The extraction was carried out according to
instructions supplied by the manufacturer.
In the remaining eight cases, RNA was extracted from a representative paraffin block containing formalin-fixed tumor tissue. In four cases (CCS 3, 4, 6, and 11) the block available for RNA extraction was from the primary tumor, in one case (CCS 5) from a local recurrence, and in three cases (CCS 2, 7, and 12) from a metastasis. The extraction was performed as directed by the manufacturer (Paraffin Block RNA Isolation Kit; Ambion, Inc., Austin, TX) with several alterations. In an effort to yield more RNA from the extraction, four 15-um sections were cut and proteinase K was used in twice the suggested volume (200 µl) for 25 minutes. To this, 2 µl of linear acrylamide were added followed by precipitation overnight at -20°C. Every effort was taken to prevent cross-contamination. For each case, a new microtome blade was used and the microtome was wiped with 10% bleach and 70% ethanol.
In all samples, RT-PCR was first performed with the outer pair of
primers consisting of a sense primer located in exon 8 of
EWS and an antisense primer in ATF1 (codon 130).
All primer sequences, annealing temperatures used, and expected product
sizes are shown in Table 1
. The reaction was carried out using the One-Step RT-PCR kit (Qiagen,
Inc., Valencia, CA) at 50°C for 30 minutes, 95°C for 15 minutes, 30
cycles of 95°C for 30 seconds, 58°C for 30 seconds, 72°C 30
seconds, and a final extension at 72°C for 7 minutes. In parallel,
each sample was amplified without reverse transcriptase in a reaction
set up with the HotStartTaq DNA polymerase kit (Qiagen), according to
package directions, as a "no RT" negative control. All reactions
also included a no RNA negative control. The adequacy of the extracted
RNA was assessed by RT-PCR, using primers for PGK
(phosphoglycerate kinase) transcripts, as described
previously.27
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. This reaction was set up using 2.5 units of AmpliTaq DNA polymerase
(Perkin Elmer, Inc., Norwalk, CT), 1X PCR buffer, 1.5 mmol/L
MgCl2 (Perkin Elmer), 200 µmol/L PCR nucleotide
mix (Boehringer Mannheim, Corp., Indianapolis, IN) and 0.6 µmol/L
each of the forward and reverse primers. Initial denaturation at
95°C for 2 minutes was followed by 95°C for 30 seconds; 64°C
for 30 seconds; 72°C for 30 seconds for 25 cycles with a 7
minutes final extension at 72°C. PCR products were electrophoresed on
a 2% agarose gel and bands were cut and purified with the Concert
rapid gel extraction system (Gibco BRL). Products were routinely
sequenced on automated sequencer.
The nested and non-nested EWS-ATF1 RT-PCR assays were
designed to detect the most common type of EWS-ATF-1 fusion
transcript, namely the EWS exon 8 to ATF1 codon
65 fusion,12
which we designate type 1 and one of the
molecular variants, namely the EWS exon 10 to
ATF1 codon 110 fusion,28
which we designate
type 2. The only negative case for these primers was also subjected to
a nested RT-PCR using nested EWS exon 7 forward
primers29
in combination with the nested ATF1
primers (Table 1)
to exclude the EWS exon 7 to
ATF1 codon 110 fusion described by Pellin et
al,30
which we designate type 3. The nested RT-PCR
protocol used was exactly as above, except for the different annealing
temperatures (Table 1)
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RT-PCR analysis for MITF mRNA expression was performed using
two sets of primers (Table 1)
. The MITF-C consensus
primers10
amplify a portion of the MITF
transcript shared by all MITF splice forms. The MITF-M
primers are designed to amplify only the melanocyte-specific isoform of
MITF, which differs from other forms by the usage of an
alternative exon 1 (exon 1M).31
RT-PCR analysis for
MITF transcripts was performed without nesting and therefore
was only considered appropriate in cases with good quality RNA. This
included three of four cases with RNA obtained from frozen tissue (CCS
9 had insufficient RNA) and one case (CCS 3) in which good quality RNA
was extracted from the archival paraffin-embedded sample.
The target amplicons of both pairs of MITF
primers included exon boundaries, to avoid amplification of trace
amounts of genomic DNA in the RNA preparations. RNA from the SK-MEL-19
melanoma cell line was used a positive control for both MITF
RT-PCR assays.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2for demographics and exact locations). All tumors were deep and were
excised by a wide-local resection. In seven cases the surgical margins
were microscopically free. The tumors in 10 cases were localized at the
time of diagnosis, while in two cases there were lung metastases at
presentation as detected by staging chest CT-scans. In 11 of 12 cases
follow-up information was available and the follow-up ranged from 5 to
204 months (mean of 44 months for survivors). Seven (63%) patients
developed local recurrences. All five tumors with positive surgical
margins eventually recurred locally. Seven of 11 (63%) patients
developed regional lymph node metastases, diagnosed at intervals of 0
to 47 months (mean of 21 months) from the primary diagnosis. Distant
metastases were identified in nine cases (82%), at time intervals
after original diagnosis ranging from 0 (three cases) to 132 months,
with a mean of 42 months. In all cases, the site of distant spread was
the lung, while additional sites such as brain, bone, adrenal gland,
and spleen were noted in one case each. Adjuvant chemotherapy
information is listed in Table 2
. At last follow-up, there were three
patients with no evidence of disease (NED), one patient alive with
disease (AWD), and seven patients dead of their disease (DOD). Only one
of the three NED patients did not develop any regional or distant
recurrences; this after a 22-month follow-up period. The other two
patients remained NED after resection of their regional (CCS 2) or
distant (CCS 1) metastasis, 9 and 136 months later. All patients alive
with or dead of disease had had distant metastases.
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. In 8 cases definite evidence of tumor invasion of tendon or
aponeurotic structures was identified microscopically. Cases CCS 6 and
CCS 10 showed a distinct microscopic appearance, with epithelioid
cells, a higher degree of nuclear pleomorphism, anaplasia, and an
alveolar pattern of growth, due to a striking loss of cell cohesion
(Figure 2)
. In some areas the tumor cells had a rhabdoid appearance, with bright
eosinophilic cytoplasm and an eccentrically located nucleus. Case CCS
10 showed in addition scattered multinucleated giant cells and evidence
of infiltration into a tendon structure.
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. All cases tested showed strong and diffuse immunoreactivity for HMB45
(cytoplasmic) and MITF (nuclear) (Figure 3)
. Two cases were negative for S-100 protein (CCS 3 and 11), but the
remaining cases were strongly and diffusely positive.
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(Figure 4)
. In case CCS 10, rare cytoplasmic granules of the size and shape of
melanosomes but lacking an internal structure were noted. In addition,
electron-dense myelin figure-like structures indistinguishable from
lysosomes were focally present in some cells. There were external
lamina and rudimentary cell junctions in four and three cases,
respectively. Numerous mitochondriae were identified in most cases. The
presence of glycogen pools was a consistent finding only in four cases.
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using the outer pair of primers for the EWS-ATF1 type 1 or
2 fusion products (Table 1)
. Of the eight cases analyzed using
paraffin-extracted RNA, four showed a faint 246-bp amplified product
using the outer primers (Figure 6)
. After the nested PCR step using the inner primers (Table 1)
, seven of
eight cases showed an amplification product of 185 bp (Figure 6)
. Thus,
in all, 11 of 12 cases (91%) contained EWS-ATF1 fusion
transcripts by RT-PCR (Table 3)
. Controls lacking RT were appropriately
negative in all 11 EWS-ATF1-positive cases. Paraffin block
case CCS 11 was negative despite repeated assays on two RNA samples
extracted from separate paraffin blocks, and despite satisfactory
results with the control RT-PCR assay for PGK transcripts.
Direct sequencing was performed in 11 of the 11 EWS-ATF1
positive cases and showed the same in-frame junction of exon 8 of
EWS to ATF1 codon 65 in all cases. No variant
fusion structure was identified. RT-PCR assays using the two pairs of
primers for the MITF transcripts showed that all 4 CCS
analyzed were positive with the consensus MITF primers, as
well as for the melanocyte-specific MITF-M transcript
(Figure 7)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The differential diagnosis of CCS includes a variety of epithelial and mesenchymal malignancies, but CCS can be particularly difficult to distinguish from the epithelioid variant of malignant peripheral nerve sheath tumor (MPNST) or from a metastatic implant from an occult cutaneous melanoma.35 A diagnosis of epithelioid MPNST is favored in the presence of a significant myxoid stroma and tumor cells in a cord-like arrangement. Epithelioid MPNST typically lacks HMB45 immunoreactivity or the presence of melanosomes on electron microscopic examination. Clinical presentation of a malignant melanoma as a soft tissue mass generally occurs in patients with a known history of cutaneous melanoma. Soft tissue is not a common site for metastatic melanoma, yet neither is it exceedingly rare. Histologically, malignant melanoma usually displays a much greater degree of cellular anaplasia than CCS and often shows intranuclear inclusions. However, unlike the distinction between epithelioid MPNST and CCS, the distinction between CCS and metastatic melanoma cannot be made in all problematic cases based on pathological grounds alone, and therefore may often require genetic confirmation. At the genetic level, malignant melanomas demonstrate a broad range of genetic alterations, most commonly involving chromosomes 1 and 5, as well as deletions of 6q.36, 37 By DNA flow cytometry, most melanomas are markedly aneuploid, while most CCS are diploid or only mildly aneuploid.38 The cytogenetic hallmark of CCS is the presence of a recurrent t(12;22)(q13;q12), resulting in the EWS-ATF1 fusion, often found as the only chromosomal abnormality. Confirming existing data in the cytogenetic literature, molecular testing shows that EWS-ATF1 is detected in almost all cases of CCS, as shown in the present study, and in no cases of cutaneous melanoma, as shown by van Roggen et al.36 In aggregate, previous cytogenetic studies and the data of van Roggen et al36 have established the specificity of the t(12;22)(q13;q12), and its resulting EWS-ATF1 fusion transcript, for CCS.
Despite this fundamental genetic difference, immunohistochemical and ultrastructural findings indicate that, like melanoma, CCS is a neuroectodermal tumor with melanocytic differentiation.3, 4, 5 Similar to previous reports, we find that the majority of CCS tumors containing the EWS-ATF1 fusion also are immunoreactive for melanoma markers, such as HMB45, and contain melanosomes by ultrastructural examination. More recently, immunoreactivity for MITF has been used as a novel marker of melanocytic lineage.7, 8, 9, 10 The MITF transcription factor is known to regulate the differentiation of melanocytes and other pigmented cells.11 MITF immunoreactivity has been previously reported in a total of 19 out of 26 CCS (73%),6, 7, 10 but none of these CCS were studied for EWS-ATF1. In the present study, all CCS cases tested showed diffuse and strong nuclear labeling for MITF, suggesting that the prevalence of MITF positivity may be higher in series consisting of genetically confirmed CCS.
Most immunohistochemical studies of MITF, including the present, have used the D5 and/or C5 monoclonal antibodies (Labvision, Fremont, CA). The precise epitopes bound by these antibodies are unknown but appear to lie in a portion of MITF encoded by both melanocyte-specific (MITF-M) and non-melanocyte-specific isoforms of MITF transcripts.11, 26 Indeed, some recent immunohistochemical studies have noted reactivity of D5 antibody with a variety of non-melanocytic normal and neoplastic cells.7, 10, 39 Therefore, we also performed RT-PCR for two portions of the MITF transcripts, a portion specific to the MITF-M isoform expressed only in melanocytes,11 and a portion shared by all isoforms of MITF. The latter primer pair had been previously used to demonstrate by RT-PCR the ubiquitous expression of MITF in a variety of normal tissues.10 We found that all four CCS analyzed were positive by RT-PCR with the consensus MITF primers, as expected from their immunoreactivity for MITF. Furthermore, we were able to confirm using the RT-PCR assay for the MITF-M isoform, that they expressed the melanocyte-specific form of MITF. MITF induces the expression of key melanocytic genes such as tyrosinase, TRP-1 and TRP-2, and appears sufficient to convert fibroblasts to melanocyte-like cells.40 Hypothetically, the aberrant expression of MITF, possibly induced by EWS-ATF1-mediated transcriptional deregulation in the mesenchymal precursor cell of CCS, may be sufficient to trigger an ectopic melanocytic differentiation cascade. Indeed, there is recent evidence for direct transactivation of the MITF-M promoter by EWS-ATF1 (David E. Fisher, unpublished data).
Detection of specific fusion transcripts in sarcomas can be particularly useful not only to distinguish CCS from other morphological mimics but also to confirm the diagnosis in the setting of an unusual histology or uncommon primary site.41 Two cases in the present study had an unusual alveolar growth pattern and rhabdoid cells with significant nuclear pleomorphism. In these cases, the detection of the EWS-ATF1 fusion transcript was useful in confirming the diagnosis.
In CCS, three hybrid transcript variants have been described so far in
the literature12, 13, 28, 30, 42, 43, 44, 45, 46
(Table 4)
. The most common fusion product consists of EWS amino acid residues 1
to 325, followed by ATF1 residues 65 to 271, seen in 87% of positive
cases (20 of 23) with fusion structure data. In the present study, all
EWS-ATF1 positive cases had this same fusion structure, as
originally described by Zucman et al,12
which we have
designated the type 1 fusion. The other two EWS-ATF1 variant
fusions are rare and were previously described as single case
reports.28, 30, 44
A fusion mRNA with a junction between
EWS exon 10 and ATF-1 codon 110 was identified by
Speleman et al28
and we now designate this as the type 2
fusion. The EWS exon 7–ATF1 codon 110 fusion,
designated here as the type 3 fusion, was recently described in two
separate case reports.30, 44
There have been too few cases
of CCS with non-type 1 EWS-ATF1 fusions to begin to comment
on possible clinical or pathological correlates. However, some of the
differences in the structure of the two variant fusion proteins could
be functionally significant. The absence of codons 65 to 109 of ATF1 in
the type 2 and type 3 fusion products would exclude the remainder of a
putative activation domain in ATF1,14
which could
hypothetically affect overall transactivation by EWS-ATF1. The absence
of the portion encoded by EWS exon 8 in the type 3 fusion
would remove the EWS IQ domain whose phosphorylation appears to enhance
EWS-ATF1 function.47
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In conclusion, the present study shows that the EWS-ATF1 gene fusion is found in the majority of CCS cases (91%) and thus provides a sensitive molecular diagnostic marker for CCS. Conventional RT-PCR methodology can be applied with good results for EWS-ATF1 detection using RNA extracted from frozen tissues. When using archival material nested RT-PCR can yield similar results, in the presence of adequate RNA. The expression of the melanocyte-specific MITF-M transcript is further evidence of the bona fide melanocytic differentiation of CCS.
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Acknowledgments |
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Footnotes |
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Supported by PO1 CA47179 to M.F.B.
Accepted for publication September 9, 2001.
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X174);
M2: 100-bp DNA ladder; ± , reverse transcriptase added/not
added (the latter representing the "no RT"
negative control).
