BIOPSIA LÍQUIDA y nuevas NGS EN CÁNCER DE PULMON

Prof. Mariano Provencio

Servicio Oncología Médica

LiquidBiopsyLabPdH (@LiquidBiopsyLab)

ctDNA

Crowley, E. et al. (2013) Nat. Rev. Clin. Oncol.

Technical challenges

• The fraction of ptDNA in total plasma is often small

• The quality of plasma is highly dependent on how it is processed

• Assaying for rare mutation detection is akin to looking for needles in haystack

Where is the mutant Pablo?

Where is the mutant Pablo?

• ctDNA shows superior sensitivity in detecting various cancers compared with circulating tumour cells (CTCs)

Detection of disease and its recurrence

Liquid biopsy may provide clinical benefit

• Detection of residual disease with liquid biopsy may enable personalised therapies earlier in

disease progression

• Serial liquid biopsies can be used to monitor disease progression and detect early recurrence

Provencio M , et al . Oncotarget. 2017 Aug 7;8(36):60291-60298. Provencio M , et al . Oncotarget. 2017 Nov 16;9(1):488-494

• ctDNA is a fraction of a fraction of the plasma1 and increases with disease progression2

Detection of ctDNA requires highly sensitive techniques

Tumor-derived nucleic acids and their detection

• The amount of ctDNA released into the blood is

dependent on several factors

Tumor grade,

histology and

vascularity1,2,3

Physiological

clearance and

degradation4,5,6

Rate of release

and cell

status3,4

Time of blood

draw and

therapy1,7,8

1. Hinrichsen, T., et al. (2016) J Lab Med. 40:313-22; 2. Bettegowda C, et al. (2014) Sci Transl Med. 6:224ra24; 3. Diaz, L.A., &

Bardelli, A. (2016) J Clin Oncol. 32:579-86; 4. Siravegna, G., et al. (2017) Nat Rev Clin Oncol. advance online publication; 5. Forte,

V.A., et al. (2016) Cancer Biol Med. 13:19–40; 6. Leung, F., et al. (2016) Clin Chem. 62:1054–60; 7. Diehl, M., et al. (2008) Nat

Med. 14:985-90; 8. Tie, J., et al. (2015) Ann Oncol. 26:1715–22.

0

100

80

60

40

20

Fre

qu

en

cy o

f cases w

ith

dete

cta

ble

ctD

NA

(%

)

Stage I (n = 49)

Stage 2 (n = 133)

Stage 3 (n = 51)

Stage 4 (n = 136)

Adapted from Bettegowda C, et al 20142

Technology

Vendrell JA et al.Circulating Cell Free Tumor DNA Detection as a Routine

Tool forLung Cancer Patient Management. Int J Mol Sci. 2017;18(2)

Diaz LA . J Clin Oncol. 2014 Feb 20;32(6):579-86.

Diaz LA . J Clin Oncol. 2014 Feb 20;32(6):579-86.

PRINCIPLE Sensitivity (MAF) Adventage Limitations

dPCR 1-0.01%

Ease of use

Robustness

Inexpensive

Only a limited

number of mutations

can be testedLimited

NGS 1-0.002%

A large

number of

mutation

can be

tested at a

time Expensive

EGFR

KRAS Unknown

A growing number of

potentially targetable cancer-related

genes are being identified

Increasing numbers

of targeted therapies are

becoming available

2004

Clinically relevant driver mutations identified in patients with lung adenocarcinoma*

*Patients referred for genomic

testing between January 2014

and March 2016 Sourced from Jordan et al. 20178

© 2017 American Association for Cancer Research

KRAS

EGFR

Unknown

2004 2016

EGFR exon 20

EGFR T790M

EGFR WT amp

EGFR sensitising

No mutations

Other drivers

PTEN loss

CDKN2A loss

BRAF non-V600E

NF1 loss

FGFR1/2

NRAS

PIK3CA

MAP2K1

ERBB2 mut

TSC1/2 loss

BRCA1/2 loss

ERBB2 amp

MET amp

MET splice

BRAF V600E

RET fusion

ROS1 fusion

ALK fusion

A growing number of potentially targetable cancer-related genes are being identified

Paciente Gen Proteína NGS (%) dPCR (%)

ARS-ALK ALK p.Gly1269Ala 0,87 0,42

AMLO-ALK ALK p.Gly1269Ala 2,99 2,81

NGS vs dPCR

Pearson´s r=0,987;p<0,0001

AF=2,81%

AF=2,99%

4 pacientes translocados:

Provencio et al submitted.

Shaw AT, et al. Resensitization to crizotinib by the lorlatinib ALK resistance mutation L1198F. N Engl J Med. 2016;374(1):54–61

Katayama R, Lovly CM, Shaw AT. Therapeutic targeting of anaplastic lymphoma kinase in lung cancer: a paradigm for precision cancer medicine. Clin Cancer Res. 2015;21:2227–35.

Friboulet L et al. The ALK inhibitor ceritinib overcomes crizotinib resistance in non-small cell lung cancer. Cancer Discov. 2014;4:662–73.

MOLECULAR PROFILING IN ALK (+) PATIENTS

Diaz LA . J Clin Oncol. 2014 Feb 20;32(6):579-86.

PRINCIPLE Sensitivity (MAF) Adventage Limitations

dPCR 1-0.01%

Ease of use

Robustness

Inexpensive

Only a limited

number of mutations

can be testedLimited

NGS 1-0.002%

A large

number of

mutation

can be

tested at a

time Expensive

Commercial NGS liquid biopsy test

Guardant 360 (Guardant Health)

FoundationOne@liquiq (Foundation

Medicine)

AVENIO ctDNA Targeted Kit, ctDNA Expanded

Kit and ctDNA Surveillance Kit (Roche

Diagnostics)

Oncomine Lung cfDNA Assay (Thermo Fisher

Scientific

NEOliquid (New Oncology)

Reveal ctDNA 28 Kit (ArcherDX)

OptiSeq™ Pan-Cancer Panel (DiaCarta)

PlasmaSELECT™-R 64 (PGDx)

Signatera™ RUO (Natera)

75.6% of oncologists reported using NGS tests in the past 12 months to guide treatment decisions

“More than 50% of oncologists in our study reported that NGS test results were difficult to interpret either often or sometimes. In addition, 25% indicated that they referred patients to other providers for NGS testing..”

* F1 CDx bait-set available Ex-US from 01 October 2018; † From date of receipt. FFPE: formalin-fixed, paraffin-embedded; HC: hybrid capture; MSI: microsatellite instability; NGS: next-generation sequencing; TMB: tumour mutational

burden; . 1. Foundation Medicine. (2018) FoundationACT Technical Specifications. Accessed Jun 2018 from

https://assets.ctfassets.net/vhribv12lmne/3SPYAcbGdqAeMsOqMyKUog/3b10e8355a717f658c82d7544503df9f/FACT_TechnicalInformation_09_RD.pdf;

2. Foundation Medicine. (2018) FoundationACT Sample patient report; 3. Foundation Medicine. (2017) FoundationACT Webpage summary. Accessed Oct 2017 from https://www.foundationmedicine.com/genomic-testing/foundation-act; 4.

Foundation Medicine. FoundationOne Liquid Technical Specifications to be published Sept 2018; 5. Foundation Medicine. (2017) FoundationOne Technical Specifications. Accessed Oct 2017 from

https://assets.ctfassets.net/vhribv12lmne/6YRrchSINOeSu48YwuesoY/0c3651c8421fa3647ccede76de9dce61/MKT-0054-02_F1_TechSpecs_digital.pdf; 6. Foundation Medicine. (2017) FoundationOne Webpage summary. Accessed Oct

2017 from https://www.foundationmedicine.com/genomic-testing/foundation-one; 7. Frampton, G.M., et al. 2013. Nat Biotechnol 31:1023-31; 8. Foundation Medicine. (2017) FoundationOneHeme Technical Specifications. Accessed Oct 2017

from https://assets.ctfassets.net/vhribv12lmne/zBxaQC12cScqgsEk8seMO/abf6133874f1e5929403f66d90c3b900/F1H_TechnicalInformation_06_digital.pdf; Foundation Medicine. (2018) FoundationOneCDx Technical Specifications.

Accessed Jun 2018 from https://assets.ctfassets.net/vhribv12lmne/4ZHUEfEiI8iOCk2Q6saGcU/671b313cb6bb85bfe861f83e31c9716d/F1CDx_TechInfo_09-03.pdf;

9. Foundation Medicine. (2017) FoundationOneHeme Webpage summary. Accessed Oct 2017 from https://www.foundationmedicine.com/genomic-testing/foundation-one-heme;

10. He, J., et al. 2016. Blood 127:3004-14.

19

FoundationOne® Liquid complements the Foundation

Medicine portfolio

FoundationOne®Liquid 1-4 Foundation®One 5-7 FoundationOne®Heme 8-10

Target patient population Solid tumours Solid tumours Haematologic malignancies & Sarcomas

Specimen type Peripheral blood FFPE tissue FFPE tissue Bone marrow aspirate Peripheral blood

Genes assayed 70 324* 406 265 (via RNA)

Genes with complete exon coverage 35 324* 406

All 4 classes of genomic alterations Yes Yes Yes

TMB No Yes Yes

MSI Yes Yes Yes

Method HC-based NGS HC-based NGS HC-based NGS

Turnaround time† < 14 days 14 days or less 14 days

* Only use authorised Specimen Collection and Shipping Boxes and refer to regional team guidance on specimen preparation and logistics. 1. Foundation Medicine.

(2017) Specimen instructions. Accessed Jun 2018 from

https://assets.ctfassets.net/vhribv12lmne/1gXe0jfhP6U60yQC2CwmIQ/66046b354aa6a26a235fd59a24464e9c/FACT_SpecimenInstructionsMA_01-05_HH_1_.pdf; 2.

Foundation Medicine. (2018) FoundationACT Technical Specifications. Accessed Jun 2018 from

https://assets.ctfassets.net/vhribv12lmne/3SPYAcbGdqAeMsOqMyKUog/3b10e8355a717f658c82d7544503df9f/FACT_TechnicalInformation_09_RD.pdf.

Provide 2 tubes of peripheral whole blood using the

Foundation One® Liquid Specimen Collection and

Shipping Box* 1

From 11 to 14 days turnaround from

sample receipt at the Foundation Medicine

laboratories2

Comprehensive report provided2

20

• For genes covered by both tissue and liquid assays, a total of 68 reportable genomic alterations were detected in 33 tissue samples:

– 75.0% (51 / 68) were also detected in temporally matched cfDNA, which includes 83.0% (44 / 53) of short variant mutations, 38.5% (5 / 13) of CNAs and 100% (2 / 2) rearrangements

• 75.0% (51 / 68) of genomic alterations detected in cfDNA were also detected in tissue

• Overall, of the 33 cases with a genomic alteration in

tissue, 87.9% (29 / 33) had at least one concordant genomic alteration in cfDNA

cfDNA: cell-free DNA; CNA: copy number amplification; ctDNA: circulating tumour DNA; NSCLC: non-small-cell

lung cancer.

Clark, T.A., et al. (2018) J Mol Diagn

Validation data shows high concordance

Number of samples profiled

FoundationACT (ctDNA) FoundationOne (tissue)

283 23,619

128 12,535

67 12,653

63 2,424

NSCLC

Breast

CRC

Prostate

Frequency

(% FoundationOne samples)

0 20 40 60 80

0

20

40

60

80

Fre

quency

(%

F

oundationA

CT

sam

ple

s)

KRAS

ALK-rearrangement

r = 0.98

TP53

TP53

TP53 TP53

KRAS

EGFR PIK3CA

PIK3CA

BRAF

ESR1 KRAS

ctDNA was in 80% of samples detected ctDNA in 86% in these cases

* F1 CDx bait-set available Ex-US from 01 October 2018; † From date of receipt. FFPE: formalin-fixed, paraffin-embedded; HC: hybrid capture; MSI: microsatellite instability; NGS: next-generation sequencing; TMB: tumour mutational

burden; . 1. Foundation Medicine. (2018) FoundationACT Technical Specifications. Accessed Jun 2018 from

https://assets.ctfassets.net/vhribv12lmne/3SPYAcbGdqAeMsOqMyKUog/3b10e8355a717f658c82d7544503df9f/FACT_TechnicalInformation_09_RD.pdf;

2. Foundation Medicine. (2018) FoundationACT Sample patient report; 3. Foundation Medicine. (2017) FoundationACT Webpage summary. Accessed Oct 2017 from https://www.foundationmedicine.com/genomic-testing/foundation-act; 4.

Foundation Medicine. FoundationOne Liquid Technical Specifications to be published Sept 2018; 5. Foundation Medicine. (2017) FoundationOne Technical Specifications. Accessed Oct 2017 from

https://assets.ctfassets.net/vhribv12lmne/6YRrchSINOeSu48YwuesoY/0c3651c8421fa3647ccede76de9dce61/MKT-0054-02_F1_TechSpecs_digital.pdf; 6. Foundation Medicine. (2017) FoundationOne Webpage summary. Accessed Oct

2017 from https://www.foundationmedicine.com/genomic-testing/foundation-one; 7. Frampton, G.M., et al. 2013. Nat Biotechnol 31:1023-31; 8. Foundation Medicine. (2017) FoundationOneHeme Technical Specifications. Accessed Oct 2017

from https://assets.ctfassets.net/vhribv12lmne/zBxaQC12cScqgsEk8seMO/abf6133874f1e5929403f66d90c3b900/F1H_TechnicalInformation_06_digital.pdf; Foundation Medicine. (2018) FoundationOneCDx Technical Specifications.

Accessed Jun 2018 from https://assets.ctfassets.net/vhribv12lmne/4ZHUEfEiI8iOCk2Q6saGcU/671b313cb6bb85bfe861f83e31c9716d/F1CDx_TechInfo_09-03.pdf;

9. Foundation Medicine. (2017) FoundationOneHeme Webpage summary. Accessed Oct 2017 from https://www.foundationmedicine.com/genomic-testing/foundation-one-heme;

10. He, J., et al. 2016. Blood 127:3004-14.

25

FoundationOne® Liquid complements the Foundation

Medicine portfolio

FoundationOne®Liquid 1-4 Foundation®One 5-7 FoundationOne®Heme 8-10

Target patient population Solid tumours Solid tumours Haematologic malignancies & Sarcomas

Specimen type Peripheral blood FFPE tissue FFPE tissue Bone marrow aspirate Peripheral blood

Genes assayed 70 324* 406 265 (via RNA)

Genes with complete exon coverage 35 324* 406

All 4 classes of genomic alterations Yes Yes Yes

TMB No Yes Yes

MSI Yes Yes Yes

Method HC-based NGS HC-based NGS HC-based NGS

Turnaround time† < 14 days 14 days or less 14 days

Brand name FoundationACT® FoundationOne® Liquid FoundationOne® Liquid

Version 1.0 2.0 3.0

Commercial launch date* May 2016 September 2018 2020**

Genes 62 70 > 70

Microsatellite instability - Yes Yes

Tumour mutational burden - - Yes

Companion diagnostic - - Yes

26

First-generation liquid biopsy test, FoundationACT, available until August 2018

Second-generation liquid biopsy test, FoundationOne Liquid, available from September 2018

* Timelines for studies may vary – refer to study protocol for more details ** Opportunity to launch earlier depending on filing timelines

Primary objective

BEP: biomarker evaluable population; bTMB: blood tumour mutational burden; cfDNA: cell-free DNA; CI: confidence interval;

HR: hazard ratio; NSCLC: non-small cell lung cancer; OS: overall survival; PD-L1: programmed death-ligand 1; PFS:

progression-free survival; SNV: single nucleotide variant; TMB: tumour mutational burden. Gandara., D., et al. (2017) Ann

Oncol 28 (suppl. 5): v460-96. 27

Retrospective clinical validation of the FMI bTMB

assay

Clinical efficacy of atezolizumab vs docetaxel in bTMB

subgroups

0 0,2 0,4 0,6 0,8 1

BEP+

≥ 10

≥ 16

≥ 20

OAK POPLAR

bTMB subgroup P

FS

HR

95% CI of *(0.68, 1.20) and **(0.73, 1.04)

* **

To test and validate a novel blood-based assay to

measure bTMB and to evaluate its association with

atezolizumab efficacy in patients with NSCLC

Methods

bTMB assay interrogates single nucleotide variants

(SNVs) in 394 genes from cfDNA in plasma and reports

score based on number of high-confidence SNVs

identified

Biomarker evaluable population (BEP) grouped by

bTMB cut points based on minimum number of SNVs:

• POPLAR – 211 patients

• OAK – 583 patients

Results

• POPLAR: Improved progression-free (PFS) and overall survival (OS)

hazard ratios with atezolizumab observed in the TMB ≥ 16 subgroup

• OAK: PFS benefit with atezolizumab vs docetaxel observed in the

TMB ≥ 10 subgroup

• TMB in blood did not correlate with PD-L1 expression

Two studies show that bTMB is associated with enriched clinical benefit with atezolizumab

Rizvi NA et al. Science. 2015;348:124-8.

Assessing TMB in liquid biopsies

Source of bias. Clonal haematopoiesis

• CHIP is defined by the presence of somatic mutation in blood or BM but without other diagnostic criteria for a haematological malignancy.

• More frequent in aged patients, patients with solid tumours • More likely to be detected with deeper sequencing approaches. • May account for false positive ctDNA sequencing results.

KR

AS

G1

2D

Hu Y et al. False-Positive Plasma Genotyping Due to Clonal Hematopoiesis.Clin Cancer Res. 2018 Mar 22.

Charles Swanton et al. Journal of Clinical Oncology 36, no.

15_suppl (May 20 2018) 12003-12003.

CHIP prevalence by age and type of Tumor

Ptashkin RN et al JAMA Oncol. 2018 Jun

5.

CHIP is Prevalent in some cancer related genes

M et al. Nat Med 2014;20:1472-

1478.

Ptashkin RN et al JAMA Oncol. 2018 Jun

5.

• Biomarker testing through ctDNA sequencing by NGS is possible.

• However, there are some concerns about false due to CHIP.

• The impact of CHIP in TMB assessment using LB has not been thoroughly evaluated

• New pipelines designed to effectively filter out germline mutations as well as mutations derived from clonal hemaotopoiesis are needed

Conclusions

Thank you!!

mprovenciop@gmail.com mariano.provencio@salud.madrid.org

LiquidBiopsyLabPdH (@LiquidBiopsyLab)