Background to HRRmtesting in prostate cancer

Z4-26721December 2020

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Contents

2

Background of prostate cancer

Treatment options for prostate cancer

Precision medicine in prostate cancer

Molecular sequencing in prostate cancer

Summary

Background of prostate cancer

The incidence of prostate cancer varies significantly worldwideEpidemiology

4 Rawla P. World J Oncol. 2019;10(2):63–89

• Prostate cancer is the second most frequent malignancy (after lung cancer) in men worldwide

• In 2018, there were approximately 1.2million new cases of prostate cancer worldwide, representing 7.1% of all cancers in men

• The incidence and mortality of prostate cancer increases with age, with the average age of diagnosis being 66 years

The diagnostic work-up for prostate cancer involves PSA levels, mpMRI, and biopsy

5mpMRI, multiparametric magnetic resonance imaging; PSA, prostate-specific antigen.1. Parker C et al. Ann Oncol. 2020;31(9):1119–1134; 2. Urology Associates. Available at: http://www.urologyassociates.net/prostate-ultrasound-and-biopsy/. Accessed November 2020; 3. Borely N and Feneley MR. Asian J Androl. 2009;11(1): 74–80. 4. NHS Website. What do cancer stages and grades mean? Available at: https://www.nhs.uk/common-health-questions/operations-tests-and-procedures/what-do-cancer-stages-and-grades-mean/. Accessed November 2020.

Diagnostic work-up for prostate cancer1 Acquiring a prostate biopsy sample2

PSA-Testing1

Men are at increased risk of prostate cancer metastasis or death if they have:• PSA>1 ng/ml at 40 years• PSA>2 ng/ml at 60 years

ESMO recommends offering early PSA testing to:1• Men >50 years• Men >45 years with family history

of prostate cancer• African-Americans >45 years• BRCA1/2 carriers >40 years One or more tissue samples

are taken from the prostate and sent to a lab for pathology analysis2

A prostate biopsy is used for tumour grading and staging as part of the diagnostic work-up3

The tumour grading describes the appearance of cancerous cells; the tumour staging describes the size of a tumour and extent of spread4

High-grade prostatic intraepithelial neoplasia (HGPIN) is the major precursor of prostate cancer1

Prostate cancer is characterised by histomorphologicalchanges1

6 *Image provided by Professor Sven Perner, Institute of Pathology, University of Lübeck1. Zynger DL et al. Int J Clin Exp Pathol. 2009;2(4): 327–338

Organoid luminal epithelium*

HGPIN (left) depicted by a large gland with multiple layers

(pseudostratified) of proliferating neoplastic cells1

High power view of HGPIN showing nuclear atypia of the pseudo-stratified neoplastic cells with

prominent nucleoli1

Prostate cancer is graded according to the type of growth pattern1

71. Kweldam CF et al. Histopathology. 2019;74(1):146–160

Prostate cancer1

Branching pattern carcinoma comprised of distinct medium-sized glandular structures with

branching outpouchings

Metastatic prostate cancer, lymph node

Metastatic prostate cancer, bone

Images provided by Professor Sven Perner, Institute of Pathology, University of Lübeck

Histological grading of prostate cancer is conducted using the Gleason grading system1-3

81. Am Cancer Soc. Understanding your pathology report- prostate cancer. https://www.cancer.org/treatment/understanding-your-diagnosis/tests/understanding-your-pathology-report/prostate-pathology/prostate-cancer-pathology.html. Accessed November 2020; 2. Natl Cancer Institute. Prostate cancer treatment (PDQ®) – patient version. Available at: https://www.cancer.gov/types/prostate/patient/prostate-treatment-pdq. Accessed November2020; 3. Kweldam CF, et al. Histopathology. 2019;74(1):146–160; 4. American cancer society. Available at: https://www.cancer.org/treatment/understanding-your-diagnosis/tests/understanding-your-pathology-report/prostate-pathology/prostate-cancer-pathology.html (last accessed November 2020)

When the pathologist examines the biopsy samples under the microscope, they will determine if the tissue architecture within the tissue looks abnormal and then assign it a pattern according to the Gleason system. The sum of the two predominant patterns determine the Gleason grade3

1

2

3

4

5

Gleason pattern range from 1 to 51 Pattern 33

Well-differentiated glands

Pattern 1 and 24

Tumour nodule of well-formed glands

Not applied in biopsies

Rarely diagnosed in radical prostatectomy specimens

9

Grade 43

Ill-formed

Abortive

Small- and large-fused

Glomeruloid

Cribriform (small and large)

Papillary

Grade 53

Single cell

Single file

Cribriform with comedonecrosis

Solid with or without comedonecrosis

1

2

3

4

5

1. Am Cancer Soc. Understanding your pathology report- prostate cancer. https://www.cancer.org/treatment/understanding-your-diagnosis/tests/understanding-your-pathology-report/prostate-pathology/prostate-cancer-pathology.html. Accessed November 2020; 2. Natl Cancer Institute. Prostate cancer treatment (PDQ®) – patient version. Available at: https://www.cancer.gov/types/prostate/patient/prostate-treatment-pdq. AccessedNovember 2020. 3. Kweldam CF, et al. Histopathology. 2019;74(1):146–160

Histological grading of prostate cancer is conducted using the Gleason grading system1-3

International Society of Urological Pathology (ISUP) grade grouping system is a more condensed stratification system and is based on the Gleason scores1

101. Epstein JI, et al. Am J Surg Pathol. 2016;40:244-252. https://www.cancer.org/treatment/understanding-your-diagnosis/tests/understanding-your-pathology-report/prostate-pathology/prostate-cancer-pathology.html. Accessed November 2020; 2. Am Cancer Soc. Understanding your pathology report – prostate cancer. Available at: https://www.cancer.org/treatment/understanding-your-diagnosis/tests/understanding-your-pathology-report/prostate-pathology/prostate-cancer-pathology.html. Accessed November 2020; 3. Prostate Cancer Foundation. Gleason score and IUSP grade group. Available at: https://www.pcf.org/about-prostate-cancer/diagnosis-staging-prostate-cancer/gleason-score-isup-grade/. Accessed November 2020.

Gleason Score ISUP Grade Group Risk Group3

≤6 Grade group 1 Low

7 (3+4)* Grade group 2 Intermediate favourable

7 (4+3)* Grade group 3 Intermediateunfavourable

8 Grade group 4 High

9 or 10 Grade group 5 High

The ISUP grade grouping system also simplifies grading into 5 categories, and makes the lowest grade a 1 (instead of a 6 as in the Gleason system), to reduce the potential for overtreating indolent cancer1

*The first number in the score represents the dominant tumour pattern and the second, the next most common pattern in the tumour.2,3

American Joint Committee on cancer staging system (8th edition)

11 1. Buyyounouski MK, et al. CA Cancer J Clin. 2017;67(3):245-253

Risk group definitions vary between international guidelines

12

Both ESMO and the European Association of Urology (EAU) define risk groups according to clinical stage, Gleason score, and PSA levels1,2

Risk Group ESMO Definition1 EAU Definition2

Low• T1-T2a AND• Gleason score ≤6 AND• PSA ≤10 ng/mL

• T1-T2a AND• Gleason score <7 (ISUP grade 1) AND• PSA <10 ng/mL

Intermediate• T2b AND/OR• Gleason score 7 AND/OR• PSA 10-20 ng/mL

• T2b OR• Gleason score 7 (ISUP grade 2,3) OR• PSA 10-20 ng/ml

High• ≥T2c OR• Gleason score 8-10• PSA >20 ng/mL

Localised:• T2c OR• Gleason score 8-

10• PSA >20 ng/mL

Locally advanced:• T3-T4 or N+• Any Gleason

score (any ISUP grade)

• Any PSA

1. Parker C, et al. Ann Oncol. 2015;26:v69-v77; 2. Mottet N, et al. EAU-ESTRO-ESUR-SIOG guidelines on prostate cancer. Published March 2017

Treatment options for prostate cancer

Staging for prostate cancer Choice of treatment depends on the stage of prostate cancer1

14

aIn addition to PSA level and MRI results, the decision to biopsy or not should be made in light of DRE findings, ethnicity, age, comorbidities, free/total PSA, history of previous biopsy and patient values.ADT=androgen deprivation therapy; CT=computed tomography; DRE=digital rectal examination; GS=Gleason score; M0 CRPC=non-metastatic castration-resistant prostate cance; MRI=magnetic resonance imaging; mpMRI=multi-parametric magnetic resonance imaging; PET=positron emission tomography; PSA=prostate-specific antigen; PSMA=prostate-specific membrane antigen; 223Ra=radium-223; RP=radical prostatectomy; RT=radiotherapy1. Parker C, et al. Ann Oncol. 2020;31(9):1119–1134

Stage-matched therapeutic strategies for prostate cancer

Patients presenting with localised/locally advanced prostate cancer can be offered curative intent1

15

aAlso suitable for localised/locally advanced disease if patient not suitable for (or unwilling to have) radical treatment.bFor men with biochemical relapse and symptomatic local disease, proven metastases or a PSA doubling time of <3 months.ADT, androgen deprivation therapy; EBRT, electron beam radiotherapy; HDR, high-dose rate; HIFU,high-intensity focused ultrasound; PC, prostate cancer; PSA, prostate specific antigen; RP, radical prostatectomy; RT, radiotherapy.1. Parker C, et al. Ann Oncol. 2020;31(9):1119–1134

Localised prostate cancer treatment algorithm High-risk localised and locally advanced prostate cancer treatment algorithm

Patients presenting with metastatic prostate cancer can be offered ADT therapy + chemotherapy or other hormone therapies

16 ADT, androgen deprivation therapy; PC, prostate cancer; RP, radical prostatectomy; RT, radiotherapy.1. Parker C, et al. Ann Oncol. 2020;31(9):1119–1134.

Metastatic prostate cancer treatment algorithm1

Precision medicine in prostate cancer

Metastatic prostate cancer is often characterised by mutations in several biological pathways1,2

18

In a multi-institutional study, profiling of 150 tumours from patients with mCRPC identified aberrations in the following pathways:1

*The DNA damage response encompasses additional types of DNA damage and repairDDR=DNA damage response; mCRPC=metastatic castration-resistant prostate cancer1. Robinson D, et al. Cell. 2015;161:1215–28; 2. Abida W, et al. PNAS 2019;116:11428-436; 3. Lord CJ and Ashworth A. Nature. 2012;481:287–293; 4. O’Connor MJ. Mol Cell. 2015;60:547–560

Mutations in DDR pathways can lead to genetic instability and drive tumour

growth3,4

More recent data from a multi-institutional study profiling 444 tumours from 429 mCRPC patients shows a similar proportion of patients with DDR pathway mutations2

Click here to learn more about the relevance of each biological

pathway in prostate cancer

Metastatic prostate cancer is often characterised by mutations in several biological pathways1,2

19

In a multi-institutional study, profiling of 150 tumours from patients with mCRPC identified aberrations in the following pathways:1

*The DNA damage response encompasses additional types of DNA damage and repairDDR=DNA damage response; mCRPC=metastatic castration-resistant prostate cancer1. Robinson D et al. Cell. 2015;161:1215–28; 2. Abida W et al. PNAS 2019;116:11428-436; 3. Lord CJ and Ashworth A. Nature. 2012;481:287–293; 4. O’Connor MJ. Mol Cell. 2015;60:547–560

Mutations in DDR pathways can lead to genetic instability and drive tumour

growth3,4

More recent data from a multi-institutional study profiling 444 tumours from 429 mCRPC patients shows a similar proportion of patients with DDR pathway mutations2

Click here to learn more about the roles of each

biological pathway

XMutations in each biological pathway may influence the development of cancer

Commonly mutated pathway Relevance to prostate cancer

PI3K Pathway This pathway is thought to be important both in early- and late-stages; it is important for regulating tumour cell survival1

WNT PathwayActivation of this pathway in prostate cancer cells is thought to contribute to their survival and proliferation; in advanced disease it also plays a role in bone metastasis1,2

Cell CyclePrecursors and early carcinomas have a 7- to 10-fold increase in their rate of cellular proliferation; in advanced and metastatic cancers, the rate of cell death is also reduced by about 60%3

AR PathwayMutations in the androgen receptor (AR) allow it to respond to very low levels of androgens or to be constantly active; these mutations are seen later in disease progression1

DNA RepairStudies have identified familial mutations in genes that are crucial for recognising and repairing DNA damage; this enhances the chance of mutations, allowing disease progression4,5

1. Velcheti V, et al. Ochsner J. 2008;8:213-218; 2. Nandana S, et al. Cancer Res. 2017;77:1331-1344; 3. Abate-Shen C, Shen MM. Genes Dev. 2000;14:2410-2434; 4. Robinson D, et al. Cell. 2015;161:1215-1228; 5. Packer JR, Maitland NJ. Biochim Biophys Acta. 2016;1863:1238-1260.

The DNA damage response is a collective term for cellular activities that result from the detection and repair of DNA damage1

BRCA1 are BRCA2 are tumour suppressor genes that play a key role in DNA damage response1

20

Base excision repair

Nucleotide excision repair

Mismatch repair

Non-homologous end joining

orHomologous

recombination repair

Base mismatches, insertions,

and deletions

Double strand breaks (DSBs)

Single strand breaks

Bulkyadducts

DDR pathway2

Type of DNA damage2

DSB, double strand breaks; DDR, DNA damage response.1. O’Connor MJ, et al. Mol Cell. 2015;60:547–560; 2. Paul A and Paul S. Front Biosci (Landmark Ed). 2014;19:605-18.

Mutations in BRCA-genes are associated with a higher risk of cancer including breast cancer, ovarian cancer, pancreatic cancer, and prostate cancer2

For DSBs, there are 2 main repair options: non-homologous end-joining (NHEJ) and homologous recombination repair (HRR)1

211. Iyama T, Willson III DM. DNA Repair (Amst). 2013;12:620-636; 2. Cooper GM. The eukaryotic cell cycle. In: The Cell: A Molecular Approach. 2nd ed. Sunderland, MA: Sinauer Associates; 2000.

NHEJ HRR

• NHEJ is the major DSB repair pathway for cells because it can be employed at any point in the cell cycle

• Unfortunately, it is an error-prone process and may result in loss of some genetic information at the site of the DSB1

It involves 3 steps1:

• HRR is restricted to the S and G2 phases of the cell cycle (when DNA replication occurs and the cell prepares to divide)1,2

• Because the DNA has just been replicated, the homologous (undamaged) copy of the DNA can be used as a template to faithfully restore the original sequence of the damaged strand1

1. Recognition of broken DNA ends

DSB

2. Processing of ends to remove damage and prepare for joining

3. Rejoining of ends

DSB

Homologous copy to be used as template

BRCA1 and BRCA2 are key players in the homologous recombination repair pathway

22

1. Lord CJ, Ashworth A. Nature. 2012;481:287-294. 2. Morgan MA, et al. Essentials of radiation therapy. In: DeVita, Jr VT, et al. DeVita, Hellman, and Rosenberg’s Cancer: Principles and Practices of Oncology. 10th ed. 2015. 3. Choi M, et al. Mol Cancer Ther. 2016;15(8):1781-1791. 4. Harding SM, et al. Genomic stability and DNA repair. In: Tannock IF, et al. The Basic Science of Oncology. 5th ed. 2013. 5. Brown JS, et al. Cancer Discov. 2017;20-37.

• BRCA proteins are crucial in mediating HRR1

– BRCA1 is recruited to the site of the DSB by ATMand another protein complex and is responsible for recruiting BRCA2 to the site2,3

– BRCA2 then helps load a protein called Rad51 to the DSB site, which helps the damaged portion of DNA “invade” and match with the undamaged sister chromatid to copy it2,4

• Other key HRR proteins:

– PALB2: Assists BRCA2 in loading the Rad51 protein to the DSB site5

– CHEK2: Interacts with the ATM protein to activate the cell cycle checkpoint for the G1-S phase transition5

ATM recruits BRCA1, and then BRCA1 recruits BRCA2 to DSB2

BRCA2 recruits Rad51 to DSB site2

Rad51 allows damaged strand to “invade” complementary strand of undamaged sister chromatid2

DSB is resolved by copying DNA from sister chromatid2

Undamaged sister chromatid

Chromatid with DSB= ATM= BRCA1= BRCA2= Rad51

Up to Approximately 28% of prostate cancers have HRR gene mutations, with BRCA2 being the most prevalent gene 1–4

23

BRCA2 only 9%

BRCA1 only 1%

ATM only

CDK12 only6% CHEK2 only

1%

PPP2R2A1%

Low prevalence genes

2%Co-occcuring

genes2%

No HRR gene alterations detected

72%

Prevalence of tumour HRRm identified in the mCRPC population (n=2792; figure adapted from data produced by de Bono, et al)1

HRRm, homologous recombination repair mutation; mCRPC, metastatic castration-resistant prostate cancer.1. de Bono J, et al. Presented at ESMO 2019, 27th–1st October, Barcelona, Spain. Poster 847PD; 2. Nombela P, et al. Cancers (Basel). 2019;11(3): 352; 3. Lang SH, et al. Int J Oncol. 2019;55(3): 597–616; 4. Nicolosi P, et al. JAMA Oncol. 2019;5(4):523-528

BRCA variants can either be germline or somatic mutations depending on their origin1,2

24BRCAm, BRCA mutation1. Griffiths AAJF, et al. Introduction to Genetic Analysis, 7th edn. 2000. Available at: https://www.ncbi.nlm.nih.gov/books/NBK21894/ Accessed November 2020; 2. Neff RT, et al. Ther Adv Med Oncol. 2017;9:519–531.

Germline mutations• Occur in germinal tissue and are present in every cell in the body1

• Are inheritable and can be passed on through family lines1

• Genetic testing of family members is recommended to identify additional BRCAm carriers who are at greater risk of developing malignancies2

• Can be detected using blood samples and tumour samples, but blood can discriminate between germline and somatic mutations

Somatic mutations• Spontaneous genetic alteration acquired by a cell1

• Cell division causes the mutation to be passed on to ‘daughter’ cells1

• Somatic mutations are non-inheritable1

• Can be detected using tumour samples

GERMLINEMutation in every cell of

the body

SOMATICMutation only

within a selection of cells such as

a tumour

Overview of types of DNA damage repair mechanisms, key genetic drivers, and treatment implications

DNA-repair mechanisms are therapeutic targets in advanced prostate cancer

25 Cheng HH, Urol Oncol. 2018;36(8): 385–388.

gBRCA2m is associated with an aggressive disease phenotype1,2

Gleason score (p<0.001)1

9%

48%

34% 33%31%

34%

26%

39%

28%

50%

34%

15%

0%

10%

20%

30%

40%

50%

60%

T1 T2 T3 ≤6 7 ≥8

Tumour size (p<0.02)1

Perc

ent o

f pat

ient

s

Non-carrier (N=1,235)

gBRCA2m carrier (N=67)

1.6%6.0%

Non-carrier (N=1,235)

gBRCA2m carrier (N=67)

Nodal involvement (p=0.009)1

In a study of 1,302 patients with prostate cancer, gBRCA2m carriers had larger tumour sizes and higher tumour grades compared with non-carriers1:

26

gBRCA2m tumours are also more likely to show a pattern of intra-ductal carcinoma,

which correlates with poor prognosis2:

gBRCA2m, germline breast cancer gene 2 mutation.1. Castro E, et al. Eur Urol. 2015;68:186–193; 2. Taylor R, et al. Nat Commun. 2017;8:13671

BRCAm patients are more likely to present with metastatic disease or develop metastases within 5 years compared to non-BRCAm patients

27

10% 11%

17% 17%

28%

19%17%

11%

19%

13%

0%

10%

20%

30%

Stage I Stage IIA Stage IIB Stage III Stage IV

Noncarrier (n=1940)

BRCA1/2 Carrier (n=79)

Prop

ortio

n of

pat

ient

s (%

)

7%

23%

0

5

10

15

20

25

BRCA1/2mnon-carrier

BRCA1/2mcarrier

Prop

ortio

n of

pat

ient

s (%

)

Patients who developed metastases within 5 years(Based on 5 year MFS in localised PC patients)

(n=78)(n=1940)

p=0.0001

Stage at diagnosis (p=0.001)

BRCA1/2m non-carrier

(n=1940)

BRCA1/2m carrier(n=79)

BRCAm, breast cancer gene mutation; MFS, metasasis free survival; PC, prostate cancer.Castro E, et al. J Clin Oncol. 2013;31:1748–1757.

28

In HRD cancer cells (including gBRCAm), deficiencies in HRR lead to the use of an error prone pathway and ultimately cell death

gBRCAm=germline BRCA mutation, HRD=Homologous recombination deficient; HRR=Homologous recombination repair; PARP=Poly (ADP-ribose) polymerase O’Connor MJ, Mol Cell. 2015;60:547–560

Double strand break

Normal cell

Repair of double strand breaks via the HRR pathway and cell

survival ✓HRR deficient cancer cell

PARP

PARPi

Reliance on error prone pathways leads to accumulation of genomic

instability and cell death

Trapped PARP on single strand breaks

Increase in double strand breaks in replicating cells

PARP

PARPi

Molecular sequencing in prostate cancer

Molecular sequencing in prostate cancer can be performed using tissue, ctDNA or blood samples

DNA extraction and

processing4-6

Library construction4-6

DNA sequencing4-6

Reporting4-6

Pathology review1

Sample preparation and storage

Data and variant analysis4-6

Plasma isolation and removal of cells/debris2

Stabilised in EDTA or other cell-stabilising

reagents2

FFPE or fresh frozen

samples1

Tissue

Blood

ctDNA

30

Buffy coat isolation3

ctDNA=circulating tumour DNA; EDTA=Ethylenediaminetetraacetic acid; FFPE=formalin-fixed paraffin-embedded1. Capoluongo E, et al. Semin Oncol. 2017;44:187–197; 2. Volckmar AL, et al. Gene Chromosome Canc. 2018;57:123–139; 3. Mychaleckyj JC. J Transl Med. 2011; 9: 91; 4. Myriad myChoice Technical Information. Available at: https://myriad-web.s3.amazonaws.com/myChoiceCDx/downloads/myChoiceCDxTech.pdf. Accessed November 2020; 5. Foundation Medicine. FoundationOne®Liquid CDx. Available at: https://www.foundationmedicine.com/test/foundationone-liquid-cdx. Accessed November 2020; 6. BRACAnalysis CDx Technical Information. Available at: https://myriad-library.s3.amazonaws.com/technical-specifications/BRACAnalysis_CDx_Tech_Specs.pdf. Accessed November 2020

Germline testing requires an additional patient consent step, and tissue testing involves tissue sample acquisition and pathology review steps1-3

31 ctDNA=circulating tumour DNA1. Capoluongo E, et al. Semin Oncol. 2017;44:187–197; 2. Myriad myChoice Technical Information. Available at: https://myriad-web.s3.amazonaws.com/myChoiceCDx/downloads/myChoiceCDxTech.pdf. Accessed November 2020; 3. BRACAnalysis CDx Technical Information. Available at: https://myriad-library.s3.amazonaws.com/technical-specifications/BRACAnalysis_CDx_Tech_Specs.pdf. Accessed November 2020

Consenting

Relative time to get result

Tissue

Germline

ctDNA

Test requisition

Sample acquisition DNA extraction

Library prep Sequencing Analysis and

interpretation Reporting

Sample Acquisition Pathology review DNA extraction

Library prep Sequencing Analysis and

interpretation Reporting

Sample acquisition DNA extraction

Library prep Sequencing Analysis and

interpretation Reporting

Tissue, ctDNA and blood HRRm testing each have their own benefits and limitations

32

Tissue ctDNA BloodType of mutation detected Somatic and germline1,2 Germline only3

Sample quality • DNA quantity: medium4

• DNA quality: low4• DNA quantity: low1

• DNA quality: variable5• DNA quantity: high4

• DNA quality: high4

Turnaroundtimes (TAT) ~2–8 weeks6 ~1–3 weeks7–10 ~2–16 weeks6

Genetic counselling Patients with a positive mutation are referred for germline testing to determine whether the mutation is somatic or germline. Germline testing requires genetic counselling11,16

Requires pre- and post-test genetic counselling4

Testing benefits

• Tissue testing is currently recognised as the gold standard for tumour analysis1

• High clinical sensitivity12

• Archival tissue for tumour histology may already be available, and provides an option for testing13

• Easy to obtain sample14

• Plasma ctDNA testing shows early promise2

• Minimally invasive and easily repeatable14

• Easy to obtain samples3,4,16

• Assess familial risk3,16

• Analysis feasible in 100% of cases(detection rate)4

• Is minimally invasive and easily repeatable15

• Blood testing is currently the ‘gold standard’ used to detect large-genomic rearrangements17-19

Testing limitations

• May miss within-tumour genetic heterogeneity3

• Obtaining samples from metastases is an invasive procedure and may be challenging (cost, morbidity, and low yield)1,3,12,20

• ~31% of tests fail due to not meeting pathology results or insufficient yields of DNA after extraction21

• The frequency of large genomic rearrangements in tumour tissue remains unknown4

• Low concentrations of circulating ctDNA1,20

• Highly sensitive tests are required1,12

• May result in false-negatives or false-positives12

• Testing is limited by the availability of an adequate amount of ctDNA, particularly at early stages22

• High potential but clinical validation in prostate cancer currently very limited22

• Does not identify patients with mutations of somatic origin or capture the potentially changing genetic profile of disease progression4

• May miss the majority of those harbouringmutations of somatic origin23

ctDNA=circulating tumour DNA; HRRm=homologous recombination repair mutation; ISUP=International Society of Urological Pathology; TAT= turnaround times References: See slide notes

Tissue, ctDNA and blood HRRm testing each have their own benefits and limitations

33

Tissue ctDNA BloodType of mutation detected Somatic and germline1 Germline only3

Sample quality • DNA quantity: medium4

• DNA quality: low4• DNA quantity: low1

• DNA quality: variable5• DNA quantity: high4

• DNA quality: high4

Turnaroundtimes (TAT) ~2–8 weeks6 ~1–3 weeks7–10 ~2–16 weeks6

Genetic counselling Patients with a positive mutation are referred for germline testing to determine whether the mutation is somatic or germline. Germline testing requires genetic counselling11,15

Requires pre- and post-test genetic counselling4

Testing benefits

• Tissue testing is currently recognised as the gold standard for tumour analysis1

• High clinical sensitivity12

• Archival tissue for tumour histology, Gleason scoring and/or ISUP grading may already be available, and provides an option for testing14,15

• Easy to obtain sample1

• Plasma ctDNA testing shows early promise1

• Minimally invasive and easily repeatable1

• Proven technology in lung cancer1

• Easy to obtain samples3,4,15

• Assess familial risk3,15

• Analysis feasible in 100% of cases(detection rate)4

• Is minimally invasive and easily repeatable13

• Blood testing is currently the ‘gold standard’ used to detect large-genomic rearrangements16-18

Testing limitations

• May miss within-tumour genetic heterogeneity3

• Obtaining samples from metastases is an invasive procedure and may be challenging (cost, morbidity, and low yield)1,3,12,19

• ~31% of tests fail due to not meeting pathology results or insufficient yields of DNA after extraction20

• The frequency of large genomic rearrangements in tumour tissue remains unknown4

• Low concentrations of circulating ctDNA1,19

• Highly sensitive tests are required1,12

• May result in false-negatives or false-positives12

• Testing is limited by the availability of an adequate amount of ctDNA, particularly at early stages21

• High potential but clinical validation in prostate cancer currently very limited21

• Does not identify patients with mutations of somatic origin or capture the potentially changing genetic profile of disease progression4

• May miss the majority of those harbouringmutations of somatic origin22

ctDNA=circulating tumour DNA; HRRm=homologous recombination repair mutation; ISUP=International Society of Urological Pathology; TAT= turnaround times References: See slide notes

References1. Boerrigter E, et al. Exp Rev Mol Diagnostics. 2020;20:219–230; 2. Ratajska M, et al. Oncotarget. 2017;8:101325–101333. Cheng H, et al. ASCO Ed Book. 2018. 372–3814. Capoluongo E, et al. Semin Oncol. 2017;44:187–1975. Volckmar AL et al. Gene Chromosome Canc. 2018;57:123–1396. Provided by Diaceutics for internal use only7. Sacher AG, et al. JAMA Oncol. 2016;2:1014–10228. Guardant. Available at: https://investors.guardanthealth.com/static-files/2f4226d5-ed0b-464d-acb8-4db6999a8ce8. Accessed

November 20209. Foundation Medicine. FoundationOne®Liquid CDx. Available at: https://www.foundationmedicine.com/test/foundationone-liquid-

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The latest ESMO guidelines include recommendations for genetic testing using NGS in prostate cancer1,2

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EAU guidelines 2020

ESMO guidelines 2020

Localised disease1

• Germline testing should be considered if at least two close blood relatives on the same side of the family have been diagnosed with tumours linked to hereditary cancer predisposition syndromes

• Tissue-based molecular assays may be used in conjunction with clinic pathological factors for treatment decision making in localised prostate cancer

Metastatic disease1

• Germline testing for BRCA2 and other DDR genes associated with cancer predisposition syndromes is recommended in patients with family history of cancer and should be considered in all patients with metastatic prostate cancer

• Tumour testing should be considered for homologous recombination genes and mismatch repair defects (or microsatellite instability) in patients with mCRPC

• Patients with pathogenic mutations in cancer-risk genes identified through tumour testing should be referred for germline testing and genetic counselling

• EAU provides limited guidance on germline or somatic/tumour testing3

• Offer early PSA testing to well-informed men at elevated risk of having prostate cancer: men carrying BRCA2 mutations >40 years of age3

ESMO guidelines 2020_NGS in oncologySummary of recommendations:2

• In countries where poly ADP ribose polymerase inhibitors (PARPi) are accessible for patients with prostate cancer, it is recommended to perform NGS on tumour sample to assess mutational status of, at least, BRCA1/2

• PTEN alterations are found very frequently in mCRPC. According to the preliminary results of the Phase III with AKT inhibitors in patients with PTEN alterations, this gene could be added to the panel

• Given that they are unlikely to be cost-effective in these cases, larger panels can be used only on the basis of specific agreements with payers taking into account the overall cost of the strategy and pending a ranking of additional alterations using a valid ranking system. These panels should include DNA repair genes and MSI signature

ESMO, European Society for Medical Oncology; EAU, European Association of Urology; NGS, next generation sequencing.1. Parker C, et al. Ann. Oncol. 2020. DOI: 10.1016/j.annoc.2020.06.011. Accessed November 2020; 2. Mosele F, et al. Ann Oncol. 2020; DOI: 10.1016/j.annonc.2020.07.014; 3. EAU Prostate Cancer Guidelines. https://uroweb.org/guideline/prostate-cancer/. Accessed November 2020

Summary

36ADT=Androgen deprivation therapy; ctDNA=Circulating tumour DNA; HRR=Homologous repair pathway; mpMRI=Multiparametric MRI; NGS=Next-generation sequencing; PSA=Prostate specific antigen

Summary • Prostate cancer is the second most frequent malignancy in men worldwide• The diagnostic work-up for prostate cancer involves PSA levels, mpMRI and biopsy, which is a prerequisite for

tumour grading and staging

• Metastatic prostate cancer is characterised by genetic mutations in several biological pathways including the HRR pathway• Gene mutations in the HRR pathway can lead to genetic instability and drive tumour growth• BRCA1 & BRCA2 are key players in the homologous recombination repair pathway

• Identifying mutations in the HRR pathway are a therapeutic target in advanced prostate cancer

• Molecular sequencing in prostate cancer, performed using tissue, ctDNA or blood samples, facilitates detection of HRR mutations which may influence disease prognosis and treatment

• Currently, tissue testing is the gold-standard but ctDNA testing is a promising alternative• The latest ESMO guidelines include recommendations for genetic testing using NGS in prostate cancer

• Choice of treatment depends on the stage of prostate cancer• Patients presenting with metastatic prostate cancer can be offered ADT therapy and chemotherapy or other

hormone therapies