the role of pathology/molecular diagnostic in personalized...
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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
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Conflict of Interest
• Honoraria: Genentech, Glaxo Smith
Kline, Boehringer-Ingelheim, Medscape,
and AstraZeneca.
• Research Agreements: Genentech,
Pfizer, Astra Zeneca, Myriad, Eli-Lilly,
and Merck.
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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
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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
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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
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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
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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!
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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
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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)
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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)
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• 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
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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
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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
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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
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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
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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
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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)
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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)
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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
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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
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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)
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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)
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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)
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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
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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
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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
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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!