1

Clara Montagut, M.D., Ph.D.

University Hospital del Mar, Barcelona

Why is sensitivity so important to

detect RAS mutations with the

liquid biopsy? Results of the

Concordance study

February 25, 2017. Sysmex Scientific Symposium

National Comprehensive Cancer Network (NCCN) 20161

Proposed ESMO consensus 2015/162

“The panel strongly recommends genotyping of tumor tissue (either primary tumor or metastasis) in all patients with metastatic colorectal

cancer for RAS (KRAS exon 2 and non-exon 2; NRAS) and BRAF at diagnosis

of stage IV disease”

“The appropriate molecular analyses are to be carried out at the time of initial diagnosis of mCRC and should

comprise a full analysis of tumour RAS mutational status (KRAS: exon 2, 3 and

4 and NRAS: exon 2, 3 and 4) with a simultaneous analysis of tumour BRAF

mutational status, conducted in a validated laboratory/testing centre, to

facilitate the best diagnostic and prognostic decision making possible.”

“Turnaround time for RAS testing (expanded RAS analysis) should be

≤7 working days from the time of receipt of the specimen by the testing laboratory to the time of issuing of the final report, for >90% of specimens”

ESMO consensus 20162

NCCN clinical practice guidelines; Colon Cancer, Version 2.2016 www.nccn.org/professionals/physician_gls/pdf/colon.pdVan Cutsem E, et al. Ann Oncol 2016

RAS testing is mandatory for first line treatment decision making

3

Why is RAS testing so important?

1. Van Cutsem E, et al. J Clin Oncol 2011;29:2011–2019; 2. Van Cutsem E, et al. J Clin Oncol 2015;33:692–700; 3. Douillard J-Y, et

al. N Engl J Med 2013;369:1023–1034; 4. Heinemann V, et al. Lancet Oncol 2014;15:1065–1075; 5. Stintzing S, et al. Lancet Oncol 2016 (epub

ahead of print); 6. Bokemeyer C, et al. Ann Oncol 2011;22:1535–1546; 7. Bokemeyer C, et al. Eur J Cancer 2015;51:1243–1252; 8. Schwartzberg,

et al. J Clin Oncol 2014;32:2240–2247; 9. Erbitux® SmPC June 2014; 10. Vectibix® SmPC February 2015.

Anti-EGFR (cetuximab and panitumumab) benefits are

greater in RAS wt than KRAS (exon 2) wt or unselected

patients

RAS wt population

Overall patient population

Exte

nd

ed

ben

efi

t o

f an

ti-E

GFR

th

erap

y

KRAS (exon 2) wt population

How do we test for RAS?Tissue-based RAS testing is well established but contains challenges

Obtain

tumor

tissue block

Manual

micro-

dissection

DNA

isolation &

purification

Assessment

of DNA

quantity

Treatment

decision

Assessment

of biomarker

status

How do we test for RAS?Tissue-based RAS testing is well established but contains challenges

Obtain

tumor

tissue block

Manual

micro-

dissection

DNA

isolation &

purification

Assessment

of DNA

quantity

Treatment

decision

Invasive

procedure

Tissue not always

accessible

Assessment

of biomarker

status

Obtain

tumor

tissue block

Manual

micro-

dissection

DNA

isolation &

purification

Assessment

of DNA

quantity

Treatment

decision

Invasive

procedure

Tissue not always

accessible

Assessment

of biomarker

status

Archival tissue

Static, potentially outdated

mutation profile, often

degraded or unavailable

How do we test for RAS?Tissue-based RAS testing is well established but contains challenges

7

93%

67%

0%

20%

40%

60%

80%

100%

Concurrent biopsy Biopsy >6 monthsold

Tissue vs. Plasma NGS Correlation

Talasaz et al. 2014 Abstract e22041, J Clin Oncol 32(15_suppl)

Would you treat a patient based on a six month

old CT scan?

Obtain

tumor

tissue block

Manual

micro-

dissection

DNA

isolation &

purification

Assessment

of DNA

quantity

Treatment

decision

Invasive

procedureSelection bias

Tumor

heterogeneity

Tissue not always

accessible

Assessment

of biomarker

status

How do we test for RAS?Tissue-based RAS testing is well established but contains challenges

Archival tissue

Static, potentially outdated

mutation profile, often

degraded or unavailable

Tumor heterogeneity

Adapted from Gerlinger NEJM 2012

Emergence of mutations of resistance

RASRAS

wt

Response to anti-EGFR

treatment

RAS mutant tumor at

progression to treatmentBasal RAS wt tumor

wtRAS wt

Anti-EGFR treatment

Temporal heterogeneity - Clonalselection

Obtain

tumor

tissue block

Manual

micro-

dissection

DNA

isolation &

purification

Assessment

of DNA

quantity

Treatment

decision

Invasive

procedureSelection bias

Tumor

heterogeneity

Tissue not always

accessible

Assessment

of biomarker

status

How do we test for RAS?Tissue-based RAS testing is well established but contains challenges

Archival tissue

Static, potentially outdated

mutation profile, often

degraded or unavailable

Liquid Biopsy – RAS testing in circulating tumor (ct) DNA

Diaz&Bardelli, J Clin Oncol 2014

Bettegowda et al. C Sci Transl Med. 2014

1%

100%

10%

0.1%

0.01% BEAMing

Real-Time PCR

Pyrosequencing

Sanger Sequencing

Detection capacity(mutant DNA/ total DNA)

Tumoral DNA

(somatic mutation)

Normal DNA

(wild-Type)

Adapted from A. Vivancos

High sensitivity is required to dectect ctDNA

Patient Characteristics N 109 (%)

Age median (range) 67 (39-86)

Gender MaleFemale

76 (70%)33 (30%)

Stage at diagnosesII / IIIIV

20 (18%)89 (82%)

Primary site of diseaseRight colonLeft colonRectumUnknown

29 (26%)39 (36%)39 (36%)2 (2%)

Primary tumor resectedYesNo

60 (55%)49 (45%)

Systemic treatment before ctDNAYesNo

82 (75%)27 (25%)

Tumor site biopsyPrimaryMetastasis

96 (88%)13 (12%)

Number of metastatic sites123 or more

28 (50%)22 (39%)6 (11%)

Metastasis LocationLiverLungPeritoneumnodeOthers

80 (73%)41 (38%)25 (23%)18 (16%)8 (7%)

115 mCRC patients No patient received anti-EGFR treatment before

plasma collection The OncoBEAM™ RAS CRC assay was used to

detect RAS mutations in plasma RAS testing in tissue was done by standard-of-

care (SOC) For discordant cases, tissue samples were re-

examined with tissue BEAMing

Exon Mutation

2

G12S

G12R

G12C

G12D

G12A

G12V

G13D

3

A59T

Q61L

Q61R

Q61H

Q61H

4

K117N

K117N

A146T

A146V

Exon Mutation

2

G12S

G12R

G12C

G12D

G12A

G12V

G13R

G13D

G13V

3

A59T

Q61K

Q61R

Q61L

Q61H

Q61H

4

K117N

K117N

A146T

NRASKRAS

ACCURACY OF PLASMA RAS MUTATION TESTING FOR THERAPY SELECTION AND MONITORING OF COLORECTAL CANCER PATIENTS

Hospital del Mar, Barcelona and Complexo Hospitalario Universitario de Santiago de Compostela

Vidal et al. ESMO 2016

Positive Agreement: 96,1%

Negative Agreement: 91,4%

Overall Agreement: 93,6%

Tissue RAS Result

PlasmaRas

Result

Positive Negative Total

Positive 49* 5 54

Negative 2 53 55

Total 51 58 109

Plasma/Tissue Correlation with OncoBEAM RAS CRC

Vidal et al. ESMO 2016

24%

9%

3%2%

5%3%1%4%

49%

PLASMA RAS BEAMING

23%

9%

1%

2%

5%3%1%4%

52%

TISSUE RAS SoC

KR12

KR13

KR61

KR117

KR146

NR12

NR13+

NR61+

WT

Overview of concordance data presented at ECC and WCGC

2015:

Strengthening the evidence for use of liquid biopsy (BEAMing, Sysmex Inostics)RAS testing

Abstract No. 402, P052Hahn S, et al.

92% overall concordance

between liquid and tissue-based RAS

testing1

Abstract No. 2012, P002Jones FS, et al.

93% overall concordance

between liquid and tissue-based RAS

testing2

Fully in line with data from WCGC 20153

1. Hahn S, et al. ECC 2015 (Abstract No. 402); 2. Jones FS, et al. ECC 2015 (Abstract No. 2012); 3. Scott R, et al. WCGC 2015 (Abstract No. P-273)

Codon% plasma

Mut fraction

Site tumor biopsy

Primary tumor

resected

Days between tissue – plasma collection

Systemic treatment before ctDNA

TreatmentBest

Response

1 KRAS 13 0,2458% primary yes 71 NoFOLFOX

PanitumumabPD

2 KRAS 12 0,128% primary yes 138 No FOLFOX Cetuximab PR

3 KRAS 61 31.73% primary yes 122 No XELOX PD

4 KRAS 12 0,896% primary yes 60 No FOLFOX Cetuximab PR

5 KRAS 61 0,316% primary no 32 YesFOLFOX

CetuximabPR

RAS PLASMA WT/ TISSUE MUT

6 KRAS 12 primary no 1195 No no

7KRAS

12primary yes 39 No

FOLFOX Bevacizumab

PR

RAS PLASMA MUT / TISSUE WT

Analysis of plasma-tissue discrepant cases

Vidal et al. ESMO 2016

Tissue DNA sensitivity may be limited because samples fail to capture

tumor heterogeneity

ctDNA may be limited when tumor DNA is not shed into circulation

p 0.056p 0.007

Correlation between circulating RAS mutations frequency and clinical characteristics

p 0.001

• Patients with hepatic metastases have higher RAS ctDNA levels

• Patients with peritoneum or lung involvment have lower RAS ctDNA levels

• RAS ctDNA levels decrease after administration of chemotherapy

Vidal & Muinelo et al. Submitted

Median MAF: 2.317%

Median MAF: 0,281%

RAS mutations

(N=52)

High sensitivity is necessary to detect RAS in plasma

Vidal & Muinelo et al. Submitted

Is plasma RAS testing a good alternative to be used in clinical

practice?

Pacientes RAS wt en tejido Pacientes RAS wt en plasma

PFS for patients treated with antiEGFR was the same

for tissue RAS wt and plasma RAS wt patients (median

PFS 10.3months)

Test in

tissue

✓ High compliance – easy for the patient

✓ Low risk

✓ Fast result

✓ Accessibility

✓ Monitoring

Oncologist

Biopsy

Pathologist

Molecular

Testing

Test in

plasma

OncoRAS Sysmex liquid biopsy RAS testing in daily clinical practice

Nurse

Blood Extraction

Plasma RAS testing: clinical applications in

metastatic colorectal cancer

1. RAS testing at diagnosis to guide treatment decision

2. Monitoring resistance to anti-EGFR therapy

23

New paradigm in treatment of mCRCTREATING CLONAL EVOLUTION

Dientsmann, JCO 2013

Need to re-evaluate tumor molecular profile along the course of the

disease: liquid biopsy

Spatial and temporal heterogeneity

Monitoring mutations of resistance in the blood of patients

Misale, Nature 2012; Diaz, Nature 2012; Bettegowda, STM 2014 ; Morelli, Ann Oncol 2015

Vidal et al. ESMO 2016

OncoBEAM RAS CRC has enough sensitivity to

detect emergence of RAS mutations during anti-

EGFR therapy

Emergence of RAS mutations after antiEGFR treatment in 18 patients

with RAS wt tumors at diagnosis

Liquid biopsy RAS monitoring: Clinical Challenges

and opportunities

Dynamics of RAS emergence: What RAS mutation percentage is clinically relevant to predict early relapse?

Can RAS testing in ctDNA help identify candidates for cetuximab rechallenge?

Aim: To assess the clinical relevance of monitoring RAS, BRAF and EGFR ECD mutations by liquid biopsy from RAS wt mCRCpatients treated with cetuximab-based therapy

Diagnosis During treatment Progression Post-progression

Plasma

sample

Tumor

sampl

e

Monthly

Cetuximab-based treatment in 1st line

Baseline +3 / +6 monthsEnd

of treatment

Liquid biopsy RAS monitoring: Clinical Challenges

and opportunities

Can RAS testing in ctDNA help identify candidates for cetuximab rechallenge?

Dynamics of RAS emergence: What RAS mutation percentage is clinically relevant to predict early relapse?

Mutations in RAS emerge during anti-EGFR treatment

RASRAS

wt

Response to treatment Progression to treatmentBasal RAS wt tumor

wtRAS wt

Cetuximab rechallenge

Mutations in RAS emerge during anti-EGFR treatment

and decline when treatment is suspendend

RAS

RAS

wt

Response to treatment Progression to treatment

wtRAS wt

Off treatment

Basal RAS wt tumor

Cetuximab rechallenge

Liquid biopsy for longitudinal monitoring of RAS mutations in blood of patients

Rechallenge with cetuximab

Siravegna et al. Nat Med 2015

Rechallenge with anti-EGFR therapy

Cetuximab + FOLFIRI

R

FIRE-4 (Phase III, n=550)

Bevacizumab + FOLFOX/XELOX

Cetuximab + irinotecan/FOLFIRI

RegorafenibCetuximab + FOLFIRI

R

1st line 2nd line 3rd line

Liquid biopsy to track/identify resistance

• Primary endpoint: OS after randomization 2• Results expected: January 2022

RAS wt

mCRCBevacizumab + FP maintenance

Take-home message

- RAS testing is mandatory for first line treatment decision in mCRC

- OncoBEAM RAS testing is a less invasive, highly sensitive

alternative to tissue-based RAS testing at diagnosis

- OncoBEAM RAS is useful to monitor emergence of RAS

mutations of resistance in mCRC patients treated with anti-EGFR

- Liquid biopsy-driven clinical trials to guide treatment strategy are

ongoing and are crucial to improve patient’s outcome

Thank you

cmontagut@hospitaldelmar.cat

35

CRYSTAL: Greater patient selection based on RAS extended the benefit

with cetuximab + FOLFIRI

HR=0.69 (0.54–0.88)

p=0.0024

Δ = 8.2

months

HR=0.796 (0.67–0.95)

p=0.0093

HR=0.878 (0.77–1.00)

p=0.0419

Δ = 3.5

months

Δ = 1.3

months

1. Van Cutsem E, et al. J Clin Oncol 2011;29:2011–2019;

2. Van Cutsem E, et al. J Clin Oncol 2015;33:692–700;

3. Douillard J-Y, et al. N Engl J Med 2013;369:1023–1034;

4. Erbitux® SmPC June 2014; 5. Vectibix® SmPC February 2015.

Cetuximab + FOLFIRI (n=178)

FOLFIRI (n=189)

0.0

0.2

0.

4

0.6

0.8

1.0

Months5442 48186 12 24 30 36

28.4

20.2

0

Months

5442 48

23.5

20.0

0.0

0.2

0.4

0.6

0.8

1.0

180 6 12 24 30 36

Months5442 48

0.0

0.2

0.4

0.6

0.8

1.0

180 6 12 24 30 36

OS

es

tim

ate

19.9

18.6

Cetuximab + FOLFIRI (n=599)

FOLFIRI (n=599)

Cetuximab + FOLFIRI (n=316)

FOLFIRI (n=350)

RAS wt2KRAS exon 2 wt1ITT (unselected)1

A similar effect of increasing biomarker selection was observed in the Phase III PRIME study of 1st line FOLFOX ± panitumumab3