Biopsy >> Genes >> Profile

Correira C. et al., 2014

Low frequency ofgenetic altarations

TMPRSS2:ERG

High frequency ofgenetic altarationsHigh frequency ofgenetic altarations

Diagnostic PCA3 ? ??

Prognostic Prolaris oncotype DX (used to evaluate positive biopsies and more accurately

discriminate indolent vs. aggressive cancer at the time of diagnosis).\\

TMPRSS2:ERG ??

Predictive AR-V7 status Taxanes, abiraterone and

enzalutamide

BRCA status PARP Inhibitors

During the past 2 years, two biomarkers have been approved

by the US FDA.

proPSA as part of the Prostate Health Index (phi) by Beckman Coulter and

PCA3 as Progensa by Gen Probe.

With the advances in genomic and proteomic technologies,

several new CLIA-based laboratory-developed tests have become

available.

Oncotype DX from Genomics Health, and

Prolaris from Myriad Genetics.

Sartori and Chan., 2014

DD3

2M

654 7 981 2 3 10 11 12 13 14 15 Pr 17 M

PCA3 gene is only expressed in prostatetissue

lane 1 and 17: negative control; lane 2: testis; lane 3: seminal vesicle; lane 4: ovary; lane 5: placenta;lane 6: heart; lane 7: duodenum; lane 8: spinal cord; lane 9: spleen; lane 10: brain; lane 11: artery;lane 12: lung; lane 13: liver; lane 14: skeletal muscle; lane 15: bladder; lane M: marker; lane Pr: prostate

Bussemakers MJG, et al. Cancer Res 1999;59:5975-9

The PCA3 test showed the best diagnostic performance whencompared to tPSA and f/tPSA, facilitating the selection of high-riskpatients that may benefit from the execution of a saturationprostatic biopsy.

Moreover, the PCA3 test showed a prognostic value, bicausehigher PCA3 score values are associated to a greater tumoraggressiveness.

The PCA3 Score represents a valuable predictor of insignificant

Pca, aggressive non aggressive

PCA3 could be used to guide the selection of men suitable for

active surveillance or focal therapy

PCA3 testing can often be valuable for guiding biopsy decisions

in men suspicious of having PCa, particularly for those with ≥ 1

negative biopsy, PSA ≥ 3 ng/mL and a life expectancy ≥ 10 years

In men with low-risk PCa, the PCA3 Assay can reassure

physicians and patients in their decision for active surveillance

Prostate

WHAT is the test? A tumor gene expression assay which

produces a Genomic Prostate Score (GPS)to help guide initial treatment decisions atthe time of biopsy

WHO is the test for? Newly diagnosed men with low to

low-intermediate risk prostate cancer(GS 3+3, low volume 3+4)

WHY do the test? To improve risk stratification by

incorporating individual underlying tumorbiology

To identify appropriate patients for ActiveSurveillance (AS) or immediate treatment

13

Two spatially distinct areas of tumorrepresenting the dominant (primary)and the highest Gleason pattern foreach patient were analyzed toidentify genes which are predictiveregardless of sampled Gleasonpattern

Selected genes which predict clinicaloutcome in the face of tumorheterogeneity and biopsy under-sampling and perform well in biopsytissue

Standardized analytical platform forreliable measurement of 17-geneGPS assay in small volume tumorfrom biopsies

Analytical Performance 96% of biopsies yielded successful GPS

results

ProstateBiopsyTURPProstatectomy

14

• Genes in multiple pathwaysare consistently predictiveof aggressive prostatecancerin both RPs and biopsies– Stromal Response– Androgen Signaling– Cellular Organization– Proliferation

• Enabled definition of thefinal17-gene RT-PCR OncotypeDX GPS assay

Klein et . al. ASCO 2012

Stromal Response

Androgen Signaling

Cellular Organization

Proliferation

Stress Response

Basal Epithelia0.3 0.6 1 1.8 3.2

Std. Odds Ratio (95% CI)

Biopsy Study RP Study

Worse OutcomeBetter Outcome

15

727 candidategenes in highest

Gleasonsamples

727 candidategenes indominant

Gleason samples374 genes

predictoutcome

(dominant)

367 genespredict

outcome(highest)

Final 17 GPS Genes

• Consistent performance in biopsies

• Represent key pathways• Analytical performance

• Predict PC death, adv. path, BCR• Value beyond existing measures

288 genespredictive

regardless ofsampledGleasonpattern

PC, prostate cancer; BCR, biochemical recurrence.

288 Genes

81 Genes

17

Stromal ResponseBGN

COL1A1SFRP4

Stromal ResponseBGN

COL1A1SFRP4

ProliferationTPX2

ProliferationTPX2

Androgen SignalingFAM13C

KLK2AZGP1

SRD5A2

Androgen SignalingFAM13C

KLK2AZGP1

SRD5A2

CellularOrganization

FLNCGSN

TPM2GSTM2

CellularOrganization

FLNCGSN

TPM2GSTM2

The combination ofmultiple pathways

is morepredictive than

any single pathway

The combination ofmultiple pathways

is morepredictive than

any single pathway

Genes Associated withBetter Outcome

Genes Associated withWorse Outcome

ARF1ATP5ECLTC

GPS1PGK1

ARF1ATP5ECLTC

GPS1PGK1

Reference Genes

UCSF Database 1997-2011Consented RP patients meeting

active surveillance criteria at diagnosisLow/Intermediate risk biopsy

GS 3+3 and low volume 3+4 (fewer than 4 positive cores)Eligible Population

n = 412

17 (4%) Excluded forinsufficient RNA quality

Final Evaluable Populationn = 395

18Klein et al. Eur Uro 2014

Cullen J,1 Rosner I,2 Brand T,3 Zhang N,4 Tsiatis AC,4Ali A,1 Chen Y,1 Knezevic D,4 Maddala T,4 Lawrence

HJ,4Febbo PG,4 Srivastava S,1 Sesterhenn IA,5 McLeod D2

1Center for Prostate Disease Research, Rockville, MD; 2Walter Reed National MilitaryMedical Center, Bethesda, MD; 3Madigan Army Medical Center, Tacoma, WA;

4Genomic Health, Inc., Redwood City, CA; 5Joint Pathology Center, Silver Spring, MD

Primary aim: To determine if GPS was associated withthe time to BCR after RP

Secondary aims: To confirm that GPS can predict the likelihood of adverse

pathology at RP. To determine if GPS is associated with time to development of

metastatic disease.

Cullen J et al. Eur Urol. 2014.

Cullen J et al. Eur Urol. 2014.

23

Decision Impact 1 (N=158) showed treatment recommendations change pre and post GPS: 24% relative increase in AS recommendation 20% had a change in treatment modality recommended 26% had a change in treatment modality and/or intensity recommended

DI 1 also showed in 85% of cases doctors had higher confidence in their treatmentrecommendations after receiving the GPBadani et al. SUO 2014

Combining Biologic & Clinical InformationRefines Risk Stratification for Individual Patients

VERY LOW RISK LOW RISK INTERMEDIATE RISK

More Individualized Biologic and Clinical Risk Assessment

Likelihood of Favorable Pathology100% 30%

Population-Based Clinical Risk Assessment

VERY LOWRISK LOW RISK INTERMEDIATE RISK

GPS=884%

GPS=884%

GPS=5157%

GPS=5157%

10% (N=37) 49% (N=191) 41% (N=160)

44% (N=169)31% (N=119)

24

GPS=2575%

GPS=2575%

UCSF Validation StudyNCCN Risk Classification

• 10% Very Low-risk• 49% Low-risk• 41% Intermediate Risk

GPS Provides Biologic Risk Information

GPS

• Adds more accurate riskassessment bycombining biologicaland clinical risk factors

• Predicted whichpatients have riskconsistent with theirNCCN clinical risk group

26% (N=100)

• 35% of men in the NCCNLow-risk group had moreindolent biology andlikelihood of favorablepathology consistentwith Very Low-risk

• 10% of men in theNCCN Low-risk grouphad more aggressivebiology and likelihoodof favorable pathologyconsistent withIntermediate risk

• Identified patients in theNCCN Very Low-riskgroup who had moreaggressive biology, withlikelihood of favorablepathology consistentwith Low andIntermediate riskdisease

• Identified patients withIntermediate risk whohad more indolentbiology, predicted to beconsistent with Low-riskdisease

• Enables more accurateidentification of a largerpopulation of patientswho can moreconfidently chooseactive surveillance

• Precisely identifies apatient’s tumor biologyand refines thepopulation-based clinicalrisk assessment with amore personalized riskassessment

Cooperberg et al, AUA 2013

Oncotype DX® GPS Provides More Accurate andIndividualized Risk Assessment at Time of Biopsy

• Developed to address tumorheterogeneity and biopsyunder-sampling

• Based on multiple biologicpathways predictive ofaggressive prostate cancer

• Optimized technology for verysmall amounts of tumor fromneedle biopsies

• Independently validated incontemporary low andlow-intermediate risk patients

25

ER - ER +

Her2 - Chemo Hormonal

Her2 + Herceptin Hormonal /Herceptin

ER: Estrogen ReceptorHer2: Her2/Neu., Erb-B2

Androgen Receptor Variant 7

BRCA mutations

Androgen Receptor Variant 7

BRCA mutations

JAMA Oncology., June., 2015

They have developed a quantitative reverse-transcription polymerasechain reaction (qRT-PCR)–based assay to quantify mRNA levels for ARand for the AR-V7 splice variant in circulating tumor cells (CTCs).4

They published on a cohort of 62 patients with CRPC in which detectionof AR-V7 was associated with resistance to abiraterone and enzalutamide(with no responders in the AR-V7–positive group) and was associatedwith shorter survival.

Antonarakis et al., N Engl J Med2014

Antonarakis e. et al JAMA Oncology., June., 2015

An important observation from thepresent study is the suggestion that

taxane therapy may be moreefficacious than AR-directedtherapy for men with AR-V7–positive CRPC.

clinical outcomes did not seem todiffer significantly on the basis ofthe type of therapy used amongAR-V7–negative patients.

If these results are confirmed by additional prospective biomarker-stratifiedclinical trials, this observation might suggest that

AR-V7–positive men may fare better receiving taxanes whereas in ARV7–negative men both treatment approaches might be

reasonable.

If these results are confirmed by additional prospective biomarker-stratifiedclinical trials, this observation might suggest that

AR-V7–positive men may fare better receiving taxanes whereas in ARV7–negative men both treatment approaches might be

reasonable.

The quantitation of AR-V7 as described byAntonarakis et al. is not easy applicable

An easy test will be the quantitation ofcirculating free tumor cftRNA that is releasedfrom dying cancer cells

A useful predictive tumor Marker has to beeasy and reliable mesured

Blood sample taken in RNABCT® collection tube containingStabilizing reagent prevents cell-free RNA contamination bycellular RNA in plasma

Sample arrives in laband speendown toisolate the plasma

Plasma is stored at -80ºc

Sample is extractedon the same day asthe downstreamprocess set up dueto cftRNA instability

cftRNA is extractedfrom the plasmausing the QIAampCirculating NucleicAcid on the QIAVacsystem

Next GenerationSequencing

Treatment-naïve radical prostatectomy specimens fromAmerican and Australianpatients that spanned a range ofgrades, stages, and risk of recurrence

DNA extraction and exome sequencing H&E slides werecut from all frozen tissue blocks

To demonstrate the feasibility of a novel next-generation

sequencing (NGS) based platform that can be

used with archival formalin-fixed paraffin-embedded (FFPE)

biopsy tissue

to evaluate the spectrum of DNA alterations seen in advanced

PC

Thes results are hypothesis generating The spectrum of driver mutations observed

highlights the potential value of comprehensivegenomic profiling for targeted therapy selectionin clinical care.

This study marks an exciting step towarddesigning targeted assays to detect drivingmutations and has potential to lead to newbiomarkers, drug targets, and generalapplicability in guiding the development of futuretherapies.

Conclusions Major advances have been made in cataloguing

the genomic alterations in prostate cancer andunderstanding the molecular mechanismsunderlying the disease.

These findings raise the possibility that prostatecancer could soon transition from a poorlyunderstood, heterogeneous disease with avariable clinical course to a collection ofhomogenous subtypes, identifiable by molecularcriteria, associated with distinct risk profiles

and perhaps amenable to specific managementstrategies or targeted therapies.

(A) Schema of multi-institutional clinical sequencing project work flow. (B) Clinical trials associated with the SU2C-PCF mCRPC project. (C) Biopsy sites of the samples used for clinical sequencing. (D) Histopathology of the cohort. Representative images of morphological analysis of mCRPC

are shown along with prevalence in our cohort.

Somatic mutationare the mutation inthe cancer tissue

90% of mCRPCharbor clinicallyactionablemolecularalterations

If aberrations

of BRCA2, BRCA1, and ATM

all confer enhanced

sensitivity to PARP

inhibitors, 19.3% of mCRPC

affected individuals

would be predicted to

benefit from this therapy.

The frequency of 19%

suggest a good predictive

marker for mCRPC

If aberrations

of BRCA2, BRCA1, and ATM

all confer enhanced

sensitivity to PARP

inhibitors, 19.3% of mCRPC

affected individuals

would be predicted to

benefit from this therapy.

The frequency of 19%

suggest a good predictive

marker for mCRPC

PARPInhibition ofPARP-1preventsrecruitment ofrepair factorsto repair SSB

XRCC1

LigIII

PNK 1

pol β

Replication(S-phase)

DNA DSB

DNA SSB

Cellsurvival

Base Excision Repair Homologous Recombination

DNA Damage

PARPInhibitor

BRCAMutation

Cancer celldeath

Using our experience of Synthetic Lethality

TOPARP is an open-label, investigator-initiated phase II trial with a

novel multi-step adaptive design (CRUK/11/029).

The first part of the study (TOPARP-A) has a two-stage design

evaluating the antitumor activity of single agent olaparib in unselected

mCRPC pts (p0=0.05; p1=0.20; α=0.02; β=0.10) with a preplanned

analysis to identify a biomarker defined sensitive subgroup.

Mateo et al., AACR., 2015

Results: Fifty pts were enrolled from 7 UK centers; all had had priordocetaxel, 48 (96%) prior abiraterone and 29 (58%) prior cabazitaxel.Overall, 16 of 49 evaluable pts experienced a response (RR 32.7%, 95%CI: 20.0 to 47.5), with 11 and 4 pts having been on treatment for >6 and>12 months respectively at data cut-off.

NGS identified homozygous deletions and/or putatively deleteriousmutations in DNA repair genes in 15/49 (30.6%) evaluable pts. While amajority of these genomic aberrations occurred in BRCA2 and ATM,biallelic loss of other relevant genes, including members of the FanconiAnemia complementation group and CHEK2, were also observed.

Among these fifteen pts, 13 (86.7%) responded to olaparib. All seven ptswith BRCA2 loss (somatic [4/7] or germline [3/7]) and 4/5 pts with ATMtruncating mutations responded to olaparib.

Mateo et al., AACR., 2015

Our lab is using two platforms for NGS:

Ion PGM™ System for Next-GenerationSequencing

MiSeq illumina

Targeted Prostate Cancer Screening in BRCA1 and BRCA2 MutationCarriers: Results from the Initial Screening Round of the IMPACT StudyEurUrol. 2014 Sep; 66(3): 489–499.

Targeted Prostate Cancer Screening in BRCA1 and BRCA2 MutationCarriers: Results from the Initial Screening Round of the IMPACT StudyEurUrol. 2014 Sep; 66(3): 489–499.

GeneKor SA

Diagnostic PCA3 ? ??

Prognostic Prolaris oncotype DX (used to evaluate positive biopsies and more accurately

discriminate indolent vs. aggressive cancer at the time of diagnosis).\\

TMPRSS2:ERG ??

Predictive AR-V7 status Taxanes, abiraterone and

enzalutamide

BRCA status PARP Inhibitors

Τμήμα επιστημονικής ενημέρωσης

Νικόλαος Τσούλος

Βασιλική Φλώρου

Δημήτρης Λαχανάς

Χρυσάνθη Βιολάκη

Σπύρος Γιαννουλάκης

Σωκράτης Τουγιαννίδης

Εξυπηρέτηση πελατών

Άννα Κοκοτάκη

Μαρινέλλα Λέκα

Ελευθερία Τσακαλάκη

Θανάσης Υφαντής

LaboratoryGeorge Nasioulas PhD, CSO

Angela Apessos PhD, Group Leader NGS

Irene Papadopoulou PhD, Group Leader Oncogenetics

Jeny Bourkoula PhD, Group Leader Cytogenetics

George Tsaousis PhD, Group Leader Bioinformatics

Ageliki Tsirigoti MS

Konstantinos Agiannitopoulos MS

Vasiliki Metaxa-Mariatou MS

Georgia Pepe MS

Chrisa Efstathiadou MS