Dr. JL Rodríguez Peralto

IMPLEMENTING NEXT GENERATION

SEQUENCING IN A PATHOLOGY

LABORATORY

Madrid, Spain

NGS

Ion Torrent™ Oncomine™ Focus Assay - Implementation

experience for EGFR mutation detection

at HOSPITAL 12 DE OCTUBRE

For Research Use Only. Not for use in diagnostic procedures.

DNA Panel RNA Panel

Hotspot genes, n=35 Fusion drivers, n=23

ALK

RET

ROS1

NTRK1

NTRK2

NTRK3

FGFR1

FGFR2

FGFR3

MET

BRAF

RAF1

ERG

ETV1

ETV4

ETV5

ABL1

AKT3

AXL

EGFR

ERBB2

PDGFRA

PPARG

Copy Number Variants, n=19

AKT1ALKARBRAFCDK4CTNNB1DDR2EGFRERBB2ERBB3ERBB4ESR1FGFR2FGFR3GNA11GNAQHRASIDH1

IDH2JAK1JAK2JAK3KITKRASMAP2K1MAP2K2METMTORNRASPDGFRAPIK3CARAF1RETROS1SMO

ALKARBRAFCCND1CDK4CDK6EGFRERBB2FGFR1FGFR2

FGFR3FGFR4KITKRASMETMYCMYCNPDGFRAPIK3CA

Oncomine™ Focus Assay (Thermo Fisher)

52 genes

For Research Use Only. Not for use in diagnostic procedures.

Tumor Sample

Ion 318 Select Chip*

Ion PGM System*Next-generation

Sequencing

Ion Reporter Workflow*Automated analysis for SNVs,

indels, CNVs and gene fusions

Oncomine Assay*

DNA/RNA Kit

Positive VariantsMutations

Copy Number Variants

Gene Fusions

Oncomine Focus Assay Workflow

For Research Use Only. Not for use in diagnostic procedures.

- 31 adenocarcinomas (A)- 1 Large cell carcinoma (C)- 9 metastatic adenocarcinomas (M)

NGS-DNA (41)

Mutation HotSpot

Mutación HotSpot for validation

Amplification

A A A A A A A A A A A A A C A A A A M A A A A A M A A M A A A M A M A A M M M A M

C5 C9 C12 C14 C15 C16 C22 C24 C25 C27 C11 C10 C21 C13 C1 C2 C18 C8 C33 C34 C36 C37 C26 C4 C39 C7 C32 C38 C19 C23 C28 C17 C3 C20 C29 C6 C30 C31 C35 C40 C41

EGFR

KRAS

ALK

BRAF

AKT1

MTOR

FGFR4

MET

PIK3CA

JAK

ERBB3

BRACA1

APC

GNAQ

GNA11

KIT

CNV MYC

Mutación

EGFR Nº Cases % Cases

WT 27 65,9

DEL19 5 12,2

L858R 6 14,6

L858R/V834L 1 2,4

INS20 1 2,4

DEL19/T790M 1 2,4

NGS (41)

EGFR Nº Cases % Cases

WT 28 70,0

DEL19 5 12,5

L858R 6 15,0

INS20 1 2,5

COBAS (40)

DISCORDANT CASES

COBAS NGS

DEL19 DEL19-T790M

WT L858R

L858R L858R-V834L

7,5%, 3/40

Oncomine™ Knowledgebase Reporter Software

For Research Use Only. Not for use in diagnostic procedures.

NGS-RNA (37)

A A A A A A A A A A A A A C A A A A M A A A A A M A A M A A A M A M A A M M M A M C A A M A A A M

C5 C9 C12 C14 C15 C16 C22 C24 C25 C27 C11 C10 C21 C13 C1 C2 C18 C8 C33 C34 C36 C37 C26 C4 C39 C7 C32 C38 C19 C23 C28 C17 C3 C20 C29 C6 C30 C31 C35 C40 C41 C42 C43 C44 C45 C46 C47 C48 C49

EGFR

KRAS

ALK

BRAF

AKT1

MTOR

FGFR4

MET

PIK3CA

JAK

ERBB3

BRACA1

APC

GNAQ

GNA11

KIT

CNV MYC

ALK

METFusion

Mutación

- 27 adenocarcinomas (A)- 1 Large cell carcinoma (C)- 9 metastatic adenocarcinoma (M)

Mutation HotSpot

Mutation HotSpot for validation

Amplificaction

Unvaluable

Fusion

- 1 ALK positive confirmed by FISH- 16 ROS1 negative IQ

Oncomine™ Knowledgebase Reporter Software

For Research Use Only. Not for use in diagnostic procedures.

BEAR Project

BEAR Project - Participating Sites

BEAR Project - Samples

48 FFPE samples : each of the 8 sites provided 6 Samples :

4 samples for gene mutation analysis (DNA extraction)

2 samples for fusion transcript analysis (RNA extraction)

Previously characterized samples:

Colorectal adenocarcinoma

Lung adenocarcinoma

Melanoma

Papillary thyroid carcinoma

Pancreatic adenocarcinoma

BEAR Project - Technical Approach

- Total number of FFPE samples processed

- DNA samples: 32 in triplicate

- RNA samples: 16 in triplicate

- Material sent from samples:

- 3 sections from each sample to each processing laboratory

- Laboratories that processed samples:

- Hospital Ramon y Cajal – Madrid

- Hospital 12 de Octubre – Madrid

- Laboratorio de Dianas Terapeuticas – Madrid

- Technical support:

- FAS: Andrea Verardi, Marco Morey

- CAC: Francesco Acquadro

Oncomine™ Solid Tumour DNA and Fusion Transcript Kits

Fusion Drivers n=4

ALK

ROS1

RET

NTRK1

Hotspot Genes n=22

EGFR

ALK

ERBB2

ERBB4

FGFR1

FGFR2

FGFR3

MET

DDR2

KRAS

PIK3CA

BRAF

AKT1

PTEN

NRAS

MAP2K1

STK11

NOTCH1

CTNNB1

SMAD4

FBXW7

TP53

30 Samples Oncomine Solid Tumor DNA CE-IVD

General Filter BEAR 32

Previously characterized

Variants

Sensitivity: 100% (32/32)*In addition 23 potentially pathogenic variants

not previously tested were found

BEAR Project - Results DNA

23 New Variants

P

L1 LQ LQ

L2

L3 LQ

WT BRAF mut EGFR mut KRAS mut NRAS

P: previous characterization

L1, L2, L3: results from the 3 labs performing Ion Torrent analysis

Wild type cases

BRAF mut cases

EGFR mut cases

KRAS mut cases

NRAS mut cases

Low quality results

KRAS +TP53 mut cases

TP53 mut cases

TP53

BEAR Project - Results DNA

LQ: low quality

BRAF + EGFR mut cases

L1

30 Samples 100%

Concordance

L2

30 Samples

L1

26 Samples

L2

26 Samples

L3

26 Samples

96%

Concordance

*

*1/26 bad quality samples didn't give results

BEAR Project Results DNA- Concordance

- NGS method used shows a very strong inter-laboratory concordance of

results obtained

- A total of 55 variants were identified in the 30 processed samples

- All the expected variants (n=32) were identified (Sensitivity 100%)

- 23 potentially pathogenic extra variants (not previously tested) were identified

in the samples

BEAR Project Results DNA- Conclusions

Fundación Jiménez Diaz

Federico Rojo

Cristina Chamizo

Nerea Carvajal

Hospital del Mar

Beatriz Bellosillo

Raquel Longaron

Erica Torres

Sergi Serrano

Hospital Universitario Vall d´Hebrón

Javier Hernández Losa

Roso Mares

Rosa Somoza

Hospital Universitario Marques de Valdecilla

Javier Gómez

Miguel Angel Piris

Thermo Fisher Scientific and Technical support:

Francesco Acquadro, CAC

Andrea Verardi, FAS ; Marcos Morey, FAS

Laboratorio de Dianas Terapeuticas

Bárbara Angulo

Carolina Dominguez

Susana Hernández

Fernando López Rios

Hospital 12 de Octubre

Jose Luis Rodríguez Peralto

Yolanda Ruano

Diana Cantero

Hospital Ramon y Cajal

José Palacios

Tamara Caniego

Almudena Santón

Hospital Virgen del Rocio

Michele Biscuola

Enrique de Álava

Thermo Fisher Illumina Qiagen

Oncomine™ Tumor Mutation

Load Assay

409 genes

DNA RNAAmplicon-based

SNVs, CNVs, smallInDels

1,75 Mb

Mutational burden

Ion Reporter Software

More artefacts

TruSight® Tumor 170

170 genes

DNA RNAHybridization capture-

based

DNA: SNVs, CNVs, Indels

RNA: Fusions, SpliceVariants

0,52 Mb

Mutational burden?

Base Space®Sequence HubSoftware Data

Less artefacts

QiaseqComprehensive

Cancer Panel

275 genes

DNA RNA

Hybridization-extension-based

SNVs, CNVs, smallIndels

0,83 Mb

Mutational burden?

Insight Software

Less artefacts

(UMIs)

Genes, Input sample

and Technology

Detected alterations

Horizontal coverage

Data analysis

NGS strategies that we have tried for TMB: pros and cons

Samples

TMB

Mu

tati

on

s/M

bOncomine Tumor Mutation Load Assay

32 NSCLCs from Hospital 12 de Octubre (Madrid)

TMB Groups Mutations/Mb

Low 1-5

Intermediate 6-19

High ≥20

46% Low

44% Intermediate

9.3% High

For Research Use Only. Not for use in diagnostic procedures.

Patient # TMB MS Mutations Found VUS TotalTissue

originType of tumour

D00/MCBMMed

7/MbStable

STK11, MYC, KEAP1,

TP53

ATR, BRIP1, CEBPA,

EP300, FAM123B, IRS2, SDHA, TGFBR2, WT1

13 BrainUnknown

primary adenocarcinoma

D01/MEERHigh

62/MbStable

ERBB2, NF1, STK11,

ERRFI1, MYC, ASXL1,CDKN2A, SMARCA4,

TET2, TP53

44 54 Soft tissueUnknown

primary adenocarcinoma

D02/ERRLow

2/MbStable

MAP2K1, ARID1A,

CDKN2A/B, KDM5C, KDM6A

BRCA2, STAT2, MAP3K1,

SYK, TNFAIP3, MLL2, NSD1

12 Soft tissue

Unknown

primarysquamous cell

carcinoma

D03/MPCRLow

1/MbStable BCOR, MYB AKT1, AR, CTNNA1, FAT1 6 Lung

Lung adenoid

cystic carcinoma

D04/ARALow

4/MbStable MLL3 FANCA, WISP 3 Lung

Lung adenoid

cystic carcinoma

D08/MTLCMed

15/MbStable

STK11, CDKN2A/B,

KEAP1, TP5320 24 Lung

Lung

adenocarcinoma

D10/MARPLow

4/MbStable

KRAS, PIK3CA, APC,

ARID1A, FAM1234B, TP53

BRIP1, SMAD2, MAP3K1,

SMAD3, NOTCH1, ZNF703, POLE, PTCH1, ROS1

15 PeritoneumUnknown

primary adenocarcinoma

Illumina

TST170

AmpliSeq

CCP

QiaSeq

CCP

✓

✓

✓

✓

✓

✓

✓

✓

✓

✓

✓

✓

✓

Thermo

TML

8,5

54

4

7,5

10

y = 1.077x - 2.9544R² = 0.9648

0

10

20

30

40

50

60

70

0 10 20 30 40 50 60 70

TML T

herm

o

FoM

Comparison study of technologies using Foundation One as gold standard

• NGS platform must be previously tested before to implant in a pathology

laboratory

• NGS with a specific platforms is an effective and efficient technology

• NGS is accurate, easily to implant, cheaper and saver tissue than sequential

PCR

• NGS is an excellent and precise technology to detect actionable mutations

• Some platform reports ie. Oncomine Knowledgebase Reporter Software

facilitate the selection of target drugs and the clinical trials available for these

patients.

Conclusions

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