PORTEC trials Gynecologic Cancer InterGroup Translational Research - - PowerPoint PPT Presentation

Lessons learned from translational work on the PORTEC trials

Tjalling Bosse

Department Pathology

LEIDEN UNIVERSITY MEDICAL CENTER

Gynecologic Cancer InterGroup Translational Research Brainstorming October 2016 Lisbon

Lessons from PORTEC-1,-2, -3 and -4

#1 … #2 … #3 … #4 …

Interobsever variability in GYNpathology review is an issue

Mandatory second opinion in surgical pathology referral material: Clinical consequences of major disagreements. Manion et al., Am J Surg Path 2008

“Clinically significant diagnostic discrepancies in the field of gyneacologic pathology occur more frequently than in other areas of pathology.”

Interobserver variability in endometrial cancer

Gilks et al, AJSP 2013

Results retrospective PORTEC-2 review

Grade 62.2 0.33 1 175 49 286 81 2 158 45 33 9 3 22 6 36 10 LVSI 90.7 0.54 present 38 10 46 13 absent 328 90 320 87 N % N % % Kappa Non-endometrioid 12 3 Original Review Agreement Myometrial invasion 91.7 0.67 <50% 46 13 60 17 ≥50% 315 87 301 83 Cervical gland. inv. 93.2 0.62 present 45 12 28 8 absent 320 88 337 92

3 2 1 3 2 1

Results retrospective PORTEC-2 review

Before central review After central review

Overal Survival in PORTEC-2

• PORTEC-1: 23,6% not eligible after retrospective path. review
• PORTEC-2: 14% not eligible after retrospective path. review

Upfront pathology review PORTEC-3 (NL+UK) 8.3% not HighRisk

De Boer, IGCS 2016 poster

Pathology review in the PORTEC studies

• PORTEC-1: 23,6% not eligible in retrospective path review
• PORTEC-2: 14% not eligible in retrospective path review
• PORTEC-3: 8,3% not eligible in prospective path review

Con’s prospective path review:

• Logistics, logistics and logistics (timing, transport and communication)

Pro’s prospective path review:

• Improves clinical trials, by “clean” inclusion
• Seems especially critical in trials with toxic treatment
• Teaching tool, identifies knowledge gaps
• Prospective central biobank collection

De Boer, IGCS 2016 poster

Biobanking the FFPE blocks from the PORTEC-trials

Obstacles

• “Old trials”; blocks are destroyed or lost
• Material exhausted for other purposes
• International trials more complex

Success factors

• Central pathology review
• Local people’s willingness to help

Trial Year Inclusion Randomisation FFPE Biobank PORTEC-1 1990-97 Low Risk EBRT vs NAT 477/715 (67%) PORTEC-2 2000-06 Intermediate Risk EBRT vs BT 384/427 (90%) PORTEC-3 2007-13 High Risk EBRT vs RT+CT 430/686 (62%) PORTEC-4a 2016-… Intermediate Risk Molecular Profiles 11/11 (100%)

Lessons from PORTEC-1,-2, -3 and -4

#1 Upfront pathology review by expert gynaecological pathologists is to be preferred to ensure enrollment of the target trial population and to avoid unnecessary treatment and toxicity #2 Upfront (multi)central collection of tissue from trial patients is to be preferred to ensure an optimal trial-tumorbiobank #3 … #4 …

What are we going to do with that biobank?

Identifying prognostic and predictive biomarkers, to personalize treatment. Reducing over- and undertreatment. 1. PRACTICAL (cheap and quick) 2. EFFECTIVE IN PREDICTING BIOLOGIC BEHAVIOR 3. REPRODUCIBLE (easy)

Pathology assessment is MORE than histotype alone

The ‘Big Five’ of endometrial cancer

Pathology assessment is more than histotype

Endometrioid Serous Clearcell

Tumortype

Pathology assessment is more than histotype

Endometrioid Serous Clearcell

Tumortype

• Gr. 1 <5% solid
• Gr. 3 >50% solid

Grade

Pathology assessment is more than histotype

Endometrioid Serous Clearcell

Tumortype

• Gr. 1 <5% solid
• Gr. 3 >50% solid

Grade

Myometrial Involvement

Pathology assessment is more than histotype

Endometrioid Serous Clearcell

Tumortype

• Gr. 1 <5% solid
• Gr. 3 >50% solid

Grade

Endocervical Involvement Myometrial Involvement

Pathology assessment is more than histotype

Endometrioid Serous Clearcell

Tumortype

• Gr. 1 <5% solid
• Gr. 3 >50% solid

Grade

Endocervical Involvement Myometrial Involvement

LVSI

What is the current risk assessment?

Low Risk

• Stage I Endometrioid + gr 1-2 + <50% myometrial invasion + LVSI neg

Low Inter Risk

• Stage I Endometrioid + gr 1-2 + ≥50% myometrial invasion + LVSI neg

High Inter Risk

• Stage I Endometrioid + gr 3 + <50% myometrial invasion, regardless of LVSI status
• Stage I Endometrioid + gr 1-2 + LVSI positive, regardless of depth of invasion

High Risk

• Stage I Endometrioid + gr 3 + ≥50% myometrial invasion, regardless of LVSI status
• Stage II & microscopic stage III
• Non endometrioid (Serous or Clear cell, etc)

Adv M+

• Stage III bulky & IVa
• Stage IVB

FIGO 2009 staging used Colombo et al., Annals of Oncology 2016

• LVSI assessment is important, but poorly defined
• LVSI assessment may requires quantification to be truly prognostic

Definitions used in daily practice by 6 independent well recognized GYNpathologists

1 Cohesive aggregate of tu tumor ce cells lls located inside a vascular space (defined by the presence of an en endot

• theliu

ium lin linin ing) and preferentially juxt xtaposed to

• th

the e ves essel l wall ll 2 Ca Carcin inoma cells cells adherent of vessel wall (with en endothelia ials cells cells) 3 Definite tu tumor cells cells within an en endothelia ial lin lined ed channel and no features to suggest artifactual vascular invasion 4 Presence of tu tumor cells cells in in ly lymphatic's 's or

• r ves

essel els, which is not caused by artifacts (such as smears, retraction, etc.) 5 Tumor cell cells usually as a group or nest within a space that is covered by en endoth theli lial l cells cells and does not contain a significant number of erythrocytes 6 The presence of a tu tumor em embolus with ithin in a ves essel el (capillary or lymphatic), usually well defined, rounding up to the contour of the vessel, may or

• r may not
• t be

e attached ed to the inner surface, may include red cells or fibrin; absence of marked autolysis

19

Manuscript in preparation

Proper histologic assessment is hampered by poor fixation

LVSI definition: a focus of tumor cells within a (blood filled) space lined by endothelial cells. Exclude LVSI mimics

• Marked autolysis
• Shear/retraction artefacts
• Smear/push artefacts
• MELF-type invasion

Criteria not required but helpful in excluding mimics

• Focus outside the immediate border of the tumor
• Focus surrounded by other vessels
• Focus contains vascular associated lymphocytes
• Multifocal, preferably >3 foci

232 uterine serous carcinoma Semiquantification:

• No LVSI
• Low (< 3 vessels)
• Extensive (≥ 3 vessels)

21

Winer I et al., Int J Gynecol Pathol 2014

(Re)assessment of LVSI from pooled PORTEC-1/-2 954 (83.6%) patients from PORTEC-1 and -2 Quantification using 2-, 3- and 4-tiered definitions of LVSI

2-tiered 

No No LVSI  LVSI present

3-tiered 

No No LVSI  Mild ild: a focus of LVSI was recognized around a tumor.  Su Substantia ial: diffuse or multifocal LVSI was recognized around the tumor.

4-tiered 

No No LVSI  Min inim imal: only a few lymph vascular vessels were involved on the border of the invasive front of the tumor.  Moderate: more vessels were involved in a wider area surrounding the tumor.  Promin inent: many vessels were diffusely involved in the deeper part of the myometrium.

Bosse et al, EJC 2015

T Serosa

Substantial LVSI

T Serosa

Mild LVSI

p<0.001

Substantial: HR 6.1 (2.3-15.9)

PORTEC-1 and -2 (N= 945) Substantial LVSI only (N=46) LVSI quantification and Regional Recurrence

p=0.08

What is the current risk assessment?

Low Risk

• Stage I Endometrioid + gr 1-2 + <50% myometrial invasion + LVSI neg

Low Inter Risk

• Stage I Endometrioid + gr 1-2 + ≥50% myometrial invasion + LVSI neg

High Inter Risk

• Stage I Endometrioid + gr 3 + <50% myometrial invasion, regardless of LVSI status
• Stage I Endometrioid + gr 1-2 + LVSI positive, regardless of depth of invasion

High Risk

• Stage I Endometrioid + gr 3 + ≥50% myometrial invasion, regardless of LVSI status
• Stage II & microscopic stage III
• Non endometrioid (Serous or Clear cell, etc)

Adv M+

• Stage III bulky & IVa
• Stage IVB

FIGO 2009 staging used Colombo et al., Annals of Oncology 2016

• LVSI assessment is important, but poorly defined
• LVSI assessment may requires quantification to be truly prognostic

What is the current risk assessment?

Manuscript in preparation

26

LVSI quantification of 42 cases - interobserver

20-Oct-16 27 Insert > Header & footer

Agreement

• N. cases

% Full (6/6) 6 14% Partial (4 or 5/6) 23 55% No 13 31% 42 100%

• Intra-class correlation

coefficient (ICC) = 0.54  moderate agreement

• Case selection: PORTEC 100%

LVSI positive (21 focal, 21 substantial)

A B C D E F Level of Agreement

1 2 2 2 2 2 2 2 1 1 2 2 2 3 1 2 1 2 2 2 4 1 2 2 2 2 2 5 1 2 1 2 2 6 2 1 2 2 1 2 7 2 1 2 2 1 2 8 1 2 1 1 9 2 2 2 2 2 10 2 2 2 2 2 2 11 2 2 2 2 2 12 2 2 1 1 13 1 1 1 1 1 1 14 1 2 1 2 2 15 1 1 1 1 16 1 2 1 2 2 17 1 1 2 2 2 2 18 2 2 2 1 2 2 19 1 1 2 2 2 2 20 1 1 1 1 21 2 2 2 2 2 2 22 2 2 2 2 2 23 2 1 1 1 24 2 1 2 2 2 2 25 2 2 1 2 2 2 26 2 2 1 2 2 27 1 1 2 2 1 2 28 2 1 2 2 2 2 29 1 2 2 2 2 30 2 2 2 2 2 31 2 1 2 1 2 32 1 2 2 2 2 2 33 1 1 2 1 2 2 34 2 2 2 1 2 35 2 2 2 2 2 2 36 2 2 2 2 2 2 37 2 1 2 1 1 2 38 1 2 1 2 39 1 1 2 2 2 40 1 2 1 1 2 41 2 2 2 2 2 42 2 2 2 2

Histopathologic assesment, reproducibility is an issue

Biomarker Practical Clinically Significant Reproducible Histopath – the big 5 YES YES MODERATE

• All 5 “biomarkers” are practical is use and have some value in predicting clinical

behavior, however all 5 have one major issue and that is REPRODUCIBILITY Room for improvement

• Fixation, fixation and fixation
• Improve definitions based on golden standard (patient outcome)!

Can molecular markers overcome this issue?

Limitations

• Variable treatment (20% RT, 10% CT, 10%

RT+CT)

• Incomplete treatment information (60%

unknown!)

• Heterogenous study population (stage I 70%,

Stage II-IV: 30%)

• Incomplete follow-up data

POLE TP53 MSI Remaining

TCGA, Nature 2013

• III. Remaining

Surrogate markers can be used for the TCGA classifiers

1Bosse et al, Gyn Onc 2014; 2Stelloo et al, Annals of Oncology 2016; 3Rayner et al, Nature reviews 2016

P53 IHC a good surrogate marker for TP53 mutations 80-95% concordance1 MMRd testing by IHC is a good surrogate marker for MSI tumors 100% concordance2 Sequencing of exon 9-14 of POLE is a good marker for ultramutation >95% concordance3

IHC practical and cheap, but we need to educate…

P53 IHC: interpretation of patterns in endometrial cancer

IHC practical and cheap, but we need to educate…

MLH1 PMS2 MSH2 MSH6

Triple negative MMR are the result of a secondary events in MSH6 Single MMR loss of MSH6 is the result of pathogenic mutations Subclonal MLH1/PMS2 is the result of MLH1 promotor hypermethylation Stelloo, et al., Annals of Oncology 2016

Biomarker Practical Clinically Significant Reproducible Histopath – big 5 YES YES MODERATE P53 – IHC YES YES GOOD* MMRd – IHC YES YES GOOD* L1CAM - IHC YES YES GOOD* ER/PR - IHC YES MAYBE MODERATE

*Accurate interpretation of p53, MMR and L1CAM IHC requires a trained GYNpathologist. Histopathologic “biomarkers” in Endometrial Cancer

TCGA surrogate markers have prognostic significance

Stelloo et al, CCR 2016; Talhouk et al, Br J Cancer 2015

5 1 0 0 .0 0 .5 1 .0 T im e (ye a rs) C um ula tive probability of lo coreg ion a l re cu rre n ce P -value < 0.001

P O L E

Locoregional recurrence PORTEC-1/-2

Biomarker Practical Clinically Significant Reproducible Histopath – big 5 YES YES MODERATE P53 – IHC YES YES GOOD* MMRd – IHC YES YES GOOD* L1CAM - IHC YES YES GOOD* ER/PR - IHC YES MAYBE MODERATE CTNNB1 exon3 – Sequencing NO YES EXCELLENT** POLE - Sequencing NO YES EXCELLENT**

*Accurate interpretation of p53, MMR and L1CAM IHC requires a trained GYNpathologist. ** Accurate interpretation of POLE en CTNNB1 sequencing results requires a molecular biologist/pathologist Histopathologic “biomarkers” in Endometrial Cancer

Molecular integrated risk assesment Favourable

• - POLE mutation

Intermediate risk Endometrial Cancer

• Stage I Endometrioid + gr 1-2 + ≥50% myometrial invasion + LVSI neg
• Stage I Endometrioid + gr 3 + <50% myometrial invasion, regardless of LVSI status
• Stage I Endometrioid + gr 1-2 + LVSI positive, regardless of depth of invasion

Unfavourable

• Substantial LVSI
• >10% L1CAM expression
• p53 mutant-like

expression Intermediate

• Microsatellite unstable or
• CTNNB1 exon 3 mutation

Favourable

• Microsatellite stable and
• CTNNB1 exon 3 wildtype

Stelloo, CCR 2016

Molecular integrated risk assesment

Stelloo, CCR 2016

0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 A rea U nder the C urve

* * * * * * * *

L o co re g io n a l re cu rre n ce D ista n t re cu rre n ce O ve ra ll su rviva l

M olecular integrated ( M olecular (P A indepe

C lin ica l ( M olecular (P A independent)

Integrated model builds on existing model

Lessons from PORTEC-1,-2, -3 and -4

#1 Upfront pathology review by expert gynaecological pathologists is to be preferred to ensure enrollment of the target trial population and to avoid unnecessary treatment and toxicity #2 Upfront (multi)central collection of tissue from trial patients is to be preferred to ensure an optimal trial-tumorbiobank #3 Prognostic or predictive markers need to fulfill 3 major criteria; a) practical , b) effective in predicting biological behavior and c) reproducible #4 …

DNA polymerase epsilon – exonuclease domain mutations

Exon 9 Exon 13 Exonuclease domain Exon 9-14 Rayner and van Gool et al., Nature Reviews 2016

POLE prognostic relevance in high-grade EC

18-okt-16

• Gr. 3 POLE wild-type
• Gr. 3 POLE proofreading-mutant

5 0 1 0 0 5 0 T im e (m o n th s ) R e c u rre n c e fre e s u rv iv a l (% )

1·0 0·8 0·6 0·4 0·2 0·0 Cumulative probabilty of recurrence 1· 0· 0· 0· 0· 0· 0 50 100 Months since diagnosis 1·0 0·8 0·6 0·4 0·2 0·0 Cumulative probabilty of recurrence 1· 0· 0· 0· 0· 0· 15 10 5 Years since randomisation P=0.02 G3 POLE wild-type G3 POLE proofreading-mutant

P=0.004

1· 0· 0· 0· 0· 0· 1· 0· 0· 0· 0· 0· 15 10 5 Years since randomisation

POLE-wildtype POLE-mutant POLE-wildtype POLE-mutant

Risk of recurrence Risk of recurrence

Church et al, JNCI 2015

Multivariable HR=0·11 95%CI 0·001–0·84 , P=0.028

Stelloo et al, Mod Path 2014

POLE-EC; To treat or not to treat remains the question

• Predictive marker for response to adjuvant therapy?
• Prognostic marker for indolent behavior?

POLE probably a true prognostic marker

Cumulative probability of recurrence P=0.069

Subanalysis NAT-arm PORTEC-1/-2 POLE highly immunogenic

Validation still required!

Van Gool et al., Oncoimmunology 2015

Lessons from PORTEC-1,-2, -3 and -4

#1 Upfront pathology review by expert gynaecological pathologists is to be preferred to ensure enrollment of the target trial population and to avoid unnecessary treatment and toxicity #2 Upfront (multi)central collection of tissue from trial patients is to be preferred to ensure an optimal trial-tumorbiobank #3 Prognostic or predictive markers need to fulfill 3 major criteria; a) practical , b) effective in predicting biological behavior and c) reproducible #4 Prognostic and predictive capacity of markers are easily confused #5 …

PORTEC-4a Randomised Phase III Trial of molecular profile-based versus standard recommendations for adjuvant radiotherapy for women with early stage endometrial cancer.

Molecular integrated risk assesment

Stelloo, CCR 2016

• 55% of high-intermediate risk patients reclassified to favourable
• 15% of high-intermediate risk patients reclassified to unfavourable

5 1 0 0 .0 0 .5 1 .0 T im e (ye a rs)

D is e a s e -fre e s u rv iv a l (% )

P -va lue < 0 .001

L o w H ig h -in te rm e d ia te H igh F a vou ra ble In te rm e d ia te U n fa vo urab le

k

2 0 4 0 6 0 8 0 1 0 0

% of patients

k ) M ole cu lar inte g ra ted C lin ical

5 1 0 0 .0 0 .5 1 .0 T im e (ye a rs)

D is e a s e -fre e s u rv iv a l (% )

P -va lu e < 0 .0 01

PORTEC4a

2 1 Standard treatment recommendation based

• n clinicopathological

factors Individual treatment recommendation based

• n molecular pathology

analysis Vaginal brachytherapy

Vaginal brachytherapy (~40%) Observation (~55%) External beam radiation therapy (~5%)

Follow-up and Quality of Life

Randomisation

A B C

Think of tumor heterogeneity

Esterik et al., Manuscript in preparation

Think of interlaboratory controls

For PORTEC4a we created multiple Control TMA blocks, including positive and negative controls for all antibodies in the profile

Inclusiecriteria

• Pilot phase: molecular profile in LUMC
• Validation of same procedures in other centers
• > 3-4 in NL and in other countries
• UK and ANZGOG planning to participate

Pilot phase

Lessons from PORTEC-1,-2, -3 and -4

#1 Upfront pathology review by expert gynaecological pathologists is to be preferred to ensure enrollment of the target trial population and to avoid unnecessary treatment and toxicity #2 Upfront (multi)central collection of tissue from trial patients is to be preferred to ensure an optimal trial-tumorbiobank #3 Prognostic or predictive markers need to fulfill 3 major criteria; a) practical , b) effective in predicting biological behavior and c) reproducible #4 Prognostic and predictive capacity of markers are easily confused #5 When considering a trial design which includes additional (molecular) tests it is preferred to consult a (molecular) pathologists and think of tumorheterogeneity and interlaboratory controls

Acknowledgements

51

LUMC, dept. of Pathology

Ellen Stelloo Inge van Gool

Enno Dreef Natalja ter Haar Bastiaan Wortman Reinhardt van Dijk Jef Ubachs Reinhardt van Dijk Manouk van Esterik Vincent Smit

LUMC, dept. of Clinical Oncology

Remi Nout Carien Creutzberg

LUMC, dept. of Gynecology

Cor de Kroon

UMCG, dept. of Gynecologic Oncology

Florine Eggink Marco de Bruyn Harry Hollema Hans Nijman

The Wellcome Trust Centre for Human Genetics, Molecular and Population Genetics Laboratory, Oxford, UK

Ian Tomlinson David Church