EGFREGFRtyrosine kinase mutations and copy number in tyrosine kinase mutations and copy number in

nonnon--small cell lung cancer small cell lung cancer

0202--0202--20072007

Mariëlle Gallegos RuizMariëlle Gallegos Ruiz

Contents

Introduction

EGFR

EGFR Inhibitors

EGFR mutations Techniques

Paraffin versus Frozen (Study I)

DNA versus RNA (Study II)

EGFR copy number

CISH versus FISH (Study III)

Conclusions

Future Directions

Extracellulardomain

Trans-membranedomain

Intracellulardomain

TyrosineKinase (TK)domain

Member of a subfamily of four closely related receptors

EGFR/Her1 (ErbB-1),

Her 2/neu (ErbB-2),

Her 3 (ErbB-3)

Her 4 (ErbB-4)

Epidermal Growth Factor Receptor

EGFR overexpressed in: Non Small Cell Lung Cancer

Esophagus cancer

Breast cancer

Head and neck cancer

Prostate cancer

Glioblastoma

EGFR Signaling

EGFR inhibition is a rationale for cancer treatment

Monoclonal antibodies; block binding of ligand

EGFR tyrosine kinase inhibitors (TKIs); block signaling

Iressa (gefitinib)

Tarceva (erlotinib)

EGFR Inhibitors

TK inhibitors have shown to be effective in 10-20% of unselected patients with NSCLC

Most responses seen inAdenocarcinoma

Females

Never smokers

East-Asian patients

EGFR mutations in NSCLC

Mutation in TK domain of EGFR correlates with response to TK inhibitors in NSCLC patients (Lynch, 2004; Paez, 2004; Pao, 2004)

85% of responders are mutated in EGFR TK domain90% exon 19 deletions and exon 21 L858R point mutations

Mutations in K-ras predict primary resistance to TKIs

EGFR and K-ras mutation analysis can be of help in identifying patients most likely to respond to EGFR TK inhibitors gefitinib and erlotinib

EGFR mutations - techniques

Introduction

EGFR

EGFR Inhibitors

EGFR mutations Techniques

Paraffin versus Frozen (Study I)

DNA versus RNA (Study II)

EGFR copy number

CISH versus FISH (Study III)

Conclusions

Future Directions

PCR and direct sequencing

WT EGFR Exon 19

MUT EGFR Exon 19

Detector

Capillary

detection of labelled nucleotides

Signal

EGFR Exon 19

Deletion A746-E750

EGFR Exon 21

Substitution T>G

AA change

Leucine to Arginine

PCR and direct sequencing

Advantages

Analytical sensitivity 10-30%

Every nucleotide change can be detected

Disadvantages

Time consuming

WT EGFR Exon 19

MUT EGFR Exon 19

FAM

FAM

detection of FAM label, separation by sizeDetector

Capillary

Signal

204 bp189 bp

Mutant EGFR Exon 19

15 bp deletion

Mutant EGFR Exon 19

9 bp deletion

204 bp

195 bp

15 bp difference

204 bp

Wild Type EGFR Exon 19

9 bp difference

Separation by size

EGFR exon 19 deletions

FAM

101 bp (wild type)

92 bp (mutant)

92 101

H1975 T790M

101

WT sample

PCR RFLP based analysis

EGFR exon 20 T790M

PCR RFLP based analysis

EGFR exon 21 L858R

Exon 21 CGG

Sau 96I Sau 96I

FAMPCR

Digest with SAu96I

or

180 bp (wild type)

91+180 bp (mutant)

H3255 L858R WT sample

PCR RFLP based analysis

Advantages

Analytical sensitivity 10-30%

Easy to perform

Disadvantages

Only known mutations can be detected

Limited enzymes available

Wild Type Mutant

Single Stranded Conformation Polymorphism (SSCP)

WTG12C

WTG12A

SSCP for K-ras

Advantages

Easy to perform

Quick screening, distinction mutation/wild type

Disadvantages

Sensitivity (our experience)

Need to confirm with sequencing

Single Stranded Conformation Polymorphism (SSCP)

DHPLC WAVE system

transgenomics

Fragment collection followed by direct sequencing

Advantages

Analytical sensitivity 1-5%

Disadvantages

False positives - importance of mutations

Time consuming

Expensive equipment

DHPLC WAVE system

Transgenomics

Several methods available

Optimal method seems to be PCR and sequencing

Summary

How does source of samples (paraffin – frozen) affects mutation analysis using PCR and sequencing?

How does template (gDNA – mRNA) affects mutation analysis using PCR and sequencing?

Study I

Introduction

EGFR

EGFR Inhibitors

EGFR mutations Techniques

Paraffin versus Frozen (Study I)

DNA versus RNA (Study II)

EGFR copy number

CISH versus FISH (Study III)

Conclusions

Future Directions

Frozen tissue is most probably best source for analysis

Most tissue is preserved fixed and embedded in paraffin

Various artifactual EGFR mutations have been described

AIM: To evaluate the effect of tissue processing and preservation on EGFR and K-ras mutation analysis

Frozen versus Paraffin

Experimental set up

47 NSCLC patientscollect paraffin specimens (47)

20 fixed in neutral buffered formalin27 fixed in sublimate formalin

collect frozen specimens (47)

Cut 15 subsequent slides

Pathologist verifies tumor percentage >50% before and after cutting of slides

EGFRexon 18exon 19exon 20exon 21

PCRK-ras

exon 1exon 2

Sequence PCR products

Isolate DNAQiagen kit Trizol

PCR success in frozen vs paraffin

Performed PCR for EGFR 18-19-20-21 and K-ras 1-2

Determined success rate in paraffin versus frozen

0

20

40

60

80

100

EGFR Exon 18(248 bp)

EGFR Exon 19(225 bp)

EGFR Exon 20(325 bp)

EGFR Exon 21(301 bp)

Kras Exon 1(253 bp)

Kras Exon 2(295 bp)

DNA fragment (length)

Rat

e of su

cess

ful P

CR

am

plif

icat

ion (%

)Frozen

FPER

ate

of

succ

ess

ful P

CR

am

plifi

cati

on

(%

)

Effect of product size

K-ras exon 2 success rate in paraffin was only 19%

Shortening the product size with 60 bp increased the success rate from 19% to 61%

0

10

20

30

40

50

60

70

Kras Exon 2(295 bp)

Kras Exon 2(235 bp)

DNA fragment (lenght)

Rat

e o

f su

cess

ful P

CR

am

plif

icat

ion

(%

)R

ate

of

succ

ess

ful P

CR

am

plifi

cati

on

(%

)

Effect of type of fixative on PCR success rate

47 fixed and paraffin embedded specimens

20 fixed in neutral buffered formalin

27 fixed in sublimate formalin

Does type of fixative influences PCR success rate?

0

20

40

60

80

100

Neutral buffered formalin Sublimate formalin

Fixative

Rat

e o

f su

cces

sfu

l PC

R a

mp

lific

atio

ns

(%)

PCR successfulP = <.0001

product size

shorter product results in higher success rate

type of fixativesuccess rate in neutral buffered formalin higher than in sublimate formalin fixed samples

Summary – PCR success rate

storage time

no significant influence

time of fixation

shown by Inoue et al (1996) that fixation for 2-3 days results in failure of 56% of PCRs as compared to fixation for 1 day

not determinable in our study

Results

Sequencing EGFR exon 20 sample 3977909

GGC Gly 779

GGC Gly 779

GGC > AGC Gly 779 Ser

Frozen FPE FPE re-analyzed

13 mutations detected in EGFR

3 previously described mutations, detected in frozen and paraffin

10 not previously identified mutations detected only in paraffin specimens

repeated analysis of these samples revealed a wild type sequence

10 mutations detected in K-ras

all previously described mutations, detected in frozen and paraffin

Tsao et al (2005) reported 24 ‘novel’ EGFR mutationsNot confirmed by repeated analysis

Fixation and paraffin embedding of samples can result in deamination of cytosine residues (Marchetti, 2005)

Use of this damaged DNA as template can result in artifactual C>T or G>A transitions

Artifactual mutations

# Fixative Artifactual mutation Nucleotide Change

7876980 Sublimate Formalin Ala 722 Val C>T

1989395 Sublimate Formalin Cys 781 Cys C>T

4987459 Sublimate Formalin Leu 792 Leu C>T

0951411 Sublimate Formalin Leu 858 Leu C>T

6123993 Sublimate Formalin Leu 858 Leu C>T

5148421 Neutral Buffered Formalin Pro 733 Ser C>T

5148422 Neutral Buffered Formalin Glu 746 Lys G>A

3977909 Neutral Buffered Formalin Gly 779 Ser G>A

3977909 Neutral Buffered Formalin Glu 804 Lys G>A

3977909 Neutral Buffered Formalin Ala 822 Thr G>A

When using paraffin samples use primers which give products as short as possible

Confirm mutations by repeated analysis

Frozen tissue is the preferable source for analysis

Conclusion study I

Study II

Introduction

EGFR

EGFR Inhibitors

EGFR mutations Techniques

Paraffin versus Frozen (Study I)

DNA versus RNA (Study II)

EGFR copy number

CISH versus FISH (Study III)

Conclusions

Future Directions

Mutation analysis using PCR and sequencing on RNA as template saves time compared to using DNA as template

AIM: To determine possible differences between sensitivity of mutation analysis in genomic DNA versus mRNA

gDNA versus mRNA

Experimental set up

47 NSCLC patientscollect paraffin specimens (47)

20 fixed in neutral buffered formalin27 fixed in sublimate formalin

collect frozen specimens (47)

Cut 15 subsequent slides

Pathologist verifies tumor percentage >50% before and after cutting of slides

Isolate DNA Isolate RNA

EGFRexon 18exon 19exon 20exon 21

PCRK-ras

exon 1exon 2

Sequence PCR products

EGFRexon 18-21

PCRK-ras

exon 1-2

Sequence PCR products

Aqueous layer (RNA)

Interphase and trizol (DNA)

PCR amplification successful in 100% of samples,

in both gDNA and mRNA

Sensitivity of sequencinggDNA vs mRNA

3 EGFR mutations identified in 2 patients

P848L, detected with same accuracy in gDNA and mRNA

identified previously, not related with response to TKIs

S768I and L861Q, mutation signal higher in mRNA than in gDNA

SNP in this sample detected with same accuracy

10 K-ras mutation detected with same accuracy in

gDNA and mRNA

Sensitivity of sequencingDNA vs RNA

gDNA

CAA > CAGGln 787 Gln

CAA > CAGGln 787 Gln

RNA

gDNA

CTG > CAGLeu 861 Gln

CTG > CAGLeu 861 Gln

RNA

AGC > ATCSer 768 Ile

AGC > ATCSer 768 Ile

RNA

gDNA

Both gDNA and mRNA can be used for mutation analysis

Some EGFR mutations may be easier to identify by using mRNA as template

mutant allele overexpressed?

Conclusion study II

Study III

Introduction

EGFR

EGFR Inhibitors

EGFR mutations Techniques

Paraffin versus Frozen (Study I)

DNA versus RNA (Study II)

EGFR copy number

CISH versus FISH (Study III)

Conclusions

Future Directions

High polysomy and amplification of EGFR gene correlates with favorable outcome after treatment with gefitinib and better survival (FISH) (Capuzzo, 2005; Hirsch, 2005)

High EGFR copy number associated with longer survival in patients treated with erlotinib (FISH) (Tsao, 2005)

EGFR copy number as assessed by Real time PCR failed to obtain predictive and prognostic information (Bell, 2005; Dziadziuszko, 2005)

EGFR copy number

EGFR copy number detection using FISH can be of importance in selecting patients for treatment with TK inhibitors

FISH technique some disadvantagesFluorescent microscope needed

Fading of signal

Morphology limited

These disadvantages can be overcome by Chromogenic In Situ Hybridization, CISH

Implementing CISH to determine EGFR copy number may be of practical value

EGFR copy number FISH and CISH

Fluorescent In Situ Hybridization

Denaturation

F

Chromogenic In Situ Hybridization

DIG

Mouse-α-DIG

EGFR-probe

**

**

DAB oxidized DAB

Denaturation

Envision HRP

Collect 58 tumor samples on which CISH was successfully performed

31 paraffin samples

27 frozen samples

Apply FISH and CISH on all samples

Count 200 cells in at least three representative fields≤ 4 dots in > 40% of cells no/low genomic gain

≥ 4 dots in ≥ 40% of cells high polysomy

≥ 15 dots or clusters in 10% amplification

Experimental set up and scoring

Success rate

FISH CISH FISH CISH

Easily assessable 19 13 6 26

Difficult to assess 8 14 11 5

Not assessable 0 0 14 0

Number of samples defined by success rate

Frozen (n=27) Paraffin (n=31)

Success rates for FISH and CISH in relation to source of the sample

Reasons of FISH failure

Autofluorescence (4)

Detachment of tissue (5)

Unrecognizable morphology (4)

Low signal (1)

Reasons of difficulties CISH

High Background

Concordance between observers

Cases with different outcome between observers for CISH

Case # OB1 OB2

34 30 (-) 43 (+)

36 23 (-) 44 (+)

44 25 (-) 66 (+)

39 43 (+) 13 (-)

Percentage of cells with ≥4 copies per nucleus (CISH)

CISH concordance 93% (Kappa coefficient 0.74)

FISH concordance 100% (Kappa coefficient 1)

Concordance between techniques

Concordance observer 1: 93% (Kappa coefficient 0.64)

Concordance observer 2: 95% (Kappa coefficient 0.76)

Correlation between FISH and CISH results for both observers

Positive Negative Positive Negative

Positive 4 1 4 0

Negative 2 37 2 38

FISH

Observer 1 Observer 2

CISH

EGFR disomy

EGFR high polysomy

EGFR amplification

High concordance found between techniques and observers

CISH is a useful alternative strategy to detect EGFR gene copy number

CISH preferable in paraffin section

FISH preferable in frozen sections

Conclusion study III

Detection of EGFR and K-ras mutations

Paraffin samples

Use primers as short as possible

Confirm mutation by independent analysis

Frozen samples

Preferable for use

Detection of EGFR copy number

Paraffin samples

Use Chromogenic In Situ Hybridization

Frozen samples

Use Fluorescent In Situ Hybridization

Future directions

Department of Medical Oncology, VUMC

Karijn Floor

José Antonio Rodriguez

Giuseppe Giaccone

Department of Pathology, VUMC

Katrien Grünberg

Gerrit Meijer

Erik Thunnissen

Acknowledgements