La biopsia liquida nella diagnosi di NSCLC

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La biopsia liquida nella diagnosi di NSCLC

fbuttitta@unich.it

VI Corso Nazionale EVENTI FORMATIVI

AIOM-SIAPEC-IAP 2016 Roma 15 giugno 2016

F i a m m a B u t t i t t a

M e d i c i n a M o l e c o l a r e O n c o l o g i c a U n i v e r s i t à - C h i e t i

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Thera screen BEAMing BIOPSY

RESECTED TUMOR

CYTOLOGY VI Corso Nazionale EVENTI FORMATIVI

BIOLOGICAL MATERIALS FOR EGFR MUTATION TESTING IN NSCLC PATIENTS

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NO SURGERY 70-75%

Biopsy 25-30%

Citology 40-50%

Histological characterization

SURGERY 25-30%

Citological characterization

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CYTOLOGY

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CYTOLOGY

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CYTOLOGY

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Tissue Handling for Biomarker Testing

Turn-around time testing

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Today’s tissue-management - lung cancer 2016

HE first

Diagnostic stain TTF1 Diagnostic stain mucin

Diagnostic stain P63/p40 ALK IHC

ALK FISH

ROS1 FISH RET FISH MET FISH

DNA isolation mutation analysis RNA isolation Met exon skip

HE last VI Corso Nazionale EVENTI FORMATIVI

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AKT1 ALK BRAF CCND1

CCND2 CCND3 CCNE1 CDK2

CDK4 CDKN2A CCND1 EGFR

FGFR2 FGFr3 Her2 HRAS

KRAS MET NF1 NRAS

NTRK1 PIK3CA PTEN RB1

RET ROS1 STK11 TSC1

NGS Panel 2 Nextera

Hybridization capture

28 genes

Detect SNVs In/dels, CNV

Fusions

CRUK-SMP2 National Lung Matrix Trial

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 The lack of neoplastic tissue may be a critical issue preventing molecular characterization.

Phase 3 IPASS study

Only 42% of the patients had biopsied tissue suitable for molecular testing,

Phase 3 INTEREST study

only 31% had adequate tissue.

Personal experience

About 10-15% of tissue biopsies and 20% of cytological samples are inadequate_.

A s s e s s m e n t o f E G F R m u t a t i o n : s e l e c t i o n

o f N S C L C p a t i e n t s f o r t a r g e t e d t h e r a p y

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4.4 Avvertenze speciali e precauzioni di impiego Quando si considera l'uso di IRESSA come trattamento per il NSCLC localmente avanzato o metastatico, è importante che la presenza della mutazione dell’EGFR del tessuto tumorale sia cercata per tutti i pazienti.

Se un campione del tumore non è valutabile, allora può essere utilizzato il DNA tumorale circolante (ctDNA) ottenuto da un campione di sangue (plasma).

Devono essere usati solo test affidabili e sensibili con utilità dimostrata per la determinazione dello stato di mutazione dell’EGFR sul tessuto tumorale o ctDNA, questo al fine di evitare risultati falsi negativi o falsi positivi (vedere paragrafo 5.1).

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Liquid biopsy: advantages over tissue or cells

4) cfDNA in blood may provide a better representation of all tumor sites of the body: primary tumor and metastases.

1) less invasive,

2) repeatable over time

3) More rapid turnaround time

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Liquid biopsy: advantages over tissue or cells

4) cfDNA in blood may provide a better representation of all tumor sites of the body: primary tumor and metastases.

1) less invasive,

2) repeatable over time

3) More rapid turnaround time

Although the liquid biopsy have a great potential and could have many applications

, some critical issues must be highlighted

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C R I T I C A L I S S U E S

1) The cf-tumor DNA amount in plasma may be variable, depending on:

- Tumor type

- In a lesser extent with tumor burden

- Tumor stage (localized/diffused) and tumor grade

Assessment of genetic alteration on liquid biopsies

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Crowley, E, Nat. Rev. Clin. Oncol. , 2013

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clinical characteristics

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J Thorac Oncol. 2015;10: 603–610

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C R I T I C A L I S S U E S

2) a large amount of wild-type DNA circulates in the plasma, and only trace amounts of the mutant allele

Assessment of genetic alteration on liquid biopsies

Crowley, E, Nat. Rev. Clin. Oncol. , 2013

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Detection of EGFR Mutations in cfDNA of NSCLC patients (First studies)

Matherial Method Reference

Plasma PNA-LNA clamp Rosell et al. N Engl J Med, 2009 Plasma Mutant-enriched PCR He C et al. Int J Cancer. 2009 Plasma dHPLC Bai H et al. J clin Oncol, 2009

Plasma Microfluidic digital PCR Yung et al. Clin Cancer Res, 2009

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Circa 24% di casi discordanti

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Lung Cancer 90 (2015) 509–515

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Sensitivity and specificity: our experience

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Sensitivity and specificity: our experience

EGFR Blood Analysis Package Software

Standard EGFR mutation test

UD-NGS: more than 20.000 reads per sample

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Detection of plasma EGFR sensitizing mutations in tissue-positive patients

EGFR status Baseline Progression

Mutated 31 (72%) 11 (76%)

Wild type 12 (28%) 4 (24%)

Total 43 (100%) 15 (100%)

EGFR status Baseline Progression

Mutated 30 (70%) 11 (73%)

Wild type 13 (30%) 4 (27%)

Total 43 (100%) 15 (100%)

Detection of EGFR mutations by UD-NGS

Detection of EGFR mutations by RT-PCR

Sensitivity: 72%

Specificity: 100%

Sensitivity: 71%

Specificity: 100%

Marchetti, et al. WCLC 2015

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EGFR

MUTATIONAL TEST

Mutated Wild type

^TOTAL

Plasma samples (Baseline)

144 (81%) 33 (19%) 177 (100%) EGFR mutational status

on plasma samples of tissue-positive patients

Marchetti et al. submitted

Sensitivity and specificity: our experience

The importance of the pre-analitical phase

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COBAS QI

UD-NGS % of mutated allele

5.99 0.02

8.19 0.05 8.91 0.07 9.5 0.24 13.97 0,81 13.59 0.98 14.86 1.68 15.82 3.05 16.18 3.84 16.88 6.42 21.58 45.29 20.62 55.83

COBAS EGFR blood test quantification index (QI): % of mutant compared to WT (an internal control amplicon in the multiplex PCR)

Q u a n t i f i c a t i o n o f E G F R m u t a t i o n s i n p l a s m a . C o m p a r i s o n o f C O B A S a n d N G S r e s u l t s

COBAS QI

7.4 0.20

8.04 0.22 10.74 1.37 11.48 1.76 14.39 37 15.29 41.17

COBAS QI

1.,34 0.16 13.11 0.91 14.57 2.46 14.7 3.7 17.08 24.5 Exon 19 deletions Exon 21 (L858R) Exon 20 (T790M)

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Exon 19 (deletions)

Exon 21 (L858R)

Exon 20 (T790M)

RT-PCR RT-PCR

Cobas

NGS NGS

NGS

R =0.968

R =0.996

2

RT-PCR

Due to the different PCR approaches used.

UD-NGS: DNA amplificaton by intron primers COBAS: mutation specific primers

Exponential relationship between RT-PCR and UD-NGS data

NGS

R =0.982 ²

P< 0.0001

Exponential curves

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Quantification of EGFR Mutations in Plasma

From: Yes/No To: How much

What about the

clinical applicability and relevance of this

quantification marker?

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No information on TIME: Months? Weeks? Days ?

EGFR plasma response to TKI treatment

EGFR mutant allele

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Quantification of EGFR mutations in plasma samples of 27 previously untreated patients, carrying EGFR mutations in

primary tumors, before and after treatment with TKI.

Baseline 0 4 8 13 18 25 35 60 days Start of TKI treatment

ACCURATE MONITORING DURING THE FIRST DAY OF TREATMENTS

Marchetti et al., JTO 2015

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RAPID RESPONDERS (70% of cases)

SLOW RESPONDERS (30% of cases)

EGFR Clearing Time More than 50% reduction of EGFR levels at 14 days

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Early resistant patients

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EGFR - PLASMA RESPONSE PERCENT TUMOR SHRINKAGE (PTS) AT TWO MONTHS

mean PTS: 59.1

mean PTS: 18.3

P<0.0001

Quantification of EGFR mutations in plasma could be used as an early predictor of

clinical response to TKI

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Jonathan W. Goldman et al., University of California. Clin Cancer Res; 22(10) May 15, 2016

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Conclusions

• The EGFR plasma test may represents an alternative for EGFR testing when tissue or cell are unavailble.

• Detection of EGFR mutations is feasible and accurate

– Sensitivity: 70-83%

– Specificity: 100%

• Quantification of mutant EGFR in cfDNA may be

useful as an early predictor of clinical response to

TKI.

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EGFR quantification in plasma of NSCLC patients - potential clinical application

An accurate quantification of mutated alleles in plasma could:

a) complement or replace more expensive and invasive methods to assess response in treated patients;

b) represent a new way to compare the effectiveness of different drugs in clinical trials

c) be an additional tool to evaluate the best treatment regimen for patients.

d) allow early detection of the resistant-inducing mutations for possible changes to therapy;

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