The current status of NGS in clinical practice

(1)

14.06.2017 1

Institut für Pathologie – Charité Berlin

M. Dietel

Institute of Pathology (Rudolf-Virchow-Haus) Humboldt University, Berlin

e-mail: manfred.dietel@charite.de

The current status of NGS in clinical practice

(2)

Disclosures

• Participation in several industry-sponsored advisory boards for which honoraria were received.

• Travel costs have also been refunded

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14.06.2017 3

The goal of diagnostic pathology is to extract from the patient’s tissue as many information as possible

by applying in parallel histological, immunological (proteomic) and molecular techniques.

An analysis as complete as possible has become an increasing demand of clinical oncologists.

Today’s Challenges in Precision Medicine

(4)

Surgery

Multidisciplinary Cooperation Enables Precision Oncology

Chemotherapy

Radiotherapy Targeted therapy

Histological + molecular tumor typing (TNM, grading

gen. alterations etc.), details on progn. +

prediction of drug efficiacy

research eQA/QC

teaching

bio- tumor

clinical trials

Individual treatment decision based e.g. on

actionability of mol.

alterations, protein pattern resulting in

novel therapies

Anatomic and molecular pathology

Clinical Suspect of a primary tumor or

recurrence

IHC

In situ hybridization Diagnosis

molecular path Radiology

Tissue

Sampling

_Endoscopy

Check point

inhibitors experimental

Interdisciplinary tumour board with

oncologists, surgeons,gynecol., (mol.) pathologists,

radiologist etc.

Li qui d bi ops y

(5)

14.06.2017 5

Charité Comprehensive Cancer Centre

QBUUZK – NET of nose cavity

(6)

Gene Aberration Drug Mechanism EvL PMID VAF Histology Priority

ATM het.

FANCA het.

Mutation

ATM Mutation

Olaparib PARP 2c 26510020 0,5 / 0,48 Prostate

PALB2 G1021R

Mutation Olaparib PARP 2c 26510020 0,43 Prostate

ATR R177Q (?)

Mutation Olaparib PARP 4 0,49

BRCA2 deletion

Mutation (LOF)

Olaparib PARP 2b

2b/c

25366685 26510020

various Prostate

-

Rucaparib PARP 2c 27002934 Ovarian

Niraparib PARP 2b 27717299 Ovarian

TP53 G266R

Mutation (LOF)

Pazopanib VEGFR/PDGFR/

KIT

2c 26646755 0,87 Sarcoma

Platinum-based Cx Cytotoxic agent R2b 26899019 NSCLC

Cisplatin, Carboplatin

Cytotoxic agent R2b 11595686 Ovarian Cancer

123 Mutations, CNA 1326, estimated purity 40-45 %

(7)

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Gene Aberration Drug Mechanism EvL PMID VAF Histology Priority

IDH2 R172T

IDH2 Mutation

AG-221 IDH2-Inhibitor 2b/c Stein et al.

Blood 2015 126:323

0,44 AML

AG-881 Pan-IDH-Inhibitor n.a. Press release AGIOS Ongoing trials

-- --

R172K Olaparib PARP 3b 28148839 Glioma,

Leukemia

DNMT3A R604Q (?)

Mutation Decitabine DNMT inhibition 2c 22124213 ^0,5 Leukemia

CDKN2A R80*

(nons.)

Loss of function

Palbociclib CDK4/6 2c

3b

26715889 24495407

0,92 Breast Melanoma

CDK4 deletion

deletion CDK4/6 Inhibition CDK4/6 R4

123 Mutations, CNA 1326, estimated purity 40-45 %

Charité Comprehensive Cancer Centre

(8)

Up-coming Molecular Diagnostic

histological standard sequential NGS

diagnosis molecular diagnostics diagnostics

K/NRAS BRAF

EGFR exons 18,19, 21 cKIT

usw.

IonAmpliseq* Cancer Panel in 46 gene

(total 604 loci).

other relevant mutations no mutations ????

metastasized neuro-endocrine carcinoma, grade 3

*Ion Torrent

(9)

14.06.2017 9

Up-coming Molecular Diagnostic

histological standard sequential parallel molecular

diagnosis molecular diagnostics diagnostics

IonAmpliseq* Cancer Panel in 46 gene

(total 604 loci).

ABL APC ALK KRAS BRAF

EGFR mut exon 20 ERBB2

FGFR2 mut FGFR3

cKIT KDL mut

604 further loci…….

metastasized neuro-endocrine carcinoma, grade 3

*Ion Torrent

Iressa ⇒

FGFR-inhibitor ⇒

sorafinib/sunitinib ⇒

(10)

Integrating NGS and Diagnostic Pathology

Personalized molecular targeted cancer

treatment

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14.06.2017 11

NGS - Reliable for patient care

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Next Generation Sequencing - Costs

0 100 200 300 400 500 600 700

1 Mut 2 Mut 3 Mut 4 Mut 5 Mut 6 Mut

conv. seqc.

NGS

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14.06.2017 13

The irreplaceable role of anatomic pathology is ….

(14)

Nerv

Manual microdissection

BRAFV600mut

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14.06.2017 15

External Quality Assurance

just some aspects

(16)

Standardised amplicon Next Generation Sequencing (NGS) is a prerequisite of predictive molecular

pathology − this requires nationwide NGS ring trials to be done in 2014

Michael Hummel, Manfred Dietel

(17)

German Cancer Consortium (DKTK)

Inventory of NGS Equipments

 Selection of 5x IonTorrent PGM and

4x Illumina MiSeq

(18)

Selection of Cases and Dilutions

• 5 x breast cancer

• 5 x colon cancer

• 5 x lung cancer

• 4 x cell line dilutions (50%, 10%, 3% and 1%)

• Molecularily predefined

• Individual mutations not known to the NGS sites

Gly (GGT) → Val (GTT)

Each sequencing centre received

• 15 DNA samples from the cases

• 15 FFPE sections for local microdissection and DNA preparation

• 4 DNA samples with various cell line dilutions in tonsilar DNA

 Data from approx. 170 NGS runs

(19)

German Cancer Consortium (DKTK)

 95% of all mutations have been correctly identified by 8 participating inststutes of pathology within the

German Cancer Consortium

Results of the NGS Project

(20)

Results of the ring trials on KRAS molecular tests

Q u a l i t y in P a t h o l o g y

2006 => 15 % of 45 participants failed 2007 => 8,5 % of 55 participants failed 2010 => 7,8 % of 69 participants failed

Today 106 Institutes of Pathology passed and received

the certificate.

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14.06.2017 21

Andreas Jung, Inst. of Pathology, LMU Munic

59 of 66 participants passed  89,4%

April 2016

Q u a l i t y in P a t h o l o g y*

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Mechanisms of Acquired Resistance in EGFR-mutant NSCLC Resistant to First Generation EGFR TKIs Erlotinib/Gefitinib

R.Katayama et al.

EGFR dominant EGFR non-dominant

Resistenz gegen EGFR-TKI-Therapie wird in 50% der Fälle durch Austausch der Aminosäure Threonin zu Methionin an Position 790 des EGFR-Proteins vermittelt

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14.06.2017 23

AZD9291 (Tagrisso) – 66% ORR in T790M positive patients*

AZD9291 is not approved in Germany. The content of these slides are not for the purposes of therapeutic recommendation

*as assessed by central tumor tissue testing

(24)

cfDNA und Liquid-Biopsie

tum or ti ssue norm al t issue

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14.06.2017 25

Ring Trial EGFR T790M Tissue and Blood

Sabine Merkelbach-Bruse

(26)

T790M from tissue  specimen

Blöcke mit T790M Allelfrequenz T790M Tumorzellgehalt Auswahl f. Ringversuch

C15.18595 A 61,76% 90% Fall 9

C15 18595 D 45,27% 60% Fall 4

C15.18595 B 63,22% 60% Fall 5

C15.31873 1CRC 6,84% 20% Nicht ausgewählt

C15.31873 1ARC 12,3% 15% Fall 2

C11.26061 80% 80% Fall 7

C14.11820 1 9,8% 35% Nicht ausgewählt

Block ext1 18% 35% Test in Köln

Block ext3 58% 80% Test in Köln

Block ext4 10% 60% Test in Köln

Andere EGFR Mut

C11.16744 1 - 70% Fall 3

C11.16744 2 - 60% Fall 6

C14.7653 2B - 80% Fall 10

WT

C15.37161 A - 60% Fall 1

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14.06.2017 27

T790M mutation from blood

Preparatory steps

− selection of blood tubes and plasma preparation

− optimisation of DNA extraction from plasma

− spiking of blood specimen DNA of cell lines

− planning and logistics

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T790M from blood – pre-analyses

Selection of tubes for blood draw

6°C 22°C

Norton et al., J Clin Lab Anal 2013

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14.06.2017 29

T790M from blood – pre-analyses

Selection of devices for blood draw

Venofix Safety von Braun

(30)

T790M from blood - logistics

Versandmittel von TNT – für internen Ringversuch verwendet

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14.06.2017 31

T790M from blood - results

Fall Status T790M Sonstige Mutationen 11 c.2369C>T p.T790M c.2573T>G p.L858R 12 c.2369C>T p.T790M c.2573T>G p.L858R

13 WT PIK3CA, E545K

14 c.2369C>T p.T790M c.2573T>G p.L858R 15 c.2369C>T p.T790M c.2573T>G p.L858R 16 c.2369C>T p.T790M c.2573T>G p.L858R 17 c.2369C>T p.T790M c.2573T>G p.L858R

18 WT PIK3CA, E545K

19 c.2369C>T p.T790M c.2573T>G p.L858R 20 c.2369C>T p.T790M c.2573T>G p.L858R

1 2 3 4 5 6

wt wt wt n.a.

wt

wt wt n.a.

wt wt wt

wt

wt wt

Gesamtpunktzahl 20 erreichte Punktzahl: 20 16 16 4 20 18

(32)

T790M overall results

Results on tissue: 95% passed the ring trial Results on blood: 75% passed the ring trial

The 2nd RT of QuIP on T790M (tissue and blood)

will take place 3rd qu. 2017.

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14.06.2017 33

Routine BRCA-mutation analyses is a prerequisite for treatments with the PARP inhibitor Olaparib.

BRCA1/2 testing can be done in due time only by NGS.

Next Generation Pathology of Ovarian Cancer and …..

Up-coming challenge in companion diagnostic of ovarian cancer:

BRCA-RT

Participants: n = 34

(from D, Sv, Au)

Certified: n = 26 (77%)

(34)

The next entity will be the triple negative breast cancer (TNBC) and prostate cancer in near future.

BRCA mutation analyses can be done only by NGS.

Targeted Therapy of Ovarian, TN Breast and

Prostate Cancer

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14.06.2017 35

Mol RTs

clonality - lymphoma

BRCA1/2 - ovarian carcinoma

T790M – NSCLC (tissue and blood )

RAS - colon

BRAF - malignant melanoma

Multigene - NSCLC – planned for 1

^st

. quarter of 2017 BRAF - NSCLC – 2

^nd

qu. 2017

ROS1 - NSCLC – planned for 2

^nd

half of 2017

QuIP® – Quality Initiative of the German Society of Pathology and the German Association of Pathologists

(36)

NGS and Integrated Systems Pathology

Griffin, Nature Reviews 2004

NGS-IonTorrent

Currently NGS combined with morphology (and in some assays computational models) is the

most relevant approach to support the decision tree for optimal therapy.

In future further assays may be

added.

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Conclusions I

• Amplicon NGS appears to be a reliable tool of diagnostic pathology for detection of genetic alterations in cancer

• Our data analyses demonstrates:

– well prepared FFPE tissue is very suitable

– different instruments at different sites produce quite homogeneous results

• The molecular ring trials on tissue produced correct results in 85 - 90% of the participants,

• RT on blood demonstrated the need for improvement.

(38)

Conclusions II

• Amplicon NGS has become a standard method in many institutes of pathology.

• For a number of analyses already today it is an almost irreplaceable method, e.g.

o BRAC 1/2

o multigene analyses in NSCLC (colon, cancer)

o liquid biopsies (at least the majority of labs use NGS)

• EQA for NGS (tissue and blood) is absolutely necessary.

• Clinical need in cases without further “classical” options.

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14.06.2017 39

Will Next Generation Sequencing Replace Sanger, Pyro etc.?

Yes, it will (personal opinion)

But next generation sequencing requires

• next generation pathologists and

• next generation oncologists

and the central question

“Who interprets reliably the multiple genetic data provided?”

has to be solved.

(40)