Biomarkers and Molecular Pathways in Endometrial Cancer

Donal BrennanQueensland Centre for Gynaecological Cancer

Plan

• Why we need biomarkers in EAC

• Serum biomarkers in pre-operative triage

• The rationale for targeted therapeutics in advanced / recurrent disease

Plan

• Why we need biomarkers in EAC

• Serum biomarkers in pre-operative triage

• The rationale for targeted therapeutics in advanced / recurrent disease

Type 1 v’s Type 2 endometrial cancer

Risk FactorsExcess Estrogen – endogenous and exogenousTamoxifenDiabetesNulliparity

Risk FactorsAgeObesity?BRCA

Loss of PTENKRAS mutationsMSI p53 mutations

Genomic instability

Type 1G1/2 EAC

Type 2G3 EACSerous

Clear Cell

Pre-operative triage

Increasing incidence of endometrial cancer in women < 50 years old

Arora et al . Best Pract Res Clin Obstet Gynaecol. 2012 Jun;26(3):311-24.

Plan

• Why we need biomarkers in EAC

• Serum biomarkers in pre-operative triage

• The rationale for targeted therapeutics in advanced / recurrent disease

Biomarkers in EAC

• Limited use• None utilised for triage of patients as diagnosis• CA-125 associated with extra-uterine disease– Lacks sensitivity and specificity– Threshold – 35 v’s 65 iu– NPV < 90%

• MRI to assess myometrial invasion– Cost issues, access issues, inter-observer variability

• Lymph nodes?????????

Predicting Extra-Uterine Disease

Salvesen HB et al Lancet Oncol 2012 8(13) e353 - e361

HE4 in endometrial cancer

• HE4 is increased in EC compared to normal endometrium

• A number of single institution retrospective studies have demonstrated HE4 associated with

– advanced stage

– myometrial invasion

– poor prognosis

• No data from population based studies

• Identified in Human Epididymis

• Secreted protein

• ? role in innate immunity

• Upregulated in a number of cancers – ovary, endometrial, lung

• FDA approved in EOC

Australian National Endometrial Cancer Study (ANECS)Australian National Endometrial Cancer Study (ANECS)

• Population based, case control study

• 1497 patients recruited between 2005 and 2007

• Peri-operative serum available for 373 patients

• CA-125 and HE4 quantified using Abbott Architecture kits

• Analysis performed in clinically accredited lab

• Non-parametric Mann Whitney U test, ROC analysis, Kaplan Meier and Cox Porportional Hazards applied.

ANECS HE4 studyANECS HE4 study

Manuscript submitted

Median HE4 (IQR) [pmol/L] P Value

Median CA125 (IQR) [U/ml] P Value

All womenFIGO Stage (2009)Stage I & II (n =339) 72·7 (53·2-106·3) <0·001 14·9 (11-23) <0·001Stage III & IV (n=30) 134·6 (83-237·7) 29·4 (15·1-76·5)

GradeGrade I & II (n=291) 73·1 (53·5-114·7) 0·572 15·4 (11·1-24·1) 0·431Grade III (n=76) 82·7 (53·3-113·4) 15·5 (11·3-28·5)

Endometrioid HistologyNon Endometrioid (n=57) 81·8 (56·0-110·7) 0·765 14·9 (11-30·5) 0·496Endometrioid (n=316) 73·7 (53·6-113·4) 15·5 (11·2-24·2)

LVSIAbsent (n=276) 74·2 (53·1-111·2) 0·908 14·9 (11·2-24·8) 0·853Present (n=57) 77·9 (51·8-114·3) 15·4 (9·9-25·9)

Myometrial Invasion≤ 50% (n=289) 68·0? (52-93·6) <0·001 14·6 (11·1-21·8) <0·001> 50% (n=84) 115·3 (81·7-178·9) 25·1 (11·8-43·5)

Age< 62 (n=184) 64·0 (48·1-91·2) <0·001 15·5 (11·6-26·1) 0·295≥62 (n=189) 85·2 (61·8-136·2) 15·2 (10·5-26)

BMI (kg/m2)< 30 (n=184) 68·4 (52·5-101·3) 0·024 15·0? (10·9-26·8) 0·723>/= 30 (n=189) 81·6 (55·1-122) 15·6 (11·4-24·2)

Diabetes No (n=309) 73·1 (53·2-106·5) 0·023 15·4 (11·2-26) 0·781Yes (n=64) 91·1 (57·3-152·3) 15·9 (11·8-26·3)

HypertensionNo (n=231) 69·8 (50-101·6) 0·001 15·2 (11·2-24·9) 0·653Yes (n=137) 82·7 (60-131·1)   15·8 (11·6-26·6)  

Association with Clinicopathological FactorsAssociation with Clinicopathological Factors

Manuscript submitted

Predicting Outer Half Myometrial Involvement

All Patients (n=373)

AUC HE4 – 0.76 (0.70-0.81)

CA-125 – 0.65 (0.57-0.72)

Endometrioid Histology (n = 316)

AUC HE4 – 0.76 (0.70-0.83)

CA-125 – 0.62 (0.53-0.71)

Endometrioid Histology, Grade 1 and 2 (n = 280)

AUC HE4 – 0.77 (0.70-0.83)

CA-125 – 0.64 (0.55-0.73)

Applying 70pmol/L as a threshold

Manuscript submitted

P = 0.05 P = 0.001

HE4 as a Prognostic Marker

Manuscript submitted

Multivariate Analysis of RFS

Manuscript submitted

• Largest study of HE4 in EC to date

• First population based study

• Elevated HE4 is associated with an aggressive phenotype

• HE4 is a superior to CA125 in predicting outer-half myometrial invasion

– HE4 < 70 pmol/L, ? avoid lymphadenectomy

• HE4 is an independent predictor of OS and RFS

HE4 Summary

Manuscript submitted

Where next?

• What is the functional role of HE4 in endometrial cancer.

• What effect does progesterone treatment have on HE4?

• Can HE4 be used to monitor patients on hormonal therapy

• Are age-specific references needed?

• Is HE4 elevated in recurrent disease ?

– Particularly if it was normal at initial presentation

• Can HE4 be used to monitor for recurrence?

HE4 may be beneficial in pre-operative triage Can we apply targeted therapeutic in advanced /

recurrent disease

HE4 may be beneficial in pre-operative triage Can we apply targeted therapeutic in advanced /

recurrent disease

Plan

• Why we need biomarkers in EAC

• Serum biomarkers in pre-operative triage

• The rationale for targeted therapeutics in advanced / recurrent disease

Historical PerspectiveHistorical Perspective

• 18th century – Biology• 19th century – Chemistry• 20th century – Physics / Computer Science• 21st century – Molecular Medicine

Principles of Molecular MedicinePrinciples of Molecular Medicine

• Successful new therapies will be based on a foundation of precise molecular understanding of disease

• Translational science must adopt a multidisciplinary approach that requires substantial infrastructure

• Human subjects are an essential early component in the evaluation of new drug candidates

Charles Sawyers J Clin Invest 2009

The Targeted Therapeutic Era to Date

• Molecular Diagnostics are urgently required• Oncogene addiction

– Only relevant in certain tumour types

• Repeat analysis during treatment– Inclusion of translational arm in early phase trials– Pathway diagnostics

Adv Genomics Genet. ; 2012(2): 33–47

Somatic Mutations in Endometrial Cancer

Classification of EAC based on somatic mutation profiling

Type 1 Type 2Type 1 – PTEN, PI3K, KRAS commonGrade III – higher incidence of p53 and PI3K mutations , less CTNNB1, KRAS Type II – p53 mutations common, PTEN mutations rare

Cheung L W et al. Cancer Discovery 2011;1:170-185

Alterations in PI3K pathway seen in 80% of endometrioid cancersLoss of MMR protein expression associated with higher mutation prevalence

Somatic Mutations in Endometrial Cancer

FGFR2 Her2

PI3KKRAS

PI3K

PTENCTNNB1

p53

Type 1 Type 2

What does and unsupervised approach add ?

• Similar mutation frequencies for normal candidates - PTEN, p53, PI3K, CTNNB1

• 10 novel tumour suppressor genes – ARID1A, INHBA, KMO, TTLL5, GRM8, IGFBP3, AKTIP, PHKA2, TRPS1, and WNT11

• 2 novel oncogenes – ERBB3 and RPS6KC1

Liang H et al. Genome Res. 2012;22:2120-2129Wu J N , and Roberts C W Cancer Discovery 2013;3:35-43

Loss of ARID1A expression in primary human cancersLoss of ARID1A expression in primary human cancers

? Haploinsufficient effect

ARID1A regulates PI3K activity in Endometrial CancerARID1A regulates PI3K activity in Endometrial Cancer

Liang H et al. Genome Res. 2012;22:2120-2129

RAS and PI3K pathwaysRAS and PI3K pathways

www.biooncology.com

Downstream Effects of PI3K and KRAS mutations Downstream Effects of PI3K and KRAS mutations

Distinct downstream signaling events are activated in PI3K pathway- and KRAS-mutant EC

Cheung L W et al. Cancer Discovery 2011;1:170-185

KRAS as a therapeutic targetKRAS as a therapeutic target

• Best molecular targets are aberrantly activated kinases that are inhibited by selective small-molecule inhibitors.

• Oncogenic Ras proteins have reduced GTPase activity

• Restoring enzymatic function is a major challenge

• Prognostic relevance of KRAS mutation remains inconsistant

• MEK inhibitors have shown some promise in other tumour types

PI3K Pathway as a therapeutic targetPI3K Pathway as a therapeutic target

Salvesen H B et al. PNAS 2009;106:4834-4839 Slomovitz B M , and Coleman R L Clin Cancer Res 2012;18:5856-5864

How do we define activity of PI3K pathway?How do we define activity of PI3K pathway?

• Major issue for trial design

• Clinically valid molecular diagnostic is urgently required

• Options include – PTEN IHC

– pAKT

– Stathmin IHC

– Sequence based assay

– Combination assay

Djordjevic et al Mod Pathology 2012

Targeting PI3K / AKT / mTORTargeting PI3K / AKT / mTOR

Slomovitz B M , and Coleman R L Clin Cancer Res 2012;18:5856-5864

Temsirolimus in recurrent o4 metastatic EACTemsirolimus in recurrent o4 metastatic EAC

Oza A M et al. JCO 2011;29:3278-3285

62 patients, weekly temsirolimus 25mg

•Chemo-Naïve group (Group A)– 69% SD or better

– Median PFS – 9.7 months

•Chemo-treated group (Group B)– 48% SD or better

– Median PFS 3.2 months

•Toxicity– Rash, fatigue, mucositis, pneumosisitis

– Higher dose reduction in chemo-treated group

•Unable to identify any predictor of response

Phase 1 and phase 2 trials of targeted therapies for endometrial cancer

>100 trials registered investigating PI3K inhibition26 trials open in endometrial cancer4 are biomarker enriched

Rapalogues / PI3K inhibitors in breast and gynae cancersCombined with various other regimensResponse rate 30% in patients with PI3KCA mutationResponse rate 10% in patients with wild type PI3KCA

Predicting response to PI3K inhibition

p85

p110

AKT

mTORC1

S6K4EBP1

PTEN

mTORC2

Rapalogs

AKT inhibitors

PI3K inhibitors

Dual mTORInhibitors

Approach to identify predictive biomarkersApproach to identify predictive biomarkers

Brennan et al Nat Rev Cancer 2010

Coexistent KRAS mutation predicts resistanceCoexistent Her2 amplifcation predicts responsePTEN mutant cells are sensitive to PI3Kb inhibitorsPTEN loss may predict response to AKT inhibitors

Must identify addicted v’s dependent populations

Future for Targeted Therapies in Endometrial Future for Targeted Therapies in Endometrial CancerCancer

• Biomarker enriched, adaptive trial design

• Repeat biopsy • Tumour heterogeneity• Circulating free DNA

Leary et al Sci Tran Med 2012

• Confirmation of pathway inhibition• Identify mechanisms of resistance• Combination therapy

– Vertical – RAF / MEK inhibition in melanoma– Horizontal – MEK + PI3K / PI3K + PARP– Endocrine therapy– Cytotoxics

ER SignallingE2 Growth Factor

e.g. EGF or IGF-1

ERE ER Responsive Gene

ER ER

P P

Classical Ligand Dependent Pathway

mRNA Protein

TissueResponse

AP-1 Target Gene

Jun

ER

P

mRNA Protein

Fos

GenomicPathway

ER

MISS

MAP KinaseActivation

Ligand IndependentPathway

Brennan et al Oncogene 2012

PRER

IGF-R

PR, PS2 Inhibition of proliferation

PI3K

AKT

mTOR

PTEN LKB1

AMPK

p-S6K

Inhibition of

Transcriptio

n

PR response genes

Metformin

IGF 1/II

E2

P

ER response genes

Rationale for combining PI3K inhibition and endocrine Rationale for combining PI3K inhibition and endocrine therapiestherapies

Conclusions

• Why we need biomarkers in EAC• Fertility sparing therapy,• Obesity

• Serum biomarkers in pre-operative triage• HE4 shows some promise

• The rationale for targeted therapeutics in advanced / recurrent disease• PI3K inhibitors are unlikely to have a role as single

agents• Combination, biomarker enriched studies may offer

best chance of clinical response