the role of pathology/molecular diagnostic in personalized...
TRANSCRIPT
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!