The Role of Pathology/Molecular

Diagnostic in Personalized Medicine

Ignacio I. Wistuba, M.D.

Jay and Lori Eissenberg Professor in Lung Cancer

Director of the Thoracic Molecular Pathology Lab

Departments of Pathology and

Thoracic/Head & Neck Medical Oncology

M. D. Anderson Cancer Center

Conflict of Interest

• Honoraria: Genentech, Glaxo Smith

Kline, Boehringer-Ingelheim, Medscape,

and AstraZeneca.

• Research Agreements: Genentech,

Pfizer, Astra Zeneca, Myriad, Eli-Lilly,

and Merck.

3

Consideration for Lung Cancer

Molecular Testing

• In advanced tumors, tissue availability is

limited

• For testing, different types of tumor samples

are available: biopsy vs. cytology

• Molecular testing is required for patients’

treatment

• Algorithm for small tissue samples utilization

has been developed

• Tissue sample must represent the setting of

the disease

Types of Tumor Specimens In Lung Cancer

Surgical Resection

Histology

Advanced Tumor

Core Needle

Biopsy (CNB)

Formalin-fixed and

Paraffin-embedded (FFPE)

Fine Needle

Aspiration (FNA)

Endobronchial Ultrasound

(EBUS) or Pleural Fluid

Alcohol-fixed

Alcohol-fixed

Alcohol-fixed –

Cell Block

Traditional

Molecular Testing for NSCLC - 2012

Adenocarcinoma

Squamous

Large Cell

Unknown

FGFR1

Amp

EGFRvIII

PI3KCA

EGFR TK

DDR2

BRAF AKT

VEGFR HER2

EPHA/B

PDGFR

FGFR

INSR

PI3K

MAPK

KRAS

EGFR

ALK

Unknown

Adenocarcinoma

Squamous Cell Ca

RET

Adapted from W. Pao and N Girard, Lancet Oncol, 2011

ROS1

Lung Cancer Targeted Therapy

Landscape Change – 2012

Adenocarcinoma Frequency Available TKIs

- EGFR mutation 15% Erlotinib/Gefitinib

- ALK-EML4 fusion 3% Crizotinib

- MET amplification 5% Met inhibitors

- KIF5B-RET fusion 1% Ret inhibitors

- ROS1-FIG fusion 2% Crizotinib

- PI3KCA mutation 5% PI3K inhibitors

- HER2 mutation 2% Her2 inhibitors

Squamous Cell Carcinoma

- FGFR1 amplification 22% FGFR TKIs

- EGFRvIII mutation 5% EGFR TKIs

- PI3KCA mutation 5% PI3KCA inhibitors

- DDR2 mutation 3% Dasatinib & Nilotinib

What’s the problem?

I gave you at least 10 cells!

Fine Needle

Aspiration (FNA) Core Needle

Biopsy (CNB)

Advanced Tumor

Tissue is the Emperor -

For diagnosis, the pathologist needs some!

Diagnostic Algorithm for Small Biopsy and

Cytology Specimens

Tumor Positive

Biopsy Cytology

Squamous

Morphology

IHC p63/p40 (+)

Adenoca

Morphology

IHC TTF1 (+)

LCNEC SCLC

Morphology

IHC NE (+) Morphology

Morphology

IHC (-)

NSCLC-NOS

Molecular Testing: EGFR mutation, ALK Fusion

EGFR Mutations in Lung Cancer

18

19

21

20

C- helix

P- loop

A- loop

Deletions - 46%

L858R - 39%

Duplications/

Insertions - 9% N-lobe

C-lobe

Extracellular

domain

Regulatory

domain

ATP binding

cleft TK

Domain

Deletion 746E-750A Wild-Type

Exon 19 – 15bp Deletion

CTG858CGG Wild-Type

Exon 21 – L858R Mutation

Sanger Sequencing (sensitivity: ~20%mutant allele)

• Biopsy:

- FFPE

- Frozen

• Cytology:

- Smears

- Cell blocks (FFPE)

EML4-ALK Fusion in NSCLC

ALK 29.3

EML4 42.3

2p23 region

FISH Test: “Break-apart Probe”

9 variants

described

Positive Cell:

Two signals separation

Positive Case:

>15% Cells Positive

(50-100 cells)

ALK Immunohistochemistry (Clone D5F3)

EML4-ALK Fusion (+) EML4-ALK Fusion (-) Courtesy of Dr. Y. Yatabe

• Biopsy:

- FFPE

• Cytology:

- Cell blocks

(FFPE)

• Test: EGFR (exons 18-21) mutations and ALK fusion

• Histology: All tumors w/adenocarcinoma component, and in small

samples NSCLC-NOS and other histologies (incomplete sampling)

• Specimen: Upfront collection of as much tissue as possible at

diagnosis

• Consider re-biopsy:

• If diagnostic sample is inadequate for molecular testing

• At time of recurrence, or disease progression on targeted

therapy

• Metastasis vs. primary:

• Most accessible site (tissue quality is more important)

• Test metastasis if developed after therapy

IASLC/AMP/CAP guidelines in draft and NCCN Clinical Guidelines NSCLC v2 2012

Practical Considerations for Molecular

Testing of Lung Cancer

IASLC/AMP/CAP guidelines in draft

NCCN Clinical Guidelines NSCLC v2 2012

Practical Considerations for Molecular

Testing of Lung Cancer - Reports

• Samples availability for testing:

• In house: less than 24 hours

• Outside: less than 3 days

• Quality control by pathologist:

• At least 500 cells

• 50% tumor (vs. no-malignant) cells, and gross dissection

recommended for enrichment

• 50 cells/slide

• Molecular Test: No specific platform is recommended

• Report:

• 10 days max

• Indicate platform

• Indicate suboptimal fixation in the report

Types of Gene Mutation Assays

• PCR-based Sanger Sequencing

• PCR-based Pyrosequencing®

• Real-time PCR DxS® Test

• PCR-based SNaPshot® (Applied Biosystem)

• PCR-based Mass ARRAY SNP Sequenom, Inc

• Next-Generation of Sequencing (NGS)

Multiplex and Flexible Tests

Multiplexed Mutation Assays Multiplex PCR Tumor Tissue

Resected Specimen Core Biopsy

SNaPshot® (Applied Biosystem)

Dias-Santagata, EMBO Mol Med 2:146, 2010

Sensitivity:10% mutant allele / ~20ng DNA/multiplex reaction

Mass ARRAY SNP - Sequenom, Inc

Use of Cytological Material for Molecular

Diagnosis of Lung Cancer

• EGFR/KRAS mutation and ALK fusions

• FNA cell blocks, fluids, endobronchial ulstrasound (EBUS),

and archival slides, all have been used successfully

• Touch preps done to ascertain the adequacy of core biopsy

material

Study Specimen Test N % Suitable

Smouse, Can Cyt, 2009 Routine EGFR mut 12 92

Schuubiers, JTO, 2010 EBUS - FNA EGFR–KRAS mut 35 77

Sakairi, CCR 2010 EBUS - FNA ALK fusion 109 100

Rekhtman, JTO, 2011 Routine EGFR–KRAS mut 128 98

Navani, AJRCCM, 2012 EBUS - FNA EGFR mut 119 90

Courtesy of M. Zakowski (modified) , New York MSKCC

NSCLC Molecular Diagnosis

Tumor (CNB)

~10% Sensitivity

Multiplex PCR ~20ng DNA/multiplex reaction

FFPE DNA

Extraction

Sequenom™ (BRAF: G464-G1391)

Wild-type Mutant

Next-Generation of

Sequencing (NGS): DNA- & RNA-seq

NGS as a Single Platform to Evaluate Multiple

Alterations (200-400 Genes) Tumors

• Mutation detection

• DNA copy number detection

• Translocations/gene fusions

• RNA-seq: gene expression, alternative splicing

Characteristics:

• High coverage: multiple (~500x) reads of the same sequence to gain

confidence in result

• Critical when ratio of neoplastic to non-neoplastic cells is low

• Allows signal to be sifted from the noise

• Examination of reads in both directions to rule out artifacts

• Confirm or rule out sequence variant using an additional method

(e.g. Sanger)

Illumina HiSeq 2000 Illumina MiSeq Ion Torrent PGM

Current:

300 – 600 Gigabases

6 – 11 days

1.5 Gigabases

1 day

1 Gigabase

6 hours

Emerging: Illumina HiSeq 2500 Ion Torrent Proton

Next Generation of Sequencing

Human Genome in a Day

Courtesy of P. Bunn, Colorado (UCCC)

Tissue Availability in Advanced NSCLC

Chemo-naïve

Bone Liver Adrenal

Adapted from Herbst et al, N Engl J Med 359:1367, 2008

Refractory to

Chemotherapy

Resistance to

Targeted Therapy

Bone Liver Adrenal

Bone Liver Adrenal

Re-biopsy

MD Anderson BATTLE Program

Stage IV

Untreated

Stage IV

Refractory

Stages I-III

Resected

BATTLE-FL (=300) (started 6/2011, n=29)

PIs: E. Kim –

J. Heymach

BATTLE (n=324) (completed, 11/2009)

PIs: E. Kim –

R. Herbst

BATTLE-Prevention (in preparation)

PIs: E. Kim –

S. Swisher

BATTLE (n=400)

(started 6/2011, n=93)

PI: V.

Papadimitrakopoulou

EML-

ALK

Fusion –

EGFR

Μut

exclusio

n

Stage 1: (n=200)

Adaptive Randomization

by KRAS Mutation Status

Primary endpoint: 8-week disease control

Stage 2: (n=200)

Refined Adaptive Randomization

“Best” discovery markers/signatures

Statistical modeling, biomarker selection

BATTLE-1 and -2 Trial Schemas

Sorafenib

Erlotinib

+AKTi

MEKi

+AKTi

Erlotinib

Stage 1: (n=97)

Equal Randomization

Sorafenib

Bexarotene

+Erlotinib

Vandetanib

Erlotinib

Stage 1: (n=158)

Adaptive Randomization

11 Molecular Marker

Analysis (14 days)

BATTLE-1 BATTLE-2 Protocol enrollment

Biopsy performed Protocol enrollment

Biopsy performed

Kim et al (Cancer Discovery 2011) and V. Papadimitrakopoulou (unpublished)

BATTLE-1 and -2 Tissue Collection and

Molecular Analysis

Sample/Marker BATTLE-1 BATTLE-2 Tissue Cores 2-3/case (1 frozen) 5/case (3 frozen)

Cytology (FNA) No Yes

Protein (IHC) Yes (n=5) Yes (n=6)

Gene Copy # (FISH) Yes (n=2) No

Mutation Yes (3 genes) Yes (9 genes)

mRNA-Affy Array Yes

(3 signatures

developed)

Yes

(Test BATTLE-1: WEE, EMT,

Sorafenib; and develop new)

Proteomic (RPPA) Yes Yes

MicroRNA Array No Yes

Next-gen Sequencing No Yes

(RNA-Seq/DNA Targeted Mut)

Kim et al (Cancer Discovery 2011) and V. Papadimitrakopoulou (unpublished)

Core Needle Biopsy (CNB)

CT

Adequacy Biopsies for

Molecular Profiling (DNA, RNA

and Proteins) in NSCLC

Refractory Tumors:

Tissue Quality Control for Molecular Testing

by Pathologist: Refractory NSCLC

SCC

BATTLE-1 = 270/324, 83%

(3 CNBs and no FNA)

Necrosis Fibrosis

BATTLE-2 (3/2012) = 74/77, 96%

(5 CNBs and FNA)

Fig. 1 The frequency of observed drug resistance mechanisms.

Modified from Sequist L V et al. Sci Transl Med 2011;3:75ra26-75ra26

Mechanisms of Resistance to EGFR TKIs

in Lung Adenocarcinoma

Unknown

(30%)

EGFR T790M

Mutation

(49%) SCLC

Features

(14%)

EMT Change

(14%)

PI3KCA Mut

(2%) MET Ampl

(2%)

Adenocarcinoma

SCLC

H&E Synaptophysin

H&E Synaptophysin

Fig. 1 The frequency of observed drug resistance mechanisms.

Doebele RC et al, Clin Cancer Res2012 Mar 1;18(5):1472-82. Epub 2012 Jan 10

Mechanisms of Resistance to ALK TKIs

in Lung Adenocarcinoma

NSCLC: Re-biopsy Diagnosis

At Tumor Progression

FNA and Cell Block

Molecular Testing: Mutation, Copy

Number Analysis, Gene Expression, etc

Histology Diagnosis &

Quality Control for Molecular Testing

Core Needle Biopsy

Molecular Testing in Lung Cancer How Do We Deal With Pathologists?

• Advocating for and/or providing enough tissue

• Being reasonable on the request (enough tissue

available for histology and molecular diagnosis)

• Guiding on the important question: tumor (yes/no),

histology type and molecular change, to prioritize

tissue

• Reassure that the material will be returned and the

information will be shared

• Being nice!