Molecular Diagnosis in Pathology: The Bridge to the 21st Century
Moderators: Dr. Ricardo Lloyd and Dr. George Kontogeorgos
Section 1 -
Molecular Diagnosis of Soft Tissue Tumors
Marc Ladanyi, M.D.
Memorial Sloan-Kettering Cancer Center
New York, NY, USA
Certain sarcomas are characterized by specific recurrent chromosomal translocations,
biologically similar to those seen in leukemias. These chromosomal translocations produce highly
specific gene fusions, of which the specificity for and prevalence in selected sarcomas are such that
they have come to define these entities
Two key concepts in translocation sarcomas are first,
that these sarcomas contain their fusion gene from their earliest presentation and do not show benign or
premalignant phase; and secondly, the fusion gene is present in all tumor cells and is expressed
throughout the clinical course. The pathobiology of the oncogenic fusion proteins involves in almost all
instances either transcriptional deregulation (most) or aberrant signaling (some). The fusion genes
produced by these translocations most often encode chimeric transcription factors that cause
transcriptional deregulation . In broad terms, chimeric transcription factors are thought to
deregulate the expression of specific repertoires of target genes, possibly providing multiple oncogenic
hits analogous to the multistep process of epithelial carcinogenesis.
All of the major cytogenetically described translocations in sarcomas have by now been cloned . A
recent study that used network modeling to analyze the relationship of all known gene fusions in human
cancer suggests that it is likely that all or most genes involved in multiple gene fusions (such as EWS, TFE3, MLL) have
already been identified and that only rare or variant gene fusions remain to be identified . Indeed,
uncommon gene fusions, some of them variants of known fusions, continue to be described. For instance,
rare "Ewing sarcoma-like tumors" have recently been reported with the following fusions: EWS-POU5F1 ,
CIC-DUX4 , and BRD4-NUT . The latter fusion is not novel as it is already know to be
associated with aggressive midline carcinomas . In addition, we have recently identified a novel
EWS-SP3 fusion in a "Ewing sarcoma-like tumor" (L. Wang, M. Ladanyi, et
al., submitted). Other recently described variants of known sarcoma fusions include PAX-NCOA1 in alveolar rhabdomyosarcoma  and EWS-CREB1 in clear cell sarcoma .
Regarding the perennial question of how and why translocations arise
an interesting recent
bioinformatic analysis of the sequence and structure of all genes involved in translocations provides
compelling new insights . Comparing 268 genes involved in translocations to 9406 control genes, the
authors found striking differences in overall gene size, average intron length, and length of the longest
intron, all three of which were significantly greater in the former group. They did not find any
differences in the presence of so-called recombinogenic sequence elements. These data support the
concept that the intronic breaks that lead to specific recurrent chromosomal translocations in cancer are
largely random events (risk of breaks is simply proportional to intron length) that become fixed through
natural selection if they provide a growth advantage to the cell. Other factors that can be incorporated
into this model include the increased "availability" of the genes for rearrangement that is created by
the open chromatin conformation associated with gene transcription or replication , and the
unexpected proximity of some translocation partner genes due to the 3 dimensional arrangement of
chromosomes in the nucleus. Evidence for the latter phenomenon has been presented in hematologic and
but not yet in sarcomas.
A broader role for gene fusions in human cancers?
Specific intergenic rearrangements or gene fusions represent, in terms of numbers of different genes
involved, the single most common type of somatic genetic alteration in human cancer. Over 75% of genes
somatically altered in human tumors are translocated . Biologically, these gene fusions operate
either by promoter substitution or the formation of aberrant chimeric proteins. Although these types of
translocations have been historically associated mainly with leukemias, lymphomas, and sarcomas, and have
been less often detected in carcinomas, Mitelman and colleagues have provocatively argued that this
skewed distribution may be at least partly artefactual . Their argument is based on the observation
that in every tumor type, the numbers of specific translocations described is proportional to the number
of cases successfully karyotyped. Carcinomas, being far more difficult to karyotype than leukemias,
lymphomas, and sarcomas, have so far yielded few specific translocations. Until recently, such
translocations had only been described in select types of less common carcinomas, including thyroid
certain types of renal
as well as a few other rare carcinomas
However, this field changed dramatically in late 2005 with the first report of recurrent fusions
involving ERG and other ETS family genes in
prostate cancer . The most common fusion, TMPRSS2-ERG, is present in
approximately 50% of prostate cancers, a striking proportion confirmed by others . These findings
have invigorated the notion proposed by Mitelman and colleagues that many more fusion genes remain to be
found within the complex karyotypes of carcinomas .
Molecular diagnosis of sarcoma translocations – practical aspects
The necessity of molecular testing in modern sarcoma diagnosis has recently been the subject of
Based on these studies, molecular confirmation of translocation status
seems more often useful in diagnosing synovial sarcomas (useful in approx. 50%) than Ewing sarcomas
(useful in approx. 10%). The two main diagnostic methods for sarcoma translocations are
reverse-transcriptase PCR (RT-PCR) and fluorescent in situ hybridization (FISH)
. RT-PCR, as an
RNA-based assay, is susceptible to failure due to poor RNA quality. As a PCR-based assay, it is also
susceptible to false-positives due to PCR cross-contamination. It is critical to include two types of
contamination controls: controls lacking only the template RNA (to detect contamination of the PCR
reagents) and controls lacking only the reverse transcriptase (to detect contamination of the patient RNA
sample). Although RT-PCR is more sensitive and provides more detailed fusion information than FISH, it
is less adaptable to paraffin material and frozen tissue is definitely preferred. Another important
consideration for RT-PCR assays is that they require extensive knowledge of the specific exons involved
by the gene fusions and of the variability in exon composition of some fusions. Recently, real-time
RT-PCR, which employs highly sensitive fluorescent detection of PCR products as they are generated ("in
real-time"), has emerged as an improved strategy for RT-PCR detection of sarcoma fusion transcripts in
archival pathology material , with the added benefit of avoiding the cross-contamination risks of
other high sensitivity PCR approaches such as nested PCR.
FISH is a DNA-based assay. Given the relatively better preservation of DNA than RNA in paraffin
material, it is generally more adaptable to paraffin material than RT-PCR, yet frozen tissue is also
preferable for FISH. False-positives in FISH can arise from overinterpretation . Split signal FISH
assays can be overinterpreted because of occasional separation of the signals in some normal cells.
Fused signal FISH assays are susceptible to false-positives due to occasional random juxtaposition of
signals. In both types of FISH assays, conservative thresholds for positivity need to be carefully
established . Several sarcoma FISH probe pairs are commercially available, including EWS, FUS(TLS), CHOP, FKHR, and SYT
(Abbott-Vysis , USA). EWS and SYT probe pairs
labeled for chromogenic in situ hybridization (CISH) are also on the market (Invitrogen, USA). These
probe pairs are all based on a split signal FISH assay design and thus only document gene rearrangement,
not specific gene fusions.
RT-PCR and FISH are complementary methods for detecting sarcoma translocations. The use of one or the
other as the first line approach often reflects differences in local expertise. Most studies comparing
both techniques in the sarcoma setting suggest that optimal diagnostic accuracy can be achieved when both
The College of American Pathologists now offers regular proficiency testing for
sarcoma translocation detection by FISH and RT-PCR.
Translating molecular diagnostic markers into IHC markers
Many translocations can be converted into IHC assays based on the phenomenon of discordance in the
expression levels of the amino- and carboxy-terminal ends of the product of gene B in tumors with A-B
gene fusions, or the markedly aberrant expression of the carboxy-terminal encoded by gene B in the
context of these fusion proteins, or their expression in aberrant cell types or aberrant cellular
compartments. This has been confirmed by westerns and has been used as a basis for the IHC detection of
oncogenic fusion proteins . For instance, we have established the latter approach for the detection
of the EWS-WT1 protein  and fusions involving
TFE3 and TFEB
tumors harbor a variety of ALK gene fusions in many or most cases, and IHC
for the ALK carboxy-terminal end has become a widely used adjunct in diagnosis
for converting molecular translocation detection into an IHC assay is provided by certain gene fusions
where a 5' exon of gene B that is not normally translated becomes translated in the context of the fusion
protein and therefore represents a novel peptide sequence for antibody generation. This approach has
recently been demonstrated for TLS-CHOP and EWS-CHOP detection in myxoid liposarcomas .
Other types of molecular diagnostic markers in sarcomas
Although recurrent translocations are by far the most widely used markers, certain other genetic
alterations are so closely associated with specific sarcomas that they can also form the basis for
confirmatory assays. This is the case with KIT (and PDGFRA) mutations in GISTs . However, KIT immunoreactivity in GIST is related
to tumor cell lineage more than to the underlying mutation. Co-amplification of MDM2 and CDK4 in well-differentiated and
dedifferentiated liposarcomas due to 12q amplification has also emerged as a potentially useful marker in
Recently, this has been translated into a robust IHC assay
mutations involving the WNT pathway (APC or beta-catenin) are characteristic
of (but not entirely specific for) aggressive desmoid-type fibromatosis. These result in nuclear
beta-catenin accumulation readily detected by IHC
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