a brief discussion on molecular and cytogenetic updates on soft tissue tumours

1. CYTOGENETIC AND MOLECULAR
PATHOLOGY OF SOFT TISSUE
TUMOURS
PRESENTER:DR.KAVVYA R
PSG MEDICAL COLLEGE

2. INTRODUCTION
• To aid in doubtful histopathological diagnosis
• Unusual clinical presentations
• Improved diagnostic accuracy
• More accurate definition of disease entities
• Therapeutic molecular targets

3. GENOMIC ABNORMALITIES

4. GENOMIC ABNORMALITIES

5. SOMATIC MUTATIONS
Neoplastic cells
Driver mutations
Self sufficient growth signals
Reduced sensitivity to inhibitors
Evasion of apoptosis &angiogenesis

6. • Driver mutations : oncogenes, tumor suppressor
genes & caretaker genes
• Passenger mutations : little or no impact on
tumorigenesis
• Important mechanisms: Small genetic variants,
chromosomal imbalances (copy number changes)
and gene fusions
SOMATIC MUTATIONS

7. Small Genetic Variants
• Mutations affecting a single nucleotide(SNV)
• Insertion/deletions
• Telomerase reverse transcriptase enzyme SNVs in
myxoid liposarcoma and solitary fibrous tumor
• KIT and PDGFRA genes in GIST is significant in pattern
and TKI dose

8. Small Genetic Variants
PDGFRA-mutant GISTs - limited expression of KIT

9. Chromosomal Imbalances
• Structural or numeric rearrangement-
quantitative deviation from the normal state
• Numeric-gain or loss of individual
chromosomes (aneusomy) to gain or loss of
one or more copies of the entire genome
(ploidy shift)

10. • Schwannoma - inactivation of the NF2 gene,
which maps to chromosome band 22q11
• Spindle cell lipomas- partial deletions with
chromosome 13q14
• Lipoblastoma -gene fusions activating PLAG1,
maps to chromosome 8
Chromosomal Imbalances

11. Chromosomal Imbalances
Karyotype-Polysomy chr.8 FISH analysis-abnormal chr.8

12. Gene Fusions
Structural chromosome rearrangements
Reshuffles the genetic material
juxtapositioning of two genes
Translation of a chimeric protein
Neoplasia

13. Gene Fusions

14. Gene Fusion Tumor Type Frequency
(%)
ACTB-GLI1 Pericytoma with t(7;12) 100
ASPSCR1-TFE3 Alveolar soft part sarcoma 100
SERPINE1-FOSB Pseudomyogenic
hemangioendothelioma
100
COL1A1-PDGFB Dermatofibrosarcoma protuberans >95
EWSR1-WT1 Desmoplastic small round cell tumor >95
FUS-DDIT3 Myxoid liposarcoma 95
NAB2-STAT6 Solitary fibrous tumor >95
Gene Fusions

15. Chromosome banding analysis
• Excellent screening method for detecting both
numeric and structural chromosome
aberrations
• Performed on cells in mitosis, more at the
metaphase
• Fresh tumor tissue, obtained within 2 to 4
days after sampling.
• Cells can be cultured, typically for 1 to 7 days,
to achieve metaphase

16. Chromosome banding analysis

17. Chromosome banding analysis

18. Genomic Arrays
• Genomic imbalances (i.e., gains and losses of
chromosomal segments) in tumor cells
• Detected by hybridizing extracted DNA known
as probes
• The signal intensity depends on the amount of
DNA attaching to a certain probe
• Fails to identify balanced chromosomal
rearrangements

19. FISH
– Directed technique that detects all rearrangements
of a particular locus
– Does not provide insight into the specific fusion
variant or type
– Cannot distinguish between two tumors that
harbor rearrangements of the same locus
– Fusion or combination FISH can help
– Can be applied to various preparations of fresh and
fixed material

20. FISH
HMGA2 gene in atypical lipoma BAP for the EWSR1 gene

21. Gene Panels
• Predesigned gene sequencing panels for mutations in
genes or parts of genes
• The number of genes in the panels varies from less than
20 to more than 4000.
• The main benefits of gene panels are that they require
low input of DNA or RNA
• Gold standard for identifying mutations that predict
response to therapy Ex:GIST

22. RT-PCR
– Directed technique that demonstrates a specific
fusion type and variant
– Can demonstrate a specific fusion transcript for
diagnosis or prognosis
– Can be adapted to track minimal residual disease
– Various fresh and formalin-fixed tissues can be
used

23. Next Generation Sequencing(RNA)
– Can look for many translocations at once
– Virtually all fusions partners for the same gene
can be detected
– Technique is very sensitive
– Sample preparation is more complex
– Bioinformatic interpretation is involved
– Various fresh and formalin-fixed tissues can be
used

24. – Many genes assessed for mutations, insertions,
and deletions and copy number
– alterations
– Must make sure that the gene panel is appropriate
for sarcoma
– Translocations are more challenging to detect with
this technique
– Very small amounts of DNA are needed
– Various fresh and formalin-fixed tissues can be
used
Next Generation Sequencing(DNA)

25. Interdependence of Factors

26. When to Consider Molecular Diagnostics?
• Small biopsy
• Poor histologic preservation
• Unusual morphologic features
• Discordant immunohistochemical results
• Uncharacteristic clinical features:
– Patient age or gender
– Rare site of involvement
• Extensive treatment effect
• Necessary for definitive treatment
• Provides prognostic information:
– Tumor natural history
– Response to treatment
• Rarely encountered entity for diagnosing pathologist

27. CHARACTERISTIC GENETIC CHANGES
• Clinically relevant aspects
• Diagnostic purposes, treatment stratification,
and targeted therapies
• Tumors are grouped according to major
lineage of differentiation

28. Fibroblastic/Myofibroblastic Tumors
• Nodular Fasciitis and Related Tumors
– Only Few cases have chromosomal aberration
– > 90% show MYH9-USP6 gene fusion
• Desmoplastic fibroblastomas
– Have pseudodiploid karyotypes
– translocations of 11q with different partner chromosomes.
– FISH analysis
– IHC: FOSL1
• Calcifying aponeurotic fibroma
– FN1-EGF fusion gene
– upregulation of EGF mRNA

29. Fibroblastic/Myofibroblastic Tumors
• Superficial fibromatoses Desmoid Type Fibromatosis
– Mostly of normal Karyotype
• Giant cell fibroblastoma (GCF) and dermatofibrosarcoma
protuberans (DFSP)
– In addition to overlapping morphologic features, they
share the same pivotal driver mutation, COL1A1-
PDGFB gene fusion(t(17;22)).

30. Fibroblastic/Myofibroblastic Tumors
• Solitary fibrous
tumor (SFT)
– No chromosomal
Abberations
– By transcriptome or
WES approaches,
NAB2-STAT6 fusion is
detected.
– Difficult to detect by
FISH and RT-PCR

31. • Low-grade fibromyxoid sarcoma (LGFMS)
and sclerosing epithelioid fibrosarcoma (SEF)
– t(7;16)  FUS-CREB3L2 fusion
Fibroblastic/Myofibroblastic Tumors

32. Adipocytic Tumors
• Conventional lipoma
– chromosome banding : 2/3 rd cases show clonal
aberrations
• translocations of chromosome bands 12q13-15 (66%)
• loss of material from chromosome arm 13q(15%)
• supernumerary ring chromosomes (5%)
• structural rearrangements of band 6p21 (5%)

33. • Angiolipomas
– lack chromosome-level aberrations
– only recurrent somatic events are SNVs affecting
the PRKD2 gene
• Chondroid lipoma
– tumor-specific t(11;16)C11orf95-MKL2 gene fusion
• spindle cell/pleomorphic lipomas
– 44 to 46 chromosomes, with monosomies or partial
deletions
– chromosomes 13 and 16
Adipocytic Tumors

34. Adipocytic Tumors
• Hibernomas
– chromosome bands 11q12-q13
Whole chromosome paint probe for chromosome 11

35. Adipocytic Tumors
• Atypical lipomatous tumors:
– The essential genetic event is amplification of
genetic material from the long arm of
chromosome 12.
– Cytogenetics:supernumerary ring chromosomes,
• Dedifferentiated liposarcoma:
– no specific genetic marker distinguishing from ALT
– amplification of genes (JUN in 1p32
and MAP3K5 in 6q23) involved in the Jun-pathway

36. • Myxoid liposarcoma
– t(16:12)  FUS-DDIT3 fusion gene
Adipocytic Tumors

37. Skeletal Muscle Tumors
• Embryonal rhabdomyosarcoma: different
constitutional predisposing mutations, affecting
Hedgehog, RAS, PIK3CA, and DNA repair pathways
• Most common chromosomal imbalance is +8
• Embryonal RMS shares deregulation pathway with
alveolar rhabdomyosarcoma

38. • Epithelioid hemangiomas: recurrent ZFP36-FOSB
fusion-increased expression of FOSB gene
• Strong nuclear staining can be demonstrated for
FOSB gene
• Epithelioid hemangioendothelioma: WWTR1-
CAMTA1 fusion, with breakpoints in exons 3 or 4 and
8 or 9
Vascular and Perivascular Tumors

39. Vascular and Perivascular Tumors

40. Ewing Sarcoma and Undifferentiated Sarcomas
• Ewing sarcoma
– first solid tumor types found to be characterized
by gene fusions.
– tumor-specific t(11;22)(q24;q12) EWSR1-FLI1
– FLI1 belongs to the ETS family of transcription
factors

41. Ewing Sarcoma

42. Genetic Disorders Associated with SFT

43. Genetic Disorders Associated with SFT

45. References
• SEVENTH EDITION- Enzinger & Weiss’s Soft Tissue Tumors
• WHO classification of soft tissue and bone tumours-fifth
edition
• Bridge JA. The role of cytogenetics and molecular diagnostics
in the diagnosis of soft-tissue tumors. Mod Pathol. 2014
Jan;27 Suppl 1:S80-97. doi: 10.1038/modpathol.2013.179.
PMID: 24384855
• Robbins &Cotran Pathologic basis of disease –SOUTH ASIA 9th
edition