Soft Tissue Tumors

• Difficult diagnosis
• Impact on prognosis and treatment
• H and E ± immunohistochemistry
• Increasing role of genomic tools
Genomic abnormalities in sarcomas

• Reciprocal translocations
• Gene amplifications
• Mutations
• Deletions
 Which type of tissues can be used ?

• Frozen tissues are the best

. < 20 % of STT are frozen
. rarely available in second opinion practice
• Fixed and paraffin-embedded tissues
. always available
. allow retrospective studies
. limitations
 Limitations of paraffin-embedded
tissues

• Fragmentation of nucleic acids due to fixation

• Type and time of fixation
• Fragments to be amplified < 200 bp
 New method of fixation

• Deshydratation with assistance of microwave

• Automates (Sakura, Milestone)
• Advantages: nucleic acids of good quality
rapidity
no formalin
Genomic diagnosis on paraffin-embedded
tissues

• Which DNA lesions can be detected ?

• Which techniques can be used ?
• Which tumor types can be diagnosed ?
Genomic abnormalities in sarcomas

• Reciprocal translocations
• Gene amplifications
• Mutations
• Deletions
Translocations in soft tissue sarcomas

• Synovial sarcoma t (X;18)

• Alveolar rhabdomyosarcoma t (2;13) and t (1;13)
• PNET t (11;22) ; t (21;22) ;…
• Myxoid liposarcoma t (12;16) and t (12;22)
• Myxoid chondrosarcoma t (9;22) ;…
• Clear cell sarcoma t (12;22)
• Desmoplastic round cell tumor t (11;22)
• DFSP t (17;22)
• Infantile fibrosarcoma t (12;15)
• Alveolar soft part sarcoma t (X;17)
• Low grade fibromyxoid sarcoma t(7;16)
Detection of translocations in paraffin-
embedded tissues

• RT - PCR
• FISH
 B2MGlob
GAPDH SSX
SSX
 Real-time PCR analysis
Advantages

• Real-time analysis and quantitative

• Automated analysis in a single tube
→ decrease in the risk of contamination
• Rapid, sensitive and specific
RT-PCR on paraffin-embedded tissues
Clinical applications

• Synovial sarcoma
• Alveolar rhabdomyosarcoma
• Clear cell sarcoma
• ES myxoid chondrosarcoma
• Intra-abdominal desmoplastic tumor
• Infantile fibrosarcoma
 RT-PCR in synovial sarcomas
Guillou et al - Human Pathol 2001
• 221 cases :
86 synovial sarcomas
91 sarcomas
31 benign mesenchymal tumors
13 malignant non-mesenchymal tumors
• Specificity : 100 %
• Sensitivity : 96 %
Should molecular testing be required for
diagnosing synovial sarcoma ?
Coindre et al, Cancer 2003

• Prospective study on 204 cases

• Diagnostis of SS certain or probable (48%) :
SSX+ in 91%
• Diagnosis of SS possible (52%) :
SSX+ in 29%
• Useful for spindle cell sarcomas
round cell tumors
epithelial like tumors
 Synovial sarcomas
Usefulness of molecular biology

Diagnosis of SS possible : 107 cas - 26 SSX+

• Spindle cell sarcomas : 49
10 SSX+ Initial diagnosis : MPNST (5)
• Round cell sarcomas : 34
7 SSX+ Initial diagnosis : PNET (3)
• Epithelial like tumors : 24
9 SSX+ Initial diagnosis : carcinoma (5)
myoepithelioma (3)
epithelioid fibroS (1)
 Diagnostic impact of RT-PCR in
synovial sarcomas

• RT-PCR is necessary in 30 % of cases :

• Spindle cell and rare site
• Poorly diff and outside limbs
• Monophasic epithelial
26-year old man
tumor in the lung 8 cm

• EMA +
• CD99 +

SSX
 45 year old man
Tumor of the leg, 7 cm

S100
 Impact of SYT-SSX fusion type on the
prognosis of synovial sarcoma

Ladanyi et al – Cancer Research 2002

• 243 patients
• SSX1 (n=147) - 5-year OS : 53%
• SSX2 (n=91) - 5-year OS : 73%
• Patients with localized disease (n=133) – Cox regression
SSX type is the only prognostic factor for OS
 Impact of SYT-SSX fusion type on the
prognosis of synovial sarcoma

Guillou et al – French Sarcoma Group

• 182 patients
• SSX1 (n= 120) - 5-year MFS : 48%
• SSX2 (n= 62) - 5-year MFS : 50%
• Patients with localized disease (n=114) – Cox regression
Grade is the first prognostic factor
 SOFT TISSUE SARCOMAS : Synovial sarcomas and Grade (n = 125)

0,9
M
E
T 0,8
A
S
T
grade 2 (n=56)
0,7
A
S
I
0,6
S

F
R 0,5
E
E
0,4
S grade 3 (n=69)
U
R 0,3
V
I
V 0,2
A
L
0,1 p = 1.9x10-5

1 2 3 4 5 6 7 8 9 10 11 12 13
years
RT-PCR in alveolar rhabdomyosarcomas

• Poor prognosis
• Intensive chemotherapy
• Histology + myogenin
• t(2;13) or t(1;13) in 85 % of cases
 Rhabdomyosarcoma study and PAX

• Bordeaux - Lausanne - Lyon - Villejuif

• 109 cases (paraffin-embedded)
- 44 PAX+ (38 PAX3 et 6 PAX7)
- 52 negative
- 13 non interpretable
• 24 cases (frozen) : concordance
 Rhabdomyosarcoma study and PAX
Concordance with histology

• 45 alveolar RMS:
39 PAX+ on 42 interpretable (95%)
• 64 embryonal RMS:
5 PAX+ on 55 interpretable (9%)
3 microbiopsies
2 dense RMSE
 Rhabdomyosarcoma study and PAX
Concordance with myogenin

• Myogenin < 50% of cells +

35 cases on 98 interpretable
PAX always -
embryonal RMS
• Myogenin > 50% of cells +
63 on 98 interpretable : 44 PAX+
46 alveolar RMS
17 embryonal RMS
 Rhabdomyosarcoma study and PAX
Recommandations

• RMS with myogenin + on < 50% cells:

molecular study unuseful
• RMS with myogenin + on > 50% cells:
- histology of embryonal RMS
molecular study necessary
- histology of alveolar RMS
molecular study useful (PAX3/7)
 myogenin

18-month old girl

Endonasal tumor

PAX-3
 Ewing sarcoma/PNET

• Poorly differentiated round cell tumor

• Immunohistochemistry is non-specific
• Atypical forms (age, location, morphology)
• Importance of detection of specific translocation
 Genetic heterogeneity of translocations
in Ewing sarcoma/PNET

• t(11;22) (q24;q12) EWS-FLi1 85-90 %

• t(21;22) (q22;q12) EWS-ERG
• t(7;22) (p22;q12) EWS-ETV1
• t(17;22) (q12;q12) EWS-E1AF
• t(2;22) (q33;q12) FEV-EWS
Molecular heterogeneity of t(11;22) (q24;q12)
(EWS-FLi1) in Ewing sarcoma/PNET

• EWS-FLi1 type1 (exon 7 of EWS/exon 6 of FLi1)

EWS-FLi1 type 2 (exon 7 of EWS/exon 5 of FLi1)
…..
→ up to 18 types of chimeric transcript
• Necessity of a long PCR product (800 bp)
 Detection of translocation in Ewing
sarcoma/PCR on paraffin-embedded tissue

• RT-PCR : low sensitivity ( < 50%)

100% with new fixation
• FISH
 Advantages of FISH

• Can be applied to interphase cells

• On fresh or fixed samples
• Overnight procedure
 EWS probes (Zymed)
EWS-FLI1 PNET
Chromosome
EWS-ERG PNET
22
EWS-ETV1 PNET
p EWS-E1AF PNET
EWS-FEV PNET
EWS c

EWS t
EWS-CHN Myxoid Chondrosarcoma
q EWS-WT1 Desmoplastic Round CT
EWS-ATF1 Clear Cell Sarcoma
PNET Clear cell sarcoma

CISH
Genomic abnormalities in sarcomas

• Reciprocal translocations
• Gene amplifications
• Mutations
• Deletions
 Amplifications in soft tissue sarcomas

• Comparative Genomic Hybridization (CGH)

• Complex losses and gains of chromosomal
material (leiomyosarcoma/MFH)
• Simple amplification :
12q 13-15 in well-diff/dediff.liposarcomas
 Amplification of MDM2/CDK4 in
liposarcomas

• Well-differentiated liposarcomas
• Dedifferentiated liposarcomas
• Value and limitation of immunohistochemistry
Detection of amplification on paraffin-
embedded tissue

• FISH
• Quantitative PCR
• CGH
Well-differentiated liposarcoma Lipoma

FISH – MDM2
 • Relative quantification
normal fat mdm2
• Internal control gene
albumin (albumin)
• Number of copies of a
gene

lipoma liposarcoma mdm2

albumin
 Amplification of MDM2/CDK4
Retroperitoneal tumors

• Pleomorphic MFH and poorly-differentiated

sarcomas
• Inflammatory MFH
• Pleomorphic rhabdomyosarcomas
• Malignant mesenchymomas
• Some leiomyosarcomas
 Amplification of MDM2/CDK4
Well-differentiated adipous tumors

• Well-differentiated liposarcomas
• Versus: lipomas with secondary changes
spindle cell/pleomorphic lipomas
fibrolipomas
lipoblastomas
 84 year woman. 2 cm tumor
nodule in the pharynx

FISH and IHC : mdm2 and cdk4 -

Pleomorphic lipoma

MDM2
 75 year old man
3 cm tumor of the pharynx
 MDM2

IHC : MDM2 +
FISH : ampli MDM2
Spindle cell liposarcoma
Genomic abnormalities in sarcomas

• Reciprocal translocations
• Gene amplifications
• Mutations
• Deletions
 KIT mutations in GIST

Hirota et al, Science 1998;279: 577-80

• Immunohistochemistry of KIT positive in 46/49 GIST

(94%)
• 5/6 with mutation of KIT
• Possible origin from cells of Cajal
• Mutated forms of KIT show a TK activity in vitro
 GIST and mutation of KIT

C-kit or CD117
• growth factor receptor
• tyrosine kinase type 3 activity
if presence of ligand
• GIST :
- mutation of KIT
- mutation of PDGFR alpha
- no mutation
Mitogenic
signal
 Mutations of KIT and PDGFRA in GIST

KIT (80%) PDGFRA (7,5%)

Global frequence of
mutations : 87,5%

Exon 9 (11%)
Membran
Exon 11 (67,5%) Exon 12 (0,9%)
Exon 13 (0,9%) Exon 14 (0,3%)

Exon 17 (0,5%) Exon 18 (6,3%)

Heinrich et al. Hum Pathol. 2002;33:484. Cytoplasm
Corless et al. Proc Am Assoc Cancer Res. 2003;44. Abstract
 Mutations of KIT and PDGFRA in
GIST

• DNA from frozen or paraffin tissues

• PCR for exons 9, 11, 13, 17 of KIT and
exons 12, 14 and 18 of PDGFRA
• DHPLC (mutation screening)
• Sequencing of the mutated exon
 Mutations of KIT in GIST

Anatomo-clinic characteristics

• KIT exon 9
– Small intestine in 95% of cases
– More frequent in malignant forms
• KIT exon 11
– No particularity
 Mutations of PDGFRA in GIST

• Mutations of PDGFRA in 5-7% of GIST

– Only one mutation in a tumor
– 35% on non-mutated KIT GIST KIT show a mutation of
PDGFRA
• Target of KIT activated by mutated PDGFRA
• Anatomo-clinic characteristics
– Mainly gastric
– Epithelioid form with low mitotic activity
(81% <5 mit/50HPF)
– Good pronosis (83,5% with benign outcome)

Heinrich et al, Science 2003;299: 708-10

Lasota et al, Lab Invest 2004;84: 874-83
 Prognosis of GIST with Glivec and types of
mutation

100 KIT exon 11 (n=85)

Overall Survival (%)

90
80 KIT exon 9 (n=23)
70
60
50
40 No mutation (n=9)
30
20
10
0
0 100 200 300 400 500 600 700 800
Days
Heinrich et al. J Clin Oncol.
2003;21:4342.
 Response to Glivec and mutation of GIST

• KIT exon 11 (67%) : good esponse

(83,5% de RP)
• KIT exon 9 (18%) : intermediate response (47,8% de
RP)
• No mutation and PDGFRA D842V (14%) : poor
response

Heinrich et al. J Clin Oncol. 2003;21:4342.

 Molecular biology in GIST

Practical interest

• Diagnosis of negative KIT (IHC) GIST

• Prediction of response to Glivec
• Future therapeutic choice ?
 Molecular classification of sarcomas

• Sarcomas with reciprocal translocation (30%)

• Sarcomas with simple amplifications (15%)
• Sarcomas with gain of function mutations (GIST)
• Sarcomas with complex abnormalities
Leiomyosarcomas
Pleomorphic rhabdomyosarcomas
Pleomorphic liposarcomas
Osteosarcomas
Myxofibrosarcomas
MFH and poorly diff sarcomas
 Conclusions
• Diagnosis of soft tissue tumors : HE ± IHC
• Importance of genomic tools : translocations,
amplifications, ….
• Most techniques are applicable on PET
• Problem of sarcomas with complex genomic