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