The Clinical Relevance of Sarcoma Translocations

1
The Clinical Relevance of Sarcoma
Translocations
Marc Ladanyi, M.D. Chief, Molecular Diagnostics
Service Department of Pathology Memorial
Sloan-Kettering Cancer Center New York, NY, USA
2

• Outline of Talk
• The Clinical Relevance of Sarcoma Translocations
• Diagnosis
• Prognosis
• Therapeutic targets

3
Selected fusion genes in sarcomas biological
overview
4
Molecular Diagnostics Service Department of
Pathology Memorial Sloan-Kettering Cancer Center
Laboratory of Diagnostic Molecular Genetics
Laboratory of Diagnostic Molecular Pathology
Laboratory of Clinical Cytogenetics
Molecular Marrow Transplant Engraftment Monitori
ng
Germline Cancer Predisposition Testing
Conventional cytogenetics and FISH
Molecular Oncology
HER2 FISH in breast CA (all other FISH in
Cyto-genetics)
Kinase Mutation Testing (EGFR, KRAS, JAK2, FLT3)
LymphomaClonality Assays
Leukemia Translocation Testing for Diagnosis and
Minimal Disease Monitoring
Other Molecular Prognostic Markers (1p/19q in
glioma)
Sarcoma Translocation Testing for Diagnosis
150-200/yr
5
Molecular Diagnostic Testing in Sarcomas Most
common indications

• Undifferentiated small round cell sarcomas
  ES/PNET vs DSCRT vs poorly diff synovial sarcoma
• Rhabdomyosarcoma confirmation of alveolar
  subtype
• Spindle cell sarcomas confirmation of monophasic
  synovial sarcoma
• Confirmation of otherwise typical cases in
  unusual age or in uncommon sites or with some
  uncommon features

6
Pediatric sarcomas confirmed in older adults
(gt50) by translocation data
7
Sarcomas presenting in unusual primary
sitesconfirmed by translocation data
Sarcoma type
Primary Sites
Ewing Sarcoma / PNET
Kidney Skin Lung
Ovary Uterus Pancreas
Breast Dura Bowel
Adrenal
Brain Parotid Hand
Desmoplastic Small Round Cell Tumor (DSRCT)
Synovial sarcoma
Prostate Lung Heart
Peritoneum Kidney Tongue
8
Sarcoma cases with unusual histology or
immunophenotype confirmed by translocation data
Sarcoma type
Unusual feature
cytokeratin weak or negative
MIC2/CD99 prominent myxoid change
Ewing Sarcoma / PNET
cytokeratin negative non-desmoplastic
Desmoplastic Small Round Cell Tumor (DSRCT)
neural features prominent myxoid
change calcification / ossification
Synovial sarcoma
Alveolar RMS
solid variant
9
Chromosomal Translocations Producing Gene
Fusions Common Detection Methods
10
Chromosomal Translocations Producing Gene
Fusions Detection Methods

• Reverse-transcriptase PCR
• RNA-based assay
• susceptible to failure due to poor RNA quality
• susceptible to false-positives due to PCR
  contamination
• more sensitive and specific than FISH
• adaptable to paraffin material frozen tissue
  preferred

11
Fusion structure variability an important
consideration in RT-PCR assays
Many different combinations of exons from EWS and
FLI1 can fuse to produce an in-frame functional
fusion protein. RT-PCR assays must be designed
to detect most or all EWS-FLI1 and EWS-ERG fusion
types.
12
Fluorescent in situ hybridization to detect
translocations 2 types
- split signal FISH 2 probes flanking one of the
breakpoints are separated by the translocation
der(17)t(X17)(p11.2q25)
TFE3 (p11.2)
ASPL (q25)
X
17
13
Fluorescent in situ hybridization to detect
translocations 2 types
- fused signal FISH one probe flanking one
breakpoint, the other flanking other breakpoint,
are brought together by the translocation
der(17)t(X17)(p11.2q25)
TFE3 (p11.2)
ASPL (q25)
X
17
14
Chromosomal Translocations Producing Gene
Fusions Detection Methods

• Reverse-transcriptase PCR
• RNA-based assay
• susceptible to failure due to poor RNA quality
• susceptible to false-positives due to PCR
  contamination
• more sensitive and specific than FISH
• adaptable to paraffin material frozen tissue
  preferred
• Fluorescent in situ hybridization
• DNA-based assay
• split signal FISH assay only documents gene
  rearrangement, not specific fusion
• fused signal FISH assays susceptible to
  false-positives due to occasional random
  juxtaposition of signals
• generally more adaptable to paraffin material
  than RT-PCR
• frozen tissue still preferred
• RT-PCR and FISH are complementary methods
• Use of one or the other as the first line dx
  approach often reflects differences in local
  expertise

15
Use of IHC to detect translocation-associated
fusion proteins

• Fusion proteins produced by fusion of gene A to
  gene B can be detected by IHC for the B portion
  of the fusion protein through one of three
  mechanisms
• Higher expression level
• Example TFE3
• Expression in inappropriate cell type
• Example WT1, ALK
• Expression in inappropriate cellular compartment
• Example ALK

16
Use of IHC to detect translocation-associated
fusion proteins

• Example Application of TFE3 C-terminal antibody
  to detect TFE3 fusion proteins due to much higher
  expression level than native TFE3
• Translocations involving TFE3 at Xp11 and 5
  possible translocation partners
• Found in some renal carcinomas of children and
  young adults and in alveolar soft part sarcomas
  (ASPS)

1
2
3
6
4
5
7
8
TFE3
5UT AD bHLH LZ
3UT
fusion points
ASPL
5UT
UBX 3UT
6
4
5
7
8
ASPL-TFE3 type 1
5UT bHLH
LZ 3UT
17
Application of TFE3 C-terminal antibody to detect
ASPL-TFE3 fusion proteins
Alveolar Soft Part Sarcoma
The ASPL-TFE3 fusion and other TFE3 gene fusions
result in prominent nuclear TFE3
immunoreactivity, using a polyclonal antibody
directed against the portion of TFE3 downstream
of the fusion point.
18
Application of ALK C-terminal antibody to detect
ALK fusion proteins
IMT
Cytoplasmic immunoreactivity ALK fusion
19
Application of WT1 C-terminal antibody to detect
EWS-WT1 fusion protein in desmoplastic small
round cell tumor
Nuclear immunoreactivity for WT1 in desmoplastic
small round cell tumor
20
Sarcoma translocations are specific, but

• Biologically, specific gene fusions are
  tumor-specific and appear necessary in the
  pathogenesis of specific cancers ?they are
  therefore near-perfect tumor markers.
• but..
• Practically, technical limitations or errors in
  detection of these translocations can lead to
  false-positives or false-negatives.

21
Promiscuous translocations translocations
described in more than one specific tumor type
Sarcoma translocations are specific, but

• FUS-ERG / t(1621) in
• Ewings sarcoma (rare subset)
• AML (rare subset)
• EWS-ATF1 / t(1222) in
• Clear cell sarcoma (most cases)
• Angiomatoid fibrous histiocytoma (some)
• EWS-CREB1 / t(222) in
• Clear cell sarcoma (some cases)
• Angiomatoid fibrous histiocytoma (most)

• ETV6-NTRK3 / t(1215) in
• Congenital fibrosarcoma (all cases)
• AML (rare cases)
• Secretory breast carcinoma (all cases)
• ALK fusions (various) in
• Inflammatory myofibroblastic tumor (most cases)
• Anaplastic large cell lymphoma (most cases)
• ASPL-TFE3 / t(X17) in
• Alveolar soft part sarcoma (all cases)
• Specific subset of pediatric renal carcinomas

22

• Outline of Talk
• The Clinical Relevance of Sarcoma Translocations
• Diagnosis
• Prognosis
• Therapeutic targets

23
Translocation testing to determine prognostic
categoriesMajor settings Level of evidence

• Ewings EWS-FLI1 type 1 vs others
• Modest effect, prob not clinically useful
• No other associations
• Synovial SYT-SSX1 vs SYT-SSX2
• Modest effect, not consistently demonstrated
  prob not clinically useful
• Associations with monophasic vs biphasic type
  (strong), sex (SSX2?gt?), site (SSX1limb)
• Rhabdo PAX3-FKHR vs PAX7-FKHR
• Clear prognostic effect
• Association with age (PAX3 older)

24
Translocation testing to determine prognostic
categories Example of pediatric rhabdomyosarcoma
NEG
PAX7
ARMS
ERMS
PAX3
25
ERMS
26
ARMS
solid
alveolar
27
Translocation testing to determine prognostic
categories Example of pediatric rhabdomyosarcoma

Significantly poorer survival (P lt 0.0001) for
alveolar RMS cases with t(213) as compared to
RMS cases without a translocation (mostly
embryonal RMS)
ERMS
ARMS
Anderson J, et al., Br J Cancer 85831-5, 2001
28
Importance of PAX3-FKHR vs PAX7-FKHR distinction
among ARMS
1.0
Overall Survival for Metastatic ARMS on IRS-IV
Sorensen PH et al., J Clin Oncol. 2002 20
2672-9
0.9
0.8
PAX7- FKHR
0.7
0.6
0.5
Proportion
PAX3 vs PAX7 p 0.0015
0.4
0.3
0.2
0.1
PAX3- FKHR
0.0
0
1
2
3
4
5
6
7
8
9
10
Overall Survival (years)
29

• Outline of Talk
• The Clinical Relevance of Sarcoma Translocations
• Diagnosis
• Prognosis
• Therapeutic targets

30
Sarcoma Translocations as Therapeutic targets

• Translocations creating fusion kinases or
  deregulated growth factors are sensitive to
  specific kinase inhibitors

31
Sarcoma Translocations as Therapeutic targets

• Translocations creating chimeric transcription
  factors (CTF)
• Transcription factors poor therapeutic targets
• Efforts to directly target CTFs still
  experimental or early
• Interference with key protein-protein
  interactions EWS-FLI1 and RNA Helicase
• Toretsky et al., Cancer Res 665574-81 (2006)
• Reduction of CTF protein stability EWS-FLI1 and
  cytosine arabinoside
• Stegmaier et al., PLoS Med 4e122 (2007)

32
Sarcoma Translocations as Therapeutic targets

• Translocations creating chimeric transcription
  factors (CTF)
• Transcription factors poor therapeutic targets
• Alternative therapeutic approach inhibit kinase
  pathways that are directly or indirectly
  activated by the CTF and that contribute to
  sarcoma growth
• Example of MET tyrosine kinase in alveolar soft
  part sarcoma

33
Alveolar Soft Part Sarcoma

• Rare soft tissue sarcoma
• FM 32
• age range 15-35
• highly chemotherapy-resistant
• site within or next to skeletal muscle
• typical presentation thigh mass

Alveolar Soft Part Sarcoma (ASPS)
34

• Discovery
• 2000 cloning of a specific gene fusion encoding
  an aberrant transcription factor, ASPL-TFE3,in
  alveolar soft part sarcoma (ASPS), a rare sarcoma
  of uncertain lineage
• 2001 identification of ASPL-TFE3 in a previously
  unrecognized subset of renal carcinomas
• Translation
• 2003 development of diagnostic
  immunohistochemical assay for tumors with
  ASPL-TFE3 or other TFE3 gene fusions now widely
  used
• 2004 Characterization of renal carcinomas with
  ASPL-TFE3 and other TFE3 gene fusions leads to
  delineation as separate entity in current WHO
  classification
• 2007 analysis of genes directly upregulated by
  ASPL-TFE3 and other TFE3 fusion proteins
  identifies MET as a transcriptional and
  therapeutic target

ASPS

1
553
ASPL
UBX
311
1
575
TFE3
bHLH -LZ
AD
296
261
1
2
1
311
591
ASPL-TFE3 type1
bHLH -LZ
1
311
626
ASPL-TFE3 type2
bHLH -LZ
AD
35

• Discovery
• 2000 cloning of a specific gene fusion encoding
  an aberrant transcription factor, ASPL-TFE3,in
  alveolar soft part sarcoma (ASPS), a rare sarcoma
  of uncertain lineage
• 2001 identification of ASPL-TFE3 in a previously
  unrecognized subset of renal carcinomas
• Translation
• 2003 development of diagnostic
  immunohistochemical assay for tumors with
  ASPL-TFE3 or other TFE3 gene fusions now widely
  used
• 2004 Characterization of renal carcinomas with
  ASPL-TFE3 and other TFE3 gene fusions leads to
  delineation as separate entity in current WHO
  classification
• 2007 analysis of genes directly upregulated by
  ASPL-TFE3 and other TFE3 fusion proteins
  identifies MET as a transcriptional and
  therapeutic target

ASPS

Renal CA
36

• Discovery
• 2000 cloning of a specific gene fusion encoding
  an aberrant transcription factor, ASPL-TFE3,in
  alveolar soft part sarcoma (ASPS), a rare sarcoma
  of uncertain lineage
• 2001 identification of ASPL-TFE3 in a previously
  unrecognized subset of renal carcinomas
• Translation
• 2003 development of diagnostic
  immunohistochemical assay for tumors with
  ASPL-TFE3 or other TFE3 gene fusions now widely
  used
• 2004 Characterization of renal carcinomas with
  ASPL-TFE3 and other TFE3 gene fusions leads to
  delineation as separate entity in current WHO
  classification
• 2007 analysis of genes directly upregulated by
  ASPL-TFE3 and other TFE3 fusion proteins
  identifies MET as a transcriptional and
  therapeutic target

ASPS

Renal CA
37

• Discovery
• 2000 cloning of a specific gene fusion encoding
  an aberrant transcription factor, ASPL-TFE3,in
  alveolar soft part sarcoma (ASPS), a rare sarcoma
  of uncertain lineage
• 2001 identification of ASPL-TFE3 in a previously
  unrecognized subset of renal carcinomas
• Translation
• 2003 development of diagnostic
  immunohistochemical assay for tumors with
  ASPL-TFE3 or other TFE3 gene fusions now widely
  used
• 2004 Characterization of renal carcinomas with
  ASPL-TFE3 and other TFE3 gene fusions leads to
  delineation as separate entity in current WHO
  classification
• 2007 analysis of genes directly upregulated by
  ASPL-TFE3 and other TFE3 fusion proteins
  identifies MET as a transcriptional and
  therapeutic target

38

• Discovery
• 2000 cloning of a specific gene fusion encoding
  an aberrant transcription factor, ASPL-TFE3,in
  alveolar soft part sarcoma (ASPS), a rare sarcoma
  of uncertain lineage
• 2001 identification of ASPL-TFE3 in a previously
  unrecognized subset of renal carcinomas
• Translation
• 2003 development of diagnostic
  immunohistochemical assay for tumors with
  ASPL-TFE3 or other TFE3 gene fusions now widely
  used
• 2004 Characterization of renal carcinomas with
  ASPL-TFE3 and other TFE3 gene fusions leads to
  delineation as separate entity in current WHO
  classification
• 2007 analysis of genes directly upregulated by
  ASPL-TFE3 and other TFE3 fusion proteins
  identifies MET as a transcriptional and
  therapeutic target

39
Oncogenic TFE3 fusion proteins activate MET
signaling by transcriptional upregulation
HGF

• ASPL-TFE3 stimulates MET kinase activity and
  downstream signaling

MET
Membrane
p
Cytoplasm
p
RAS
p
Autophosphorylation
Crk
p
Gab1
p
c-Raf
p
PI3-k
p
U0126
p
PHA665752 (Pfizer)
MEK1/2
p
Akt
LY294002
PD98059
p
p
MAPK/Erk1/2
?
Met
Nucleus
Met
p
Met
ASPL-TFE3
MET
p
p
p
p
ASPS and renal carcinomas with TFE3 fusions
Transcription
MYC
Stat3
cell growth adhesion motility invasion
genes
Tsuda M, Davis IJ, Argani P, Shukla N, McGill GG,
Nagai M, Saito T, Laé M, Fisher DE, Ladanyi M.
Cancer Res 67919-929, 2007.
40
Effects of MET inhibitor PHA665752 on cell
viability
ASPL-TFE3 RCC line (no ASPS line available)
Other sarcomas
PD98059 (MEK inhib.)
HS-SY-II
FU-UR1
1.2
FU-UR1
RH30
A673
Fuji
1
RH30
0.8
A673
IB Met
0.6
HS-SY-II
0.4
IB actin
Fuji
0.2
0
0
10
20
50
100
PHA665752
(µM)
1.2
LY294002 (PI3K inhib)
1.2
1
PHA665752
1
0.8
0.8
0.6
0.6
viability
0.4
0.4
0.2
0.2
0
0
10
20
50
100 (µM)
0
0
0.05
0.1
0.2
0.5
1
10
20
50
(µM)
41

• Discovery
• 2000 cloning of a specific gene fusion encoding
  an aberrant transcription factor, ASPL-TFE3,in
  alveolar soft part sarcoma (ASPS), a rare sarcoma
  of uncertain lineage
• 2001 identification of ASPL-TFE3 in a previously
  unrecognized subset of renal carcinomas
• Translation
• 2003 development of diagnostic
  immunohistochemical assay for tumors with
  ASPL-TFE3 or other TFE3 gene fusions now widely
  used
• 2004 Characterization of renal carcinomas with
  ASPL-TFE3 and other TFE3 gene fusions leads to
  delineation as separate entity in current WHO
  classification
• 2007 analysis of genes directly upregulated by
  ASPL-TFE3 and other TFE3 fusion proteins
  identifies MET as a transcriptional and
  therapeutic target
• 2008 Phase I clinical trials of new MET
  inhibitors include ASPS patients

42
Oncogenic activation of MET signaling

• Kinase domain missense mutations (activating)
• Familial and sporadic (15) papillary renal cell
  carcinomas
• rare in other cancers
• MET fusion protein (constitutively activated)
• TPR-MET (only in carcinogen-treated cell line
  MNNG-HOS)
• Autocrine signaling
• MET and HGF co-expression
• MET amplification
• Gastric cancer (10-20)
• EGFR-mut lung adenocarcinomas resistant to EGFR
  TKIs
• Aberrant transcriptional upregulation of MET
• by amplified MITF in some melanomas (MET is a
  direct target of MITF)
• by TFE3 fusion proteins in ASPS and renal
  carcinomas with TFE3 fusions

43

• Outline of Talk
• The Clinical Relevance of Sarcoma Translocations
• Diagnosis
• Prognosis
• Therapeutic targets
• Thank you !