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Characterizing the DDR pathway in acutely treated testicular germ
cell tumors
Alicia Braxton
Testicular Germ Cell Tumors
• Extremely sensitive to chemotherapy• Curable with chemo + surgery– Over 99% of patients
• Curable even after metastasis occurs– 85% success rate of treatment once metastasis
develop (5 years post treatment)
Huddart, R. A. et al . (2003). Management of Testicular Germ Cell Tumors. American Journal Of Cancer, 2(5), 325-334.
Somatic tumors are chemoresistant
• Chemotherapy resistance poses a challenge for treatment
• Accumulate mutations in DDR pathways– Ex: p53
• Infer: Compromised repair pathways chemotherapy resistance
Germ Cells avoid mutations in their DDR pathways.
Perhaps this makes the TGCT sensitive to chemotherapy treatment.
Bartkova, J. et al. (2007). DNA Damage Response in human testes and testicular germ cell tumors: biology and implications for therapy. International Journal of Andrology, 30, 282-291
Aim: What mechanisms make TGCT sensitive to chemotherapy?
If we are able to identify the mechanism conferring chemotherapy sensitivity to TGCT,
perhaps we could apply that knowledge to the treatment of more resistant somatic cell
tumors.
Types of TGCTsGonadal Germ Cell Cancers
OvarianTesticular
Seminoma Non-seminoma
Yolk SacTumor
ChoriocarcinomaTeratoma
EmbryonalCarcinomaTeratocarcinoma
Rare Most CommonCancer of Young Men
Tim Pierpont
Skeletal Muscle(Mesoderm)
Embryonal Carcinoma(Pluripotent Cell Type)
Ciliated Respiratory(Endoderm)
Neural Like(Ectoderm)
Embryonal Carcinoma(Pluripotent Cell Type)
Teratoma and Embyronal Carcinoma Components
Teratocarcinomas
Tim Pierpont
Cancer Stem Cell• 2 properties– Self renew– Differentiate
• Current cancer treatment strategy
• Target cancer stem cells: Inhibit regrowth of tumor
Tim Pierpont
A Novel Mouse Model for TGCTs
• Novel TGCT on mice 129 background• Undergone 2 oncogenic events
• Original thinking: Post-natal development of a more malignant tumor in the testes
LSL-KrasG12D STOP 1
G12D
Ptenflox Exon 5
Inactive
Active Pten Δ5 INACTIVEOncogenic!
Tim Pierpont
Lox-KrasG12D 1
G12D
ACTIVEOncogenic!Stra8-Cre
Human treatment of TGCT• Chemotherapy + Inguinal orchidectomy • Chemo: BEP protocol– Bleomyocin– Etoposide– Cisplatin
• 3-4 cycles of treatment depending on stage of cancer
• Cisplatin and etoposide introduced in late 70s/early 80s
Huddart, R. A. et al . (2003). Management of Testicular Germ Cell Tumors. American Journal Of Cancer, 2(5), 325-334.
Cisplatin
• Platinum containing chemotherapuetic agent• Covalent cross-linking of DNA double
stranded breaks apoptosis • Used in people to treat TGCT, as well as
ovarian, bladder and cervical cancers• Malignant embryonic carcinoma cells-
sensitive to Cisplatin
http://chemocare.com/chemotherapy/drug-info/cisplatin.aspx#.U80vNfldUsA
Cisplatin : Pharmacology, clinical uses and adverse effects. 2012. Hauppauge, NY, USA: Nova Science Publishers, Inc.
Increased survival with Cisplatin
Tim Pierpont
Kaplan Meier Tumor Free Survival Curve
Oct4
HRP
Immunohistochemical staining
Tim Pierpont
Cisplatin decreases Oct4 cells
Tim Pierpont
Increased survival with treated allograft tumors
10k
Treated1mil
Treated100k
Treated25k
Cum
ulati
ve su
rviv
al
Days
0%
20%
40%
60%
80%
100%
0 605040302010
Tim Pierpont
We know the mice are sensitive to Cisplatin treatment: Now we are working to determine
the mechanism behind their sensitivity
What makes TGCT chemosensitive?
• Mice with tumors injected IP with Cisplatin• Tumors collected at various time points post
treatment– 6, 12, 24, 36, 48, and 96 hours
• Cassette, embed, section and fix samples to slide
• Stain using IHC techniques
Initial Markers of Interest
• Oct4- TF that promotes pluripotency– Expressed by malignant embryonic carcinoma cells
• TUNEL- tags apoptotic cells• yH2AX- identifies double stranded breaks in
DNA– shows the DNA damage
• Ki67- marks proliferating cells
Help us gauge at which time points to look closer with more specific markers: p53, pCHK1,
and other markers of DDR pathway
Damage has occurred by 12 hours
0 12 hr 96 hr48 hr24 hr
Cisplatin Tr
eatment
Cells dyin
g off, no prolife
ration
Decreased Oct4
cluste
rs
Proliferation lim
ited to
Oct4 ce
lls
Apoptosis sto
ps
6 hr
?
A new data set: 6 hours post treatment
Ki67H&E yH2AX TUNEL Oct4
Mouse 2753
Ki67 Oct4yH2AX TUNELH&E
Mouse 2557
No Oct4 6 hours post treatment
6 hr
36 hr
12 hr 24 hr
96 hr
0 24 36 48 96
Oct4
1260 24 36 48 96126Expected Actual
Oct
4
Untreated
48 hr
Two waves of damage: 6 & 36 hrs
0 24 36 48 96
yH2AX
126
6 hr
36 hr
12 hr 24 hr
96 hrNot a serial sectionExpected Actual
0 24 36 48 96126
yH2A X
Untreated
48 hr
Two waves of apoptosis: 12 & 48 hrs
6 hr
36 hr
12 hr 24 hr
96 hrExpected Actual
0 24 36 48 96
TUNEL
120 24 36 48 96126 6
TUN
EL
Untreated
48 hr
Proliferation peaks at 24 & 96hrs
0 24 36 48 96
Ki67
126
6 hr
36 hr
12 hr 24 hr
96 hr
0 24 36 48 96126Expected Actual
Ki67
Untreated
48 hr
0 24 36 48 96
Oct4
126
TUNEL
yH2AX
Ki67
Overview of trends0 24 48 72 96
TUNNEL
y-H2AX
Ki67
Previous Results
Current Results
Future Research
• Repeat TUNEL on all samples• Look into other markers of the DDR pathway– p53, pCHK1– Differentiation-Sox17
• Collect another 6 hour post treatment sample– Oct4 negative existing samples
• More acute time point– 2 hours
Thanks ToTim PierpontAmy LyndakerClaire AndersonPei Xin LimErin DaugherityKelly HumeJoanna MleczkoElizabeth MooreYashira Negrón
David KarambiziCindy LuanEllen HongZanah FrancisCharlton Tsai
Thank you!
Cornell PathologistsDonald H. Schlafer,Teresa L. Southard
Committee
Robert WeissDoina TumbarJohn Schimenti
Duke UniversityMatthew S. Cook
Blanche Capel
Collaborators