The current status of NGS in clinical practicemedia.aiom.it/userfiles/files/doc/AIOM-Servizi/slide/... · 2017-06-29 · • Amplicon NGS has become a standard method in many institutes

14.06.2017 1Institut für Pathologie – Charité Berlin

M. Dietel

Institute of Pathology(Rudolf-Virchow-Haus)

Humboldt University, Berlin

e-mail: [email protected]

The current status of NGS in clinical practice


Disclosures

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

• Travel costs have also been refunded


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


Surgery

Multidisciplinary Cooperation Enables Precision Oncology

Chemotherapy

RadiotherapyTargeted therapy

Histological + molecular tumor

typing (TNM, grading gen. alterations etc.), details on progn. +prediction of drug

efficiacy

researcheQA/QC

teaching

bio-banking

tumor registry

clinical trials

Individual treatmentdecision 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

RadiologyTissue Sampling Endoscopy

Check point inhibitors experimental

Interdisciplinary tumour board with

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

radiologist etc.Liqu

id b

iops

y


Charité Comprehensive Cancer Centre

QBUUZK – NET of nose cavity


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

PALB2G1021R

Mutation Olaparib PARP 2c 26510020 0,43 Prostate

ATR R177Q (?)

Mutation Olaparib PARP 4 0,49

BRCA2deletion

Mutation(LOF)

Olaparib PARP 2b

2b/c

25366685

26510020

various

Prostate

-

Rucaparib PARP 2c 27002934 Ovarian

Niraparib PARP 2b 27717299 Ovarian

TP53G266R

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 %



Gene Aberration Drug Mechanism EvL PMID VAF Histology Priority

IDH2R172T

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 AGIOSOngoing trials

-- --

R172K Olaparib PARP 3b 28148839 Glioma, Leukemia

DNMT3AR604Q (?)

Mutation Decitabine DNMT inhibition 2c 22124213 0,5 Leukemia

CDKN2AR80* (nons.)

Loss offunction

Palbociclib CDK4/6 2c

3b

26715889

24495407

0,92 Breast

Melanoma

CDK4deletion

deletion CDK4/6 Inhibition CDK4/6 R4

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



Up-coming Molecular Diagnostic

histological standard sequential NGSdiagnosis molecular diagnostics diagnostics

K/NRAS BRAF

EGFR exons 18,19, 21cKITusw.

IonAmpliseq* CancerPanel in 46 gene

(total 604 loci).

other relevantmutations

????no mutations

metastasized neuro-endocrine

carcinoma, grade 3

*Ion Torrent


Up-coming Molecular Diagnostic

histological standard sequential parallel moleculardiagnosis molecular diagnostics diagnostics

IonAmpliseq* CancerPanel in 46 gene

(total 604 loci).

ABLAPCALK

KRASBRAF

EGFR mut exon 20ERBB2

FGFR2 mutFGFR3

cKITKDL mut

604 further loci…….

metastasized neuro-endocrine

carcinoma, grade 3

*Ion Torrent

Iressa ⇒

FGFR-inhibitor ⇒

sorafinib/sunitinib ⇒


Integrating NGS and Diagnostic Pathology

Personalized molecular

targeted cancer treatment


NGS - Reliable for patient care


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


The irreplaceable role of anatomic pathology is ….


Nerv

Manual microdissection

BRAFV600mut


External Quality Assurance

just some aspects


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

pathology − this requires nationwide NGS ring trialsto be done in 2014

Michael Hummel, Manfred Dietel

2024

German Cancer Consortium (DKTK)

Inventory of NGS Equipments

Selection of 5x IonTorrent PGM and 4x Illumina MiSeq


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 knownto 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


95% of all mutations have been correctly identified by

8 participating inststutes of pathology within the

German Cancer Consortium

Results of the NGS Project


Results of the ring trialson 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.


Andreas Jung, Inst. of Pathology, LMU Munic

59 of 66 participantspassed 89,4%

April 2016

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


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

R.Katayama et al. Sci Transl Med. 2012 Feb 8;4(120):120ra17.

EGFR dominant EGFR non-dominant

Resistenz gegen EGFR-TKI-Therapie wirdin 50% der Fälle durch Austausch der Aminosäure Threonin zu Methionin an Position 790 des EGFR-Proteins vermittelt(T790M)

http://www.ncbi.nlm.nih.gov/pubmed/22277784


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

cfDNA und Liquid-Biopsie

cfDNA, circulating free DNA; ctDNA, circulating tumor DNA. Crowley, Nat Rev Clin Oncol 10, 472 (2013)

tum

or ti

ssue

norm

al ti

ssue


Ring Trial EGFR T790MTissue and Blood

Sabine Merkelbach-Bruse


T790M from tissue specimenBlöcke 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



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

C15.38321 2D - 80% Fall 8


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


T790M from blood – pre-analyses

Selection of tubes for blood draw

6°C 22°C

Norton et al., J Clin Lab Anal 2013


T790M from blood – pre-analyses

Selection of devices for blood draw

Venofix Safety von Braun


T790M from blood - logisticsVersandmittel von TNT – für internen Ringversuch verwendet


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


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.


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%)


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


Mol RTs

clonality - lymphomaBRCA1/2 - ovarian carcinomaT790M – NSCLC (tissue and blood )RAS - colonBRAF - malignant melanomaMultigene - NSCLC – planned for 1st. quarter of 2017BRAF - NSCLC – 2nd qu. 2017ROS1 - NSCLC – planned for 2nd half of 2017

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


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.

http://jco.ascopubs.org/content/vol23/issue8/images/large/jco12005f01.jpeg


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.


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 1/2o 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.


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.

hhjhkjInstitut für Pathologie – Charité Berlin, Campus Mitte

Institute of Pathology, Rudolph-Virchow-Haus, Charité Humboldt-Universität zu Berlin

Berliner MauerAlexander Ufer

Institut für Pathologie, Rudolf-Virchow-Haus, Charité Humboldt-Universität zu Berlin

Berliner Medizin-historisches Museum

Documents

The current status of NGS in clinical practicemedia.aiom.it/userfiles/files/doc/AIOM-Servizi/slide/... · 2017-06-29 · • Amplicon NGS has become a standard method in many institutes