Giuseppe Fornarini, U.O. Oncologia Medica 1 Ospedale Policlinico San Martino IRCCS Genova

Trattamenti della malattia con mutazioni di BRCA e altro… Background

• Prostate cancer is inheritable…but • Important to know the family history • A different type of are involved…BRCA1/2, Lynch Syndrome…others • Pts with BRCA2 mutation have 3-9x risk of PC and more lethal • Germline could be different from somatic mutation • In advanced disease clinical management does not differ • To whom should we offer germline genetic testing and when… because Germline pathogenic alterations may have both familial and therapeutic implications

To whom should we offer germline genetic testing?

Presented By Heather Cheng at 2018 ASCO Annual Meeting More recent genetic findings in advanced PC may also inform therapy

Presented By Heather Cheng at 2018 ASCO Annual Meeting

Outline

• The 6 DNA repair pathways • Prevalence of DNA repair defects in prostate cancer • DNA repair and therapeutic implications – PARP inhibitors – Hormonal therapy – Immune checkpoint inhibitors – Platinum chemotherapy

Emmanuel S. Antonarakis, MBBCh Single-Stranded (ss)DNA Repair Pathways • Mismatch repair – Base errors from DNA replication and recombination – MSH2, MSH6, MLH1, PMS2 • Nucleotide excision repair – DNA damage from UV light, polycyclic aromatic hydrocarbons – XPA-G, ERCC1-8, CSA/B, RPA, RAD23A/B • – DNA damage from alkylation, oxidation/ROS, deamination – PARP1/2/3, POLβ, MUTYH, XRCC1, MBD4, NTHL1

Mateo J, et al. Eur Urol. 2017;71:417-425. Emmanuel S. Antonarakis, MBBCh Double-Stranded (ds)DNA Repair Pathways • – DNA damage from ionizing radiation or other dsDNA injury – FANC genes, BRCA1/2, ATM, PALB2, RAD50, RAD51, NBN, GEN1, MRE11, BLM, ATR • Nonhomologous end joining – DNA damage from ionizing radiation or other dsDNA injury – XRCC4/5/6, LIG4, DCLRE1C, PRKDC, NHEJ1, POLL/M • Translesion DNA synthesis – Error-prone recovery mechanism when no DNA template – POLH, POLI, POLK, PCNA, REV1/3 (error-prone DNA polymerases)

Mateo J, et al. Eur Urol. 2017;71:417-425. Makridakis NM, et al. Front Genet. 2012;3:174. Emmanuel S. Antonarakis, MBBCh Germline Mutations in Prostate Cancer: 1 in 10 • 11.8% (82/692) of men with metastatic prostate cancer Distribution of Presumed inherited a germline DNA repair Pathogenic Germline mutation vs 4.6% of Mutations 499 men with localized disease

RAD51C Presumed Pathogenic Germline MSH6 1% 1% MRE11A Mutations MSH2 1% 1%BRIP1 1% GEN1 2% FAM175A PMS2 2% in Metastatic Cases (N = 692) NBN 2% 1% ATR 2% RAD51D PALB24% No. of % of Men 4% BRCA2 Mutations BRCA1 7% 44% BRCA2 37 5.35 CHEK2 12% ATM 11 1.59 ATM CHEK2* 10 1.87 13% BRCA1 6 0.87

*n = 534; data censored for metastatic cases with Pritchard CC, et al. N Engl J Med. 2016;375:443- inadequate sequencing. 453. Association Between Germline DNA- Repair Defects and Intraductal/Ductal Histology Distribution of Pathogenic • Germline mutations in Germline Mutations 14% (21/150) of men with CDH1 5% recurrent/advanced prostate cancer NBN 5% • Men with intraductal/ductal histology more likely to have germline mutations MSH6 5% PALB2 5%

BRCA2 43% Incidence of Pathogenic Germline BRCA1 9% Mutations CHEK2 14%

ATM 14% Intra/Ductal No P Value* Histology Intra/Ductal Histology 40% (10/25) 9% (11/125) P = .003

Isaacsson Velho P, et al. The Prostate. 2018;[Epub ahead of print]. *Fisher’s test. Emmanuel S. Antonarakis, MBBCh Effect of DDR Mutation on Treatment Responses

Presented By Carmel Pezaro at 2018 ASCO Annual Meeting Selected Trials for mCRPC with Relevance to DNA repair defects

Presented By Heather Cheng at 2018 ASCO Annual Meeting PARP Biology

• A key role in the repair of ssDNA breaks via BER pathway • Binds 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 PARP NAD+ Nicotina XRCC1 Lig3 mide + PNK pADPr Polß

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

Degeneration into Ohmoto A, et al. Onco Targets Ther. 2017;10:5195-5208. dsDNA breaks Emmanuel S. Antonarakis, MBBCh Synthetic Lethality Hypothesis

PARP function PARP function PARP function BRCA function BRCA function BRCA function

Normal cell Normal cell Cancer cell Non-BRCA mutation carrierBRCA mutation carrier BRCA mutation carrier (1 allele lost) (both alleles lost)

DNA damage DNA damage DNA damage

PARP inhibitor PARP inhibitor PARP inhibitor

PARP function PARP function PARP function BRCA function BRCA function BRCA function

Repair, Repair, Repair, Repair, Repair, Cell Death Survival Survival Survival Survival Survival Farmer H, et al. Nature. 2005;434:917-921. Bryant et al. Nature. 2005;434:913-917.

Emmanuel S. Antonarakis, MBBCh TOPARP

Presented By Carmel Pezaro at 2018 ASCO Annual Meeting TOPARP Results: Response

Presented By Carmel Pezaro at 2018 ASCO Annual Meeting

TOPARP Results

Presented By Carmel Pezaro at 2018 ASCO Annual Meeting Single Agent Trials In Progress

Presented By Carmel Pezaro at 2018 ASCO Annual Meeting Slide 17

Presented By Carmel Pezaro at 2018 ASCO Annual Meeting Combination Trials In Progress

Presented By Carmel Pezaro at 2018 ASCO Annual Meeting Clinical Data Extrapolations

Presented By Carmel Pezaro at 2018 ASCO Annual Meeting DNA Repair Defects and Hormonal Therapy Abiraterone in mCRPC With HR Deficiency • 20/80 (25%) evaluable pts with mCRPC had DNA repair defects DNA Repair Defects* PFS by DRD Status Biallelic Monoallelic 1.0 + Censored Log-rank P = .0254 BRCA2 0.8 Median, Mos (95% ATM CI) 0.6 DRD: 14.5 (11.0- BRC 19.5) A1 0.4 WT: 8.1 (5.5-11.0) RAD5 1B 0.2

RAD51 Probability of PFS C PALB2 0 3 6 9 1215 18 21 24 27 30 33 36 39 42 FANCA Pts at Mos Risk, n 2 copy Copy-neutral Frameshi Not DRD 20 2019 13 6 4 3 2 1 losses LOH ft detected WT 60 4334 20 10 5 3 0 1 copy Missense Nonsens In-frame *Data shownloss for 25 of 80 pts with exploratorye tumorindel sequencing. Hussain M, et al. J Clin Oncol. 2017;[Epub ahead of print]. Emmanuel S. Antonarakis, MBBCh Abiraterone or Enzalutamide and HR Deficiency PFS by DRD Status OS by DRD Status 100 100 Any Mutation No 80 Yes (other) 80

Yes (ATM/BRCA1/BRCA2)

60 60 OS OS (%)

PFS (%) PFS 40 40 Any Mutation No 20 20 Yes (Other) Yes (ATM/BRCA1/BRCA2)

0 0 0 6 12 18 24 0 6 12 18 24 30 36 42 48 Pts at Risk, n Mos Pts at Risk, n Mos No150 102 60 38 18 No150 145 128 75 75 52 35 22 11 Yes (other) 13 9 6 4 3 Yes (other) 13 13 13 9 8 6 5 3 0 Yes (ATM/BRCA1/ 9 8 6 4 Yes3 (ATM/BRCA1/ 9 9 8 7 6 4 4 4 0 BRCA2) BRCA2)

Antonarakis ES, et al. Eur Urol. 2018. In press. Emmanuel S. Antonarakis, MBBCh Abiraterone vs Enzalutamide and HR Deficiency • Randomized phase II Time to Progression by 1. HRR Status* HR P Value crossover study in treatment- 0 PSA > 40 ng/mL 1.50 .032 LDH > ULN 1.87 .008

naive pts with mCRPC (N = 0 ALP > ULN 1.16 .496 202) . Hemoglobin < 130 1.45 .055 8 Visceral mets 2.27 .004 0. ECOG PS 2 1.26 .327 • BRCA2- or ATM-truncating ctDNA > 2% 1.44 .086 6 mutations or rearrangements: HRR defect 5.27 < .001 0 – Somatic (ctDNA): 6/115 . (5.2%) 4 Probability Probability of PFS 0. HRR defect Yes – Germline (WBC): 8/202 2 No ctDNA unquantifiable (4.0%) 0 • Monoallelic BRCA2 or ATM 0 4 8 1 1 2 2 2 2Mos 6 0 4 8 deletion in 21 pts Pts at 8 6 4 2 1 5 3 1 Risk, n 12 6 13 91 48 5 2 0 – No TTP differences (P = .205) 01 2 2 3 0 7 0 0 0 0 0 5 4 Annala M, et al. Cancer Discov. 2018;[Epub ahead of print]. *Abiraterone + enzalutamide arms combined. Emmanuel S. Antonarakis, MBBCh DNA Repair Defects and Immune Checkpoint Inhibitors KEYNOTE-016: Responses to Pembrolizumab in MMR-Deficient

• Radiographic responses acrossTumors 12 tumor types at 20 wks (N = 86)

100

Prostate 50 Ampulla of Vater Cholangiocarcinoma Colorectal 0 Endometrial cancer Gastroesophageal -50 Neuroendocrine From Change Osteosarcoma SLD (%) Baseline Pancreas -100 Prostate Small Intestine Thyroid Prostate Unknown primary (n = 1)

Le DT, et al. Science. 2017;357:409-413. Emmanuel S. Antonarakis, MBBCh MMR Mutations in mCRPC

MSI Analysis: • 4/150 (2.7%) mCRPC pts Hypermutated vs were MSI-high, 3 of Nonhypermutated CRPC whom had MMR 0.5

MSI Positive 0 148 150 mutations (2%) 0.4 0 149 – 13 mut/Mb (Pt #149) – 0.3 MSH2 0 147 0.2 – 21 mut/Mb (Pt #147) – no 0 32, 41, 49, 67, MMR mutation 0.1 93 0 Negative

– 23 mut/Mb (Pt #148) – Loci Unstable Fraction 0 0 50 100 150 MSH2 0 0 0 Nonsynonymous – 25 mut/Mb (Pt #150) – Mutations MSH2 and MLH1

Robinson D, et al. Cell. 2015;161:1215- 1228. Emmanuel S. Antonarakis, MBBCh MMR Mutations Can Cause HRD Mutations Patient Case Gene Mutation Primary MMR mutation . MSH2 E809X* + LOH = MSI-high (> 100 mut/Mb) Secondary DNA-repair . BRCA2 . E1646fs* mutations . ERCC4 . M361fs* . ERCC5 . E474fs* . FANCM . V1336fs* . MSH6 . F1104fs*

* truncation by stop codon (X) or frameshift (fs).

• This patient should be treated with a PD-1 inhibitor, not a PARP inhibitor

Emmanuel S. Antonarakis, MBBCh MMR Defects in Prostatic Ductal Carcinoma • 4/10 (40%) had MMR mutations; 3/10 (30%) had MSI and hypermutation

Ductal Est. Tumor Total Coding Pt Hyper- Component Content MMR Gene Alteration HR Gene Alteration Muts/1.2 Mb No. mut for NGS, % From NGS, % Sequenced

1 71 30 No CHEK2 c.1100delC + LOH No 4

2 45 40 MSH2 inversion No No 4 3 65 60 No No No 4 4 30 60 MSH6 c.1900_1901del + LOH No Yes 29

MSH2-GRHL2 rearrangement + 5 97 50 No Yes 34 LOH 6 99 50 No No No 5 7 25 0 ------8 31 70 No No No 5 9 35 10 No BRCA2 c.594delT + likely LOH No 3

MLH1 exon 19+ 3’UTR 10 -- 60 No Yes 32 homozygous deletion

Schweizer MT, et al. Oncotarget. 2016;7:82504-82510. Emmanuel S. Antonarakis, MBBCh MMR Defects and Gleason Grade

Cases with MSH2 • 1.2% (14/1176) of primary loss adenocarcinomas and NEPC ControlsP = without MSH2.008 loss had MSH2 protein loss by 1200 IHC

1000

) • Pathology and MSH2 loss 2 – Primary Gleason pattern 5 800 enriched for MSH2 loss: 8% 600 (7/91)

– MSH2 loss in pts with any 400 CD8 CD8 (cells/mm other Gleason score: < 1% 200 (5/1042) – P < .05 0

Guedes LB, et al. Clin Cancer Res. 2017;23:6863-6874. Emmanuel S. Antonarakis, MBBCh DNA Repair and Platinum Chemotherapy Platinum Response in mCRPC With HR Deficiency

Pt Allele BRCA2 Mutation Mutation Type Pt 5 mos 1 1 c.9196C>T; p.Q3066X Premature stop 7 2 ABI ENZ DOC CAR 2 127 bp del in exon 11 Fs deletion 60 05 +DOX 2 1 c.8904delC; p.V2969Cfs *7 Fs deletion 04 03 2 c.2611delT; p.S871Qfs *3 Fs deletion 02 3 1 Homozygous copy loss Copy loss (ng/mL) PSA 01 * 2 Homozygous copy loss Copy loss 0 2012 2013 2014 2015 18 mos Pt ABI Pt 30 mos 1

50 1 DOC ENZ CAR CAR 3 2 CARABI CAR CAR CIS PA 0 +DOC +DOC 1 4 +DOC +DOC +ETO C 80 300 6 0 2 * 4 * 0

10 (ng/mL) PSA 2 PSA (ng/mL) PSA 0 0 0 2012 2013 2014 2011 2012 2013 2014 Clinical Treatment Course Clinical Treatment Course Cheng H, et al. Eur Urol. 2016;69:992- *Time of metastatic biopsy. 995. Emmanuel S. Antonarakis, MBBCh Platinum Response in mCRPC With FANCA Deficiency

• Near-CR to cisplatin/docetaxel in a pt with metastatic NEPC, lasting 12 mos – Genome of metastatic tumor found to be highly altered; germline FANCA mutation (S1088F) with somatic LOH also identified – In preclinical studies, loss of FANCA associated with increased cisplatin sensitivity Increased Cisplatin Sensitivity With Loss of FANCA

Cell Culture IC50 Xenograft Model

FANCA KO20.8 μM )

Model 3 130 Control 2.5 μM 1000 Vehicle 110 800 90 ControlKO2 Cisplatin 70 FANCA 600 50 GAPDH 400 30 10 200

Cell Viability (%) Viability Cell 0 0 0.01 0.1 1.0 10 100 (mm Volume Tumor 0 4 8 11 14 Days Cisplatin IC50 (μM)

Beltran H, et al. JAMA Oncol. 2015;1:466-474. Emmanuel S. Antonarakis, MBBCh HR Deficiency and Response to Carboplatin • 8/141 (5.7%) men with mCRPC had pathogenic germline BRCA2 variants PSA Response With OS With Carboplatin/Docetaxel Carboplatin/Docetaxel by by BRCA2 Carrier Status BRCA2 Carrier Status

0 1.00

BRCA2 carrier

* BRCA2 noncarrier 25 0.75 Log-rank P = .03 * 0.50 50 * 0.25 75

BRCA2 carrier Probability of OS PSA Decline (%) PSA BRCA2 noncarrier 0 100 * 0 12 24 36 48 60 72 84 96 Observations (n=141) Mos After Initiation of Pts at Risk, nCarboplatin/Docetaxel *Carriers of pathogenic germline variants in other BRCA2 133 51 14 6 5 3 2 1 1 DNA repair genes (MSH2, ATM, BLM, FANCA). noncarrier BRCA2 8 5 3 3 2 2 0 0 0 carrier Pomerantz MM, et al. Cancer. 2017;123:3532-3539. Emmanuel S. Antonarakis, MBBCh Conclusions

• Not all DNA repair lesions are created equal • Somatic (and germline) DNA repair mutations are common in prostate cancer, particularly mCRPC • HRD mutations may sensitize to PARP inhibitors, platinum agents • MMR mutations may sensitize to immune checkpoint inhibitors • The role of germline vs somatic, and single- vs double-copy inactivation, remains unclear

Emmanuel S. Antonarakis, MBBCh Grazie!!!!!