How it is done!
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Transcript of How it is done!
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Cytogenetic Insights in Cytogenetic Insights in Mesenchymal TumorsMesenchymal Tumors
Jonathan A. Fletcher, M.D.Jonathan A. Fletcher, M.D.Pathology & PediatricsPathology & Pediatrics
Brigham & Women’s HospitalBrigham & Women’s HospitalDana-Farber Cancer InstituteDana-Farber Cancer Institute
Harvard Medical SchoolHarvard Medical SchoolBoston, MABoston, MA
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How it is done!
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Mince, then disaggregate cells by overnight treatment with collagenase
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Disaggregated cells are plated as monolayer
cultures on glass slides or in
plastic flasks
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All cultures are inspected daily, to determine whether tumor cells are growing, and when metaphase harvests should be performed
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Leiomyoma: simple karyotype with t(12;14)
HMGA2(HMGIC)
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Leiomyosarcoma: G-banded karyotype
Complex!
Clonal (arrows) andnonclonal aberrations
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Example 1
Novel biologic mechanisms revealed through indentification of recurrent
cytogenetic abnormalities in mesenchymal tumors
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Aneurysmal Bone Cystand the fusion fusion oncogene
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Aneurysmal Bone Cystand the fusion fusion oncogene
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Aneurysmal Bone Cyst
• Patients are generally < 20 years old• Can recur locally, but do not become
malignant• “Primary ABC” have been generally
regarded as nonneoplastic • “Secondary ABC” associated with
– osteoblastoma– chondroblastoma– giant cell tumor– osteosarcoma
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Aneurysmal Bone Cyst
• 1999: Panoutsakopoulos et al. reported translocation t(16;17) in two ABC– neoplastic basis– recurrent oncogenic mechanism
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Aneurysmal Bone Cyst• 17p13 rearrangements in:
– “solid variants” of ABC– soft-tissue ABC
• 25% of ABC have t(16;17)• >25% of ABC have alternate
translocations, involving 17p13, but not 16q22
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Aneurysmal Bone Cyst• t(16;17)
– 17p13 gene = USP6 (Ewing’s oncogene)– 16q22 gene = CDH11 (aka “osteoblastic
cadherin”)– promoter swapping between CDH11 and
USP6• fusion of highly active CDH11 promoter to the
5’ UTR of USP6
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Aneurysmal Bone CystCorroboration of “promoter swapping”
mechanism in USP6 fusions
Translocation GenePromoter
Swapping?
t(9;17) osteomodulin YES
t(17;17) COL1A1 YES
t(1;17) TRAP150 YES
t(3;17) ZNF9 YES
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Aneurysmal Bone CystWhat is the neoplastic cell?
Mechanisms in secondary ABC?
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USP6 oncogene in ABC spindle-cells
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USP6 oncogene is not found in“secondary ABC”
Secondary ABC Associated Chondroblastoma
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USP6 or CDH11 Rearrangement in ABC
• 36 of 52 (69%) primary ABC• 0 of 17 secondary ABC
– giant cell tumor– osteoblastoma– chondroblastoma– fibrous dysplasia
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USP6 is an evolutionarily-recent fusion of the PRC17 and USP32 genes
(hominoid specific)
USP6
PRC17 USP32TBC (rabGAP) UBP
TBC (rabGAP) UBP
>95% identity
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USP6 Function: regulates endocytosis/destruction of
activated proteins
TBC (rabGAP) UBP
- inactivates rab family members
-rab function required for endocytosis of activated EGFR
-UBP protease reverses ubiquitination
- ?synergize with rabGAP function to inhibit endocytosis/proteolysis
CDH11
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USP6 Expression • Normal expression restricted to
embryonic tissues and testis• Neoplastic expression restricted to
mesenchymal tumors:– 2 of 2 osteoblastomas– 1 of 4 myofibromas– 1 of 3 Ewing’s sarcomas
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Conclusions• USP6 is overexpressed due to promoter
swapping mechanisms in most primary ABC• USP6 overexpression may stabilize
oncogenic proteins• USP6 is an evolutionarily recent gene, with
likely relevance in sarcoma• Useful models of mesenchymal tumor
biology can come from unlikely places
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Example 2Smooth Muscle Tumors
• Use of cytogenetic clues to identify clinically-relevant biologic pathways in a genetically complex disease
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Leiomyoma: simple karyotype with t(12;14)
HMGA2(HMGIC)
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Leiomyosarcoma: G-banded karyotype
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How do leiomyomas progress to malignancy?
???
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Intravenous Leiomyomatosis
Typical uterine leiomyomaBalanced t(12;14)
Intravenous leiomyomatosisUnbalanced t(12;14)Partial trisomy 12q
Paola Dal CinBrad QuadeCynthia Morton
t(12;14) with:partial trisomy 12qpartial deletion 14q
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Cytogenetic correlates for leiomyoma progression
• Vascular invasion– intravenous leiomyomatosis– unbalanced t(12;14)
• Increased proliferation– cellular leiomyoma– deletion 1p (also common in lms)
• Distant metastases– “benign metastasizing leiomyoma”– deletions of 19q and 22q
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Pulmonary Chondroid Hamartoma(HMAG2 & HMGA1 oncogenes)
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PCH: primitive mesenchymal, fat, chondroid
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PCH: primitive mesenchymal, fat, chondroid, smooth muscle
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Leiomyosarcoma/Leiomyoma: where do they start?
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Is there proof that any sarcoma arises from a differentiated mesenchymal cell?
• osteo – bone• chondro – cartilage• lipo – fat• leiomyo – smooth muscle• rhabdo – skeletal muscle• fibro – myofibroblast• “GIST” – interstitial cell of Cajal
NO
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Andre Oliveira
Paola Dal Cin
Cynthia Morton
Marisa Nucci
Anette Duensing
Chang-Jie Chen
Nora Joseph
Bryna Mcconarty
Felicity Smith
Lynn Yu
Christopher Hubert
Maureen Thyne
Vicki Derr
Stana Weremowicz
George Demetri
Christopher Fletcher
Sam Singer
Antonio Perez-Atayde
Mark Gebhardt
Andrew Rosenberg
Julia Bridge
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THANK YOU!!!