Primary bone tumors can be benign or malignant and affect people of a wide age range. Molecular profiling using techniques like next generation sequencing has helped distinguish tumors with overlapping features and identified recurrent genetic alterations specific to different tumor types. For example, chondrosarcomas commonly harbor mutations in IDH1 and IDH2, while giant cell tumor of bone has H3F3A alterations in over 95% of cases. This molecular information can provide diagnostic, prognostic and predictive insights and help guide targeted therapy development.

1. MOLECULAR PROFILING OF
OSTEOARTICULAR NEOPLASM
Dr.Manjula
1

2. BONE TUMORS
• Primary bone tumors comprise both benign
and malignant tumors affecting people with
wide age range.
• Benign are more common than malignant
tumors.
• Diagnosis heavily relay on imaging findings.
2

3. BONE TUMORS
• Thus, the diagnosis and treatment is best
accomplished with a multidisciplinary
approach consisting of surgeons, radiologists,
pathologists, and oncologists to produce
optimal patient care.
3

4. BONE TUMORS
• Traditionally, diagnosis has relied on
histopathological assessment of tumor tissue
combined with clinical and radiological
correlation.
• This has been supplemented with cytogenetic
analysis including karyotype analysis and
fluorescence in situ hybridization (FISH).
• Immunohistochemical analysis has played
little role in the diagnosis of bone tumors.
4

5. MOLECULAR GENETICS IN BONE
TUMORS
• Although recent molecular advances have
provided pathologists with specific targets
amenable to antibody interrogation in some
tumors such as GCT of bone and
chondroblastoma.
5

6. MOLECULAR GENETICS IN BONE
TUMORS
• Polymerase chain reaction (PCR) and
massively parallel next generation sequencing
(NGS) based assays have provided additional
tools for assessing molecular alterations in
bone tumors.
6

7. CLASSIFICATION
• BENIGN:Enchondroma
• Osteochondroma
• Chondroblastoma
• Chondromyxoid fibroma
• MALIGNANT:Chondrosarcoma
CHONDROGENIC
TUMORS
• BENIGN:Osteoma
• Osteoid osteoma
• Osteoblastoma
• MALIGNANT:Osteosarcoma
OSTEOGENIC
TUMORS
7

8. • Pseudomyogenic
hemangioendothelioma
• Epithelioid hemangioma
VASCULAR
TUMORS
• Aneurysmal bone cyst
• Giant cell tumor
OSTEOCLASTIC
GIANT CELL
RICH TUMORS
• Chordoma
NOTOCHORDAL
TUMORS
8

9. • Fibrous dysplasia
• Adamantinomas
MESENCHYMAL
TUMORS
• LCH
HEMATOPOIES
TIC
NEOPLASMS
9

10. INTRODUCTION
• Large scale sequencing of DNA and RNA from
bone tumors has revealed many recurrent
alterations that are exclusively meant for
different tumor types.
• This has transformed the ability of pathologists to
distinguish tumors with overlapping histologic
features.
• These are now included as a part of laboratory
diagnostic armoury.
10

11. INTRODUCTION
• Next phase of research in this area is to
determine the genetic profiles of bone tumor
which can be employed as prognostic and
predictive markers .
• If targeted therapies can be developed against
these alterations.
11

12. CARTILAGINOUS TUMORS
• They are the M/C group of primary bone
tumor.
• Osteochondroma – bone surface.
• Enchondroma - central within medullary
cavity.
• Both can transform into chondrosarcoma.
12

13. OSTEOCHONDROMA
• Exostosis - M/C benign bone tumor.
• 85% are sporadic.
• 15% are part of Autosomal Dominant, Multiple
Hereditary Exostosis Syndrome.
• Endochondral in origin.
• M/C site - metaphysis near growth plate.
13

14. • Gross image of osteochondroma; notice the
hyaline cartilage cap overlying mature
cancellous bone.
• Low power view of osteochondroma (4x);
cartilage cap lined by perichondrium,
contiguous with mature bone.
14

15. OSTEOCHONDROMA
• M/E - Cap composed of mature hyaline cartilage with
overlying fibrous perichondrium.
• Germline loss of function mutation in EXT1 and EXT2.
• Reduced expression of EXT1 and EXT2 is observed in
sporadic cases too.
• These genes encode enzymes that synthesize heparan
sulfate glycosaminoglycans(GAGs).
15

16. MULTIPLE HEREDITARY EXOSTOSES (MHE)
• MHE is caused by germline mutation in the
EXT1 (at 8q24) or EXT2 (at 11p11–12) genes.
• These genes have been implicated in the
formation of both sporadic and hereditary
osteochondromas.
• Defects in EXT1 are roughly twice as common
as defects in EXT2.
16

17. CHONDROMA
• Benign tumor of hyaline cartilage.
• Endochondral in origin.
• Mutation in IDH1 and IDH2.
17

18. CHONDROSARCOMA
• Malignant cartilage producing tumor.
• Mutation in EXT gene.
• Sporadic chondrosarcoma harbour mutation
in IDH1 and IDH2.
• Silencing of CDKN2A locus by DNA
methylation is also observed.
18

19. CHONDROSARCOMA
• Whole genome sequencing of
chondrosarcoma revealed mutation in TP53
and CDKN2A.
• Detection of IDH 1/2 helps in differentiating
dedifferentiated chondrosarcoma with
osteosarcoma component from primary
osteosarcoma of bone.
• Since the treatment of both varies.
19

20. CHONDROSARCOMA
• IDH 1 mutation in chondrosarcoma also show
expression of brachyury – the diagnostic hallmark
of chordoma.
• HEY1-NCOA2 fusion – characteristic of
mesenchymal chondrosarcoma.
• An association between 6q13–21 chromosome
aberrations and locally aggressive behavior has
been described in chondrosarcomas.
20

21. CHONDROSARCOMA
• Central conventional and periosteal cartilaginous
tumor and dedifferentiated chondrosarcoma
harbour mutation in IDH 1 or 2.
• These mutant IDH 1 / 2 fails to convert isocitrate
to alpha ketoglutarate and leads to accumulation
of D-2-hydroxyglutarate (2HG).
21

22. CLEAR CELL CHONDROSARCOMA
• Cytogenetic analysis has shown recurrent
chromosome 9 and 20 abnormalities.
• IDH mutations are absent.
22

23. Myxoid Chondrosarcoma
(Chordoid Sarcoma).
• EWSR1 and NR4A3 gene rearrangements are
observed.
23

24. Dedifferentiated Chondrosarcoma
• Harbor IDH1/2 mutations.
• At the molecular level, the process of
anaplastic transformation is accompanied by
overexpression of TP53 and HRAS mutation.
24

25. MESENCHYMAL CHONDROSARCOMA
• It is non conventional chondrosarcoma representing
2%.
• M/C in vertebral bodies in head and neck region.
• They are strongly reactive for CD 99 and are
mistaken for Ewing’s.
• Fusion transcript in HEY1-NCOA2 is recently
identified using exon expression data by rapid
amplification of cDNA PCR.
• This is also detected by FISH and RTPCR. 25

26. 26

27. MULTIPLE ENCHONDROMA
• It is a group of diseases with spectrum of overlapping
phenotype.
• M/C variant is Ollier disease.
• Second M/C is Maffucci syndrome.
• They have high risk of glioblastoma.
• >90 % have IDH 1 / 2.
• Germline alteration include PTPN11, which encodes
protein tyrosine phosphatase.
27

28. MULTIPLE ENCHONDROMA
• Non receptor type 11 and ACP5 encodes
tartrate resistant acid phosphatase.
• PTHR1 alteration is associated with multiple
enchondroma.
• IDH 1/2 mutation were first identified in 2008
in brain tumors.
• Now in recent times, a vaccine targeting
mutant IDH1 has been developed.
28

29. CHONDROMYXOID FIBROMA
• It is a benign nonconventional cartilaginous
tumor.
• Nord et al recently identified that structural
rearrangements involving promoter swapping
and gene fusion resulting in aberrant
expression of glutamate receptor gene, GRM1
which is a G protein coupled receptor.
https://jcmtjournal.com/article/view/3898 29

30. CHONDROMYXOID FIBROMA
• Cytogenetically, chondromyxoid fibroma is
characterized by rearrangements of
chromosome 6 at band q13 or q25.
• Aberrations in 6q13 map to the COL12A1
locus is also seen.
30

31. OSTEOCLAST RICH NEOPLASM
• In recent times, genetic alterations of number
of osteoclast rich lesions have been described.
• USP6 fusion transcript in 70% of ABC.
31

32. GIANT CELL TUMOR
OSTEOCLASTOMA
• H3F3A genetic alteration detected in >95% of
GCT.
• But H3F3B have never been detected in GCT
so far.
• GCT metastasing to lung showed G34W
alteration and TP53 was not detected.
32

33. GIANT CELL TUMOR
• Most cases exhibit chromosomal abnormalities,
usually in the form of telomeric association that
can involve a variety of chromosomes.
• Such as 11p, 13p, 14p, 15p, 19q, 20q, and 21p.
• Telomeric association is a rare form of
cytogenetic abnormality characterized by end-to-
end fusion of intact chromosomes.
33

34. CHONDROBLASTOMA
• >95% harbour mutation in H3F3A or H3F3B.
34

35. ANEURYSMAL BONE CYST
• USP6 fusion
transcript.
• It is most
commonly
seen in nodular
fascitis.
35

36. GIANT CELL GRANULOMA
• Did not harbour USP6 or H3F3 alteration.
• Still the genetic alteration remain to be
identified in these tumors.
36

37. BONE FORMING NEOPLASM
37

38. OSTEOID OSTEOMA
OSTEOBLASTOMA
OSTEOID OSTEOMA:
• Less than 2 cm.
• Severe nocturnal pain releived with PGE2.
OSTEOBLASTOMA:
• More than 2 cm.
• Unresponsive to aspirin.
38

39. OSTESARCOMA
• M/C primary malignant bone tumor.
• Bimodal age of distribution.
• M>F affected.
• Metaphyseal region.
• Osteosarcoma typically shows complex
karyotypes, with structural alterations (including
translocations) and numerical alterations (gain and
loss) involving multiple chromosomes.
39

40. OSTESARCOMA
Mutations in the following:
• RB mutation – 70% of sporadic cases.
• TP53 mutation.
• CDKN2A – encodes two tumor suppressor gene P16
and p14 are inactivated.
• MDM2 and CDK4 – inhibit p53 and RB function.
• Deletions and amplifications of chromosomes 3, 6,
and 8 result in gene alterations may have prognostic
significance, including LSAMP, RUNX2, and MYC.
40

41. HIGH GRADE OSTESARCOMA
• Recent studies show that in approx 20% of
cases show amplification of fibroblastic
growth receptor.
• This was found in cases which failed to
respond to chemotheraphy.
41

42. PARAOSTEAL AND LOW GRADE
CENTRAL OSTEOSARCOMA
• These are bone forming neoplasm.
• Low grade central osteosarcoma and fibrous dysplasia
cannot be easily differentiated since both have central
location.
• They are characterized by MDM2 gene amplification.
• Gene amplification can be detected by molecular
diagnostic techniques or MDM2 & CDK4 protein can be
detected by IHC if only decalcified tissue is available
42

43. FIBROUS DYSPLASIA
• Benign tumor with localized development
arrest.
• It is a mosaic disorder caused by substitution
of GNAS1 with frequent involvement of codon
201.
43

44. TUMORS OF UNCERTAIN
DIFFERENTIATION
44

45. EWINGS SARCOMA
• Malignant bone tumor characterized by primitive
round cells without obvious differentiation.
• It is a round blue cell tumor involving bone and
soft tissue mostly in children.
• It is characterized by the fusion of EWSR1 with FLI1
in 85 % cases.
• EWSR1 – chr 22 and FLI1 – chr 11.
45

46. 46
(a) H&E morphology of Ewing sarcoma/PNET.
(b) The tumor shows CD99 membranous pattern.
(c) FLI-1p (nuclear pattern, positivity by IHC.
(d) Confirmatory test by EWSR1 (22q12) dual-color, break-apart rearrangement
probe fluorescence in situ hybridization (FISH).
Separated red and green arrows demonstrate the genetic alteration, while the
two joint arrows are indicative for the intact chromosome.
INTACT
CHR

47. EWINGS SARCOMA
• About 95% of cases of Ewing family of tumors show
on cytogenetic examination the reciprocal
translocation t(11;22)(q24;q12) or t(21;22)(q22;q12),
which results in the fusion of the EWSR1 gene at
22q12 with the FLI1 or ERG genes respectively.
• The most common fusion is the one that results in
“in frame linking” of EWSR1 exon 7 with FLI1 exon 6.
• This gene encodes a chimeric protein EWS/FLI1 that
binds to chromatin and dysregulates transcription
leading to uncontrolled growth and abnormal
differentiation. 47

48. EWINGS SARCOMA
• These translocations are useful diagnostically and can be
detected by RT-PCR, can also be detected by molecular
cytogenetic analysis (FISH).
• It has been demonstrated that those tumors harboring
ERG abnormalities are frequently negative for EWSR1.
• Two other “Ewing-like” sarcomas have been identified.
• CIC-DUX4 tumors closely resemble Ewing sarcoma
histologically and immunohistochemically but are
frequently positive for WT1 and harbor the
t(4;19)(q35;q13.1).
48

49. EWINGS SARCOMA
• These tumors appear to be even more clinically
aggressive than Ewing sarcoma.
• In the remaining 5% cases EWSR1 or FUS fuses with other
ETS and non-ETS family genes such as ETV1, ETV4, ERG,
NFATC2, SMARCA or SP3.
• The other recently discovered Ewing-like tumor is
characterized by the BCOR-CCNB3 gene fusion.
• These tumors contain both round cell and spindled cell
elements and often lack the diffuse membranous CD99
immunoreactivity seen in Ewing sarcoma.
49

50. PHOSPHATURIC MESENCHYMAL TUMOR
• Rare bone tumor common in patients with long
standing osteomalacia who are resistant to
vitamin D and Calcium.
• FGF23 has been found to have a role in
phosphate homeostasis.
• Removal of this tumor results in dramatic drop in
circulating levels of FGF23 and reversal of
osteomalacia.
• Recently FN1-FGFR1 gene fusion has been
observed in 60% cases.
50

51. PSEUDOMYOGENIC
HEMANGIOENDOTHELIOMA
• Unusual tumor occuring in sites like subcutis,
deep soft tissue and bone.
• Behaves in an indolent manner with multifocality
and rarely metastases.
• IHC – immunoreactive for CK, CD31, ERG can lead
to misdiagnosis.
• But SERPINE1-FOSB fusion gene helps in
diagnosis.
51

52. EPITHELIOID HEMANGIOMA
• Locally aggressive tumor.
• ZEP36-FOSB fusion has been reported in cases
with atypical features.
52

53. CHORDOMAS
• Expansile lobulated intraosseous mass that usually
permeates the cortex and invades adjacent soft
tissue.
• Cut surface is gelatinous to chondroid
• Grossly, this chordoma has a fleshy cut surface
with invasion of the adjacent soft tissue.
53

54. CHORDOMAS
54
Low power architecture is lobular,
with fibrous bands separating
lobules.
Extracellular myxoid matrix

55. CHORDOMAS
• Chordomas commonly show hypodiploid
karyotype, frequently with loss of
chromosomes 3 (especially 3p), 4, 10, and 13.
55

56. ADAMANTINOMAS
• Adamantinomas commonly show recurrent
numerical chromosomal abnormalities, mainly
gain of chromosomes 7, 8, 12, 19, and 21.
56

57. LANGERHANS CELL HISTIOCYTOSIS
• LCH should be regarded as a neoplastic disease.
• Recently, it has been shown that a relatively high
percentage of cases of LCH harbour BRAF V600E
mutations.
• Resulting in activation of the MAPK pathway.
• A smaller percentage of lesions have mutations in
the MAP2K1 gene. 57

58. SUMMARY
58

59. BONE LESIONS MUTATION
Aneurysmal bone cyst USP6 gene rearrangement
Conventional central chondrosarcoma, high grade IDH 1/ 2 mutation
CDKN2A alteration
Nonconventional dedifferentiated central
chondrosarcoma
IDH 1/ 2 mutation
Conventional central chondrosarcoma, low grade
AND Enchondroma
IDH 1/ 2 mutation
Chondroblastoma H3F3B mutation
Chondromyxoid fibroma GRM 1 rearrangement
Epithelioid hemangioendothelioma WWTR 1 – CAMTA 1
Ewings sarcoma EWSR1 – FLI1 and EWSR1 - ERG
Fibrous dysplasia GNAS1 substitution
GCT BONE H3F3A substitution
Osteosarcoma, central high grade FGFR1 gene amplification
Osteosarcoma, central low grade MDM2 gene amplification 59

60. References
60

61. 61