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1. Molecular and Genetic Understanding of CRPC
Updates from Lab and Lessons for Clinic
Dr Alok Gupta
MD, DM,
Consultant Medical Oncologist
Max Super Speciality Hospital, New Delhi
Ex-Asst. Professor, AIIMS, New Delhi

2. Outline
 Genomic alterations in prostate cancer
 DNA damage response pathways
 DNA repair defects in advanced prostate cancer
 Therapeutic implications of DNA repair defects in advanced prostate
cancer
 HRD - PARP inhibitors and synthetic lethality
 MMR defects and immune checkpoint inhibitors
 Conclusions

3. Heritability of prostate cancer (PCa)
Family history is strong risk factor
• Nordic twin study1
• 57% heritability for PCa
• Only melanoma had higher heritability (58%)
• BUT…..identifying specific genes that contribute to this risk has proven challenging
• Genetic linkage studies in multi-case PCa families over 2 decades revealed few
candidates e.g. HOXB13 G84E mutation present in 3.1% of familial PCa2
1Mucci et al. JAMA. 2016 Jan 5;315(1):68-76
2Ewing et al. JAMA. 2016 Jan 5;315(1):68-76

4. Germline DNA repair gene mutations & Prostate Cancer
Higher risk of developing PCa
Mutated gene Increased risk of PCa Reference
BRCA2 Up to 8.6 fold Br J Cancer. 2011;105(8):1230-4
BRCA1 3.75 fold Br J Cancer. 2012;106(10):1697
ATM 2.2 fold Nat Genet. 2015;47(8):906
CHEK2 1.6 fold J Clin Oncol. 2016;34(11):1208
NBN 3.9 fold Cancer Res. 2004;64(4):1215
PALB2 ???
0.4% of metastatic PCa
N Engl J Med. 2016;375(5):443-53
MMR/Lynch 4.9 fold Genet Med. 2014;16(7):553

5. More aggressive PCa (vs. non-carriers)
• BRCA2
• Younger onset, higher T stage, higher Gleason, more LN involvement,
shorter PCa-specific survival and OS1,2
• BRCA2 or BRCA1 or ATM :
• 4-fold higher risk lethal PCa, shorter OS3
• BRCA1 :
• Higher recurrence rates, shorter PCa-specific survival4
1J Natl Cancer Inst. 2007;99(12):929
2J Clin Oncol. 2013;31(14):1748
3Eur Urol. 2017;71(5):740
4Clin Cancer Res. 2010;16(7):2115
Germline DNA repair gene mutations & Prostate Cancer

6.  11.8% (82/692) of men with metastatic
prostate cancer inherited a germline
DNA repair mutation vs 4.6% of
499 men with localized disease
Germline Mutations in Prostate Cancer: 1 in 10
Pritchard CC, et al. N Engl J Med. 2016;375:443-453.
Distribution of Presumed Pathogenic
Germline Mutations
PALB2 4%
RAD51D 4%
ATR 2%
NBN 2%
PMS2 2%
GEN1 2%
MSH2 1%
MSH6 1%
RAD51C 1%
MRE11A 1%
BRIP1 1%
FAM175A 1%
BRCA2 44%
ATM 13%
CHEK2 12%
BRCA1 7%
Gene No. of Mutations % of Men
BRCA2 37 5.35
ATM 11 1.59
CHEK2* 10 1.87
BRCA1 6 0.87
Presumed Pathogenic Germline Mutations
in Metastatic Cases (N = 692)
*n = 534; data censored for metastatic cases with inadequate sequencing.

7. References in slidenotes.
EZH2
AR gene expression
Mutation/gain/V7
PCA3
AMACR
Prostatic
intraepithelial
neoplasia
Localized
PCa
Loss of 8p
(NKX3.1)?
Activation of
β-catenin, wnt
Loss of APC
Castration-
resistant
PCa
Metastatic
PCa
Curable
Incurable
Normal
prostatic
epithelium
Epigenomic changes,
including GSTP1
hypermethylation
Inactivation of p53, RB1, CDK12
ETS gene family
activation via fusion
with TMPRSS2 or other
AR-driven genes
cMYC
Mutations in SPOP, FOXA1, epigenetic regulators
Anaplastic/
NE PCa
Loss of PTEN, 13q (RB1), 5q (CHD1),
16q, 6q, 3p (FOXP1, SHQ1)
Gain of 8q, 3q (PIK3CA)
Tx
Genomic Alterations in Prostate Cancer Progression
Compromised DNA repair

8. DNA Damage Response

9. DNA Damage Response (DDR)
 Cellular DNA is subject to continuous damage from both environmental
agents (mutagens) and endogenous sources
‒ 1000s of events/day (eg, ssDNA and dsDNA breaks, alkylation, x-linked)
 DDR has evolved to maintain DNA sequence and fidelity
‒ At least 6 different types of DNA repair processes exist to deal with wide
variety of DNA damage
 Inherited defects in DDR are among the most carcinogenic of all
hereditary syndromes (eg, Lynch syndrome, HBOC)
 Some DDR defects can be exploited for therapeutic benefit
O’Connor MJ. Mol Cell. 2015;60:547-560.

10. DNA repair gene mutations
• Evolution of next-generation sequencing has allowed germline DNA repair gene mutations
to be linked to PCa
Single strand DNA repair
pathways
Double strand DNA repair pathways
Mismatch repair (MMR) Homologous recombination (HR)
Base excision repair Non-homologous end joining (NHEJ)
Nucleotide excision repair
DNA damage response (DDR)

11. Consequences of DNA Damage
 If DNA damage correctly repaired, no consequences
 If no repair or inefficient repair due to defects in DDR genes
‒ Cell death is most common fate – unrepaired dsDNA breaks are lethal
‒ Can result in accumulation of DNA damage in the form of mutations
‒ Activation of oncogenes, inactivation of tumor suppressor genes
 Accumulation of DNA damage/mutations can lead to cancer
formation/progression
‒ Resulting cancer cells may be more susceptible to drugs that inactivate
remaining DNA repair pathways
Mateo J, et al. Euro Urol. 2017;71:417-425.

12. DNA Repair Defects in Prostate Cancer

13. DNA Repair Defects in Localized Prostate
Cancer
 Sequence analysis of localized, nonindolent prostate tumors with
similar risk profiles (N = 477)
– 200 whole-genome sequences, 277 whole-exome sequences
 47/477 (9.9%) tumors had DNA repair mutations
– FANCA (n = 9)
– ATM (n = 8)
– RAD51 (n = 7)
– CDK12 (n = 6)
– BRCA2 (n = 5)
Fraser M, et al. Nature. 2017;541:359-364.

14. DNA Repair Defects in mCRPC
 34/150 (22.7%) mCRPC pts had DNA repair alterations, many of
which were biallelic
– 19/150 (12.7%) with loss of BRCA2
– ~ 90% were biallelic, with 8/150 (5.3%) resulting from a pathogenic
germline BRCA2 mutation + a somatic event
– Recurrent biallelic loss of ATM also observed, including some
arising from pathogenic germline alterations
– Mutation events also observed in BRCA1, CDK12, FANCA,
RAD51B, and RAD51C
Robinson D, et al. Cell. 2015;5:1215-1228.

15. Genomic Landscape of Advanced Prostate Cancer
 23% of metastatic
CRPCs harbor DNA
repair alterations
 The frequency of
DNA repair
alterations
increases with
disease
progression
Robinson D et al, Cell, 2015
Robinson D, et al. Cell. 2015;161:1215-1228.

16. DNA Repair Defects in mCRPC: Enrichment Analysis in
Primary vs Metastatic Tumors
Tumor Samples (N = 918)
Primary
Metastasis
583
335
CARD11
Primary
DNA repair defects: 11%
Metastases
DNA repair defects: 21%
Significance
(Fisher’s qvalue)
Genomic Alteration Frequency
AlteredPrimarySamples(%)
Altered Metastatic Samples (%)
50
10
5
30
0
0 5 10 30 50
Amplification/mutation
Homdel/mutation
Mutation
SPOP
PTEN
KMT2C
KMT2D
MYC
FOXA1
TP53
RB1
BRCA2
AR
ZFHX3
CDK12 APC
IDH1
RYBP/FOXP1
JAK1
SPEN
IGF2R
PREX2
CTNNB1
CCND1
FAT1
MGA
MED12
USP28
ANKRD11
GNAS
ERF
CHD8
GRIN2A
RNF43
USP7 ASXL1
SAMD9
Armenia J, et al. ASCO 2017. Abstract 131.

17. Therapeutic Implications of
DNA Repair Defects in Advanced Prostate Cancer

18. PARP Biology
 PARP (polyADPribose polymerase) enzymes play a key role in the repair of
ssDNA breaks via BER pathway
 Bind directly to sites of DNA damage
 Once activated, uses NAD as a substrate to add large, branched chains of
poly(ADP-ribose) polymers (ie, PARylation) to itself and interaction partners
 Recruits other DNA repair enzymes to site of damage
DNA damage
NAD+ Nicotinamide
+ pADPr
Lig3XRCC1
Polß
PNK
PARP
Ohmoto A, et al. Onco Targets Ther. 2017;10:5195-5208.

19. PARPi Leads to Increase in dsDNA Breaks
 Inhibition of PARP
‒ Prevents recruitment of
DNA repair enzymes to
ssDNA breaks or traps
PARP on DNA
‒ Leads to failure of ssDNA
repair and accumulation
of ssDNA breaks
‒ Replication fork is arrested
at damage, produces dsDNA
breaks
Ohmoto A, et al. Onco Targets Ther. 2017;10:5195-5208.
PARP
XRCC1
DNA
Lig III
PNK 1
DNA
Polβ
During S-phase, replication fork is
arrested at site of ssDNA breaks
Degeneration into
dsDNA breaks
ssDNA
breaks
PARP inhibition

20. Synthetic Lethality Hypothesis
Farmer H, et al. Nature. 2005;434:917-921. Bryant et al. Nature. 2005;434:913-917.
Repair,
Survival
Repair,
Survival
Normal cell
Non-BRCA mutation carrier
PARP function
BRCA function
PARP inhibitor
PARP function
BRCA function
DNA damage
Repair,
Survival
Repair,
Survival
Normal cell
BRCA mutation carrier
(1 allele lost)
PARP function
BRCA function
PARP inhibitor
PARP function
BRCA function
DNA damage
Repair,
Survival
Cell Death
Cancer cell
BRCA mutation carrier
(both alleles lost)
PARP function
BRCA function
PARP inhibitor
DNA damage
PARP function
BRCA function

21. TOPARP: Trial of Olaparib in mCRPC
30 patients 20 patientsTotal: 50 patients
(49 evaluable)
Eligibility: Histologically confirmed metastatic CRPC, ECOG 0-2, no previous PARPi or platinum
Mateo J, et al. N Engl J Med. 2015;373:1697-1708.
FIRST PART:
TREATED
UNSELECTED
mCRPC pts
Randomized study
in unselected
mCRPC pts
Biomarker guided
patient selection in
next part (Part B)
END OF TRIAL
High response rate:
≥ 50% Responding
Intermediate RR
(10-50% responding)
Putative biomarker
identified (RR > 50%)
Low antitumor
activity RR < 10%

22. 49evaluable pts-RR 33%
16/49ptshadDNA repair
defect
-RR 88%

23. Radiologic PFS by Presence of Genomic
Defects in DNA Repair Genes
OS by Presence of Genomic Defects in
DNA Repair Genes
ProportionofPatients
ProportionofPatients
Mateo J, et al. N Engl J Med. 2015;373:1697-1708.
0
0.25
0.50
0.75
1.00
0 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20
Mos Since Trial Entry
Log-rank P < .001
Biomarker positive,
median: 9.8 mos
Biomarker negative,
median: 2.7 mos
0
0.25
0.50
0.75
1.00
0 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20
Mos Since Trial Entry
Log-rank P = .05
Biomarker positive,
median: 13.8 mos
Biomarker negative,
median: 7.5 mos
TOPARP-A: PFS and OS by Presence of DNA Repair
Defects
 All patients (N = 50) treated with olaparib 400 mg PO BID

24. De Felice F, et al. Drug Des Devel Ther. 2017;11:547-552.
Olaparib: Ongoing Studies in Prostate Cancer
Trial Study Design Eligibility Study Arms Endpoint Results
TOPARP Phase II Advanced CRPC Oral olaparib ORR 33% ORR
Kaufman, et al Phase II
BRCA1/2-mut adv solid
tumors (PC n = 8)
Oral olaparib ORR
PFS: 9.8 vs 2.7 mos
OS: 13.8 vs 7.5 mos
ORR in PC: 50%
NCT01972217
Randomized
phase II
mCRPC Olaparib + abiraterone Safety
PFS: 7.2 mo
OS: 18.4 mo
(ongoing study)
NCT02484404 Phase I/II
Adv/recurrent solid
tumors
Anti-PD-L1 + olaparib Safety
Recommended
dose (ongoing)
KEYNOTE 365 Phase I/II mCRPC
Anti-PD-L1 + cediranib
± olaparib
Pembro + olaparib
Safety
AEs, ORR, OS
(ongoing)
NCT02893917
Randomized
Phase II
mCRPC Olaparib + cediranib PFS
PFS, RR, OS
(ongoing)
NCT02324998 Phase I Int/high-risk PC Olaparib ± placebo
Degree of
PARPi
AEs (ongoing)

25. Mismatch Repair Defects

26. MMR Mutations in mCRPC
 3/150 (2%) had MMR mutations
 4/150 (2.7%) were MSI-high
Patient Cases With DNA Repair Defects
Reprinted from Robinson D, et al. Integrative clinical genomics of advanced prostate cancer. Cell.
2015;161(5):1215-1228. Copyright © 2015 with permission from Elsevier Inc.

27. PD-1 Inhibition in MMR-Deficient Cancers
Le DT, et al. ASCO 2016. Abstract 103. Le DT, et al. N Engl J Med. 2015;372:2509-2520.
Radiographic Response With Pembrolizumab
-100
MMR-P CRC
MMR-D CRC
MMR-D non-CRC
ChangeFromBLinthe
SumofLongest
Diameters(%)
20% increase (PD)
100
0
50
-50 30% decrease (PR)
Biochemical Response With Pembrolizumab
MMR-P CRC
MMR-D CRC
MMR-D non-CRC
ChangeinTumor
MarkerLevel(%)
Days
0% (no change)
200
0
-100
0 100 200 400300
100
Pt
PFS(%)
PFS with Pembrolizumab
Mos
MMR-D
(mPFS: NR)
100
50
0
0 3 6 12 15 18 21 24 27 30
MMR-P
(mPFS: 2.3 mos)
9
OS(%)
Mos
MMR-D
(mOS: NR)
OS with Pembrolizumab100
50
0
0 3 6 12 15 18 21 24 27 30
MMR-P
(mOS: 5.98 mos)
9

28. Pembrolizumab for MSI-H or MMR-Deficient
Cancers
 In May 2017, the PD-1 inhibitor pembrolizumab received
accelerated approval from FDA for:
– MSI-H or MMR-deficient CRC that has progressed following
treatment with a fluoropyrimidine, oxaliplatin, and irinotecan
– For ALL unresectable or metastatic MSI-H or MMR-deficient
SOLID TUMORS (pediatric and adult) that have progressed on
prior treatment and with no satisfactory alternative treatment
options
 Dosage and administration (MSI-H cancers): 200 mg IV every 3
wks
Pembrolizumab [package insert]. 2017.

29. KEYNOTE-016: Responses to Pembrolizumab in
MMR-Deficient Tumors
 Radiographic responses across 12 tumor types at 20 wks (N = 86)
Le DT, et al. Science. 2017;357:409-413.
Ampulla of Vater
Cholangiocarcinoma
Colorectal
Endometrial cancer
Gastroesophageal
Neuroendocrine
Osteosarcoma
Pancreas
Prostate
Small Intestine
Thyroid
Unknown primary
100
50
0
-50
-100
ChangeFrom
BaselineSLD(%)
Prostate
Prostate
(n = 1)

30. MMR Mutations in mCRPC
 4/150 (2.7%) mCRPC pts
were MSI-high, 3 of whom
had MMR mutations (2%)
– 13 mut/Mb (Pt #149) – MSH2
– 21 mut/Mb (Pt #147) – no
MMR mutation
– 23 mut/Mb (Pt #148) – MSH2
– 25 mut/Mb (Pt #150) – MSH2
and MLH1
Robinson D, et al. Cell. 2015;161:1215-1228.
MSI Analysis:
Hypermutated vs Nonhypermutated CRPC
FractionUnstableLoci
0 500 1000 1500
0.10
0
0.30
0.20
0.50
0.40
Nonsynonymous Mutations
Negative
MSI Positive
149
147
148 150
32, 41, 49, 67, 93

31. Conclusions
 DNA repair mutations are common in prostate cancer, particularly mCRPC
‒ Both somatic and germline mutations can lead to DNA repair defects
 20% to 30% of metastatic CRPCs harbor alterations in DNA repair genes
 HR DNA repair mutations sensitize to PARP inhibitors
‒ “Synthetic lethality” hypothesis in action
 MMR mutations are rare...but may sensitize to immune checkpoint
inhibitors

32. THANK YOU