Download Breakthroughs in Molecular Diagnostics and Monitoring

Breakthroughs in Molecular Diagnostics
and Monitoring
Domnita Crisan, MD, PhD.
Clinical Pathology
William Beaumont Hospital
“Everything in medicine is genetic-or influenced
by genetics – with the possible exception of
trauma, and I’m not entirely sure about that”.
Francis Collins, MD, PhD., Head of the National
Center for Human Genome Research
1
The role of B-catenin and c-myc in the
inhibition of epithelialization and wound
healing.
Stojadinovic O, et. at. Am. J. Pathol. July 2005.
2
Molecular Pathology in Clinical Practice
Fundamental in almost every aspect of healthcare
delivery:
„ diagnosis
„ prognosis
„ therapeutic choice
„ therapeutic outcome monitoring
„ prediction of disease risk
„ beginning-of-life choices
„ patient and specimen ID
„ molecular epidemiology
1953
Major Breakthroughs in DNA
Technology
1962
Wilkins, Max Perutz (X-ray analysis of
hemoglobin), Crick, Steinbeck, Watson,
John Kendrew (myoglobin and the structure
of proteins)
Rosalind Franklin
X-Ray difraction
image of DNA
Beaumont DNA Symposium 2006
Modified from Dan Farkas, Ph.D.
3
Major Breakthroughs in DNA
Technology
1958 DNA Polymerases: Kornberg & others
„ 1966 Elucidation of the Genetic Code:
„
Nirenberg, Khorana, Jacob, Monod
„
1970 Reverse Transcriptase: Baltimore, Temin &
Mizutani
„
1970 Restriction Endonucleases: Nathans,
Smith, Wilcox, & others
1975 Southern Blotting: Southern
„ 1977 DNA Sequencing: Sanger, Maxam & Gilbert
„
Early Molecular Diagnostics
The 1980s
Transition from “Revolution” in Molecular
Biology to Clinical Applications
4
Manual DNA/RNA Extraction, Gel
Electrophoresis and Southern Blots
•Highly labor intensive
•Very long turnaround time (7-10 d)
•Extremely poor reimbursement
•Replaced by new techniques
Beaumont DNA Symposium 2006
Modified from Dan Farkas, PhD
Major Breakthroughs in the 1980s
1985 Restriction fragment length
polymorphism analysis
1985 Invention of PCR
1986 Development of fluorescence in situ
hybridization (FISH)
1988 Discovery of thermostable DNA
polymerase: PCR automation
5
Polymerase Chain Reaction
„ Described in 1985 by Kary Mullis
„ Nobel Prize in 1993
„ Amplifies DNA up to approximately
35 kb
„ Multiple cycles of
- DNA denaturation
- Primer annealing
- Polymerase extension
PCR Amplification Process
Template DNA
Denaturation and
Primer Annealing
5’ 5’
Extension of
Primers
6
PCR Changed Everything
PCR Moved Molecular Diagnostics Forward
Exponentially:
„ Menu (Hematology, Oncology, Genetics,
Infectious Diseases, Paternity Testing, Forensics,
etc.)
„ Reimbursement (new codes/new
reimbursement/better economic model for labs)
„ Progress towards automation
„ PCR “wannabes” (competition = good)
Major Breakthroughs in the 1990s
and Beyond
1992
1993
1996
2001
2003,
Conception of real time PCR
Discovery of structurestructure-specific endonucleases for cleavage
assays
First applications of DNA microarrays
First draft versions of the human genome sequence
April 14
Completion of the human genome sequencing announced in a
Joint Proclamation of the International Human Genome
Sequencing Consortium.
“We, the Heads of Government of the United States of America,
the United Kingdom, Japan, France, Germany, and China, are
proud to announce that scientists from our six countries have
completed the essential sequence of three billion base pairs of
DNA of the human genome, the molecular instruction book of
human life.”
life.”
7
History of Genome Project – the
summit - 2001
HGP consortium publishes its working draft in Nature
(15 Feb)
Celera publishes its draft in Science (16 Feb).
Complete sequence of the entire set of human chromosomes
8
“Omics”
The Next Generation
Beyond the GENOME
„
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„
TRANSCRIPTOME
PROTEOME
METABOLOME
KINOME
PHARMACOGENOME
NUTRIGENOME
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REAL TIME QUANTITATIVE PCR
„
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„
„
„
Arguably THE big development in molecular
diagnostics in the last five years
Quantitative
Combines Amplification with Detection
Fast
Automated
Kits don’t necessarily require molecular MTs
Small footprint
10
DNA Microarrays
Shift from the static standard genomics to
functional genomics
„ Gene expression profiling:
transcriptomics (which of the 30,000
genes are expressed and at what levels)
„ Endpoint of transcriptomics = Proteomics
„
– to monitor gene translation
– currently only 1% of proteins have 3Dstructure determined
Microarray Applications
Monitoring of gene expression: global gene
expression to determine transcriptional
profile = transcriptome as the major
determinant of cell phenotype and function
„ Gene expression can yield important
pathogenesis and prognostic information
„ Key advance: cluster analysis
„
11
Chronic Lymphocytic Leukemia
AmpliChip CYP450 Array
Array consists of:
•2D6 gene: 27 alleles (7 are
duplications)
•2C19 gene: 2 alleles
Beaumont DNA Symposium 2006
Courtesy of Dan Farkas, PhD
12
William Beaumont Hospital
Royal Oak Campus
Beaumont Reference
Laboratory
13
William Beaumont Hospital
Royal Oak, Michigan
Department of Clinical Pathology
Molecular Pathology Services
• INHERITED DISORDERS
• GENETIC RISK FACTORS
• MOLECULAR DETECTION OF
INFECTIOUS AGENTS
• MOLECULAR HEMATOLOGY
• PHARMACOGENOMICS
Molecular Center of Excellence (MCOE)
Award 2004
• FIRST MCOE IN MICHIGAN
• 30 MCOE LABORATORIES IN US
(SECOND MCOE LAB IN MICHIGAN – 2006)
Inherited Disorders
„
Inherited thrombophilia
– Factor V. Leiden
– Prothrombin mutation
– MTHFR genotyping (2 mutation detection)
„
„
Hereditary hemochromatosis (2 mutation
detection)
Cystic Fibrosis: ACOG panel of mutations,
extended panel (40 + 4)
14
Inherited Disorders, cont’d
„
Ashkenazi Jewish Panel
–
–
–
–
–
–
–
–
–
Bloom Syndrome
Canavan disease
Cystic fibrosis
Familial dysautonomia
Fanconi anemia
Gaucher disease
Mucolipidosis type IV
NiemannNiemann-Pick disease
TayTay-Sachs disease
Genetic Risk Factors for
Cardiovascular Disease
APO E genotype
„ ACE genotype
„ Angiotensinogen genotype
„ Angiotensin II receptor type I genotype
„ Glycoprotein Pl A1/A2 genotype
„ MTHFR for Hyperhomocysteinemia
genotyping (2 mutation detection)
„
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Infectious Diseases
„
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Chlamydia trachomatis
Neisseria Gonorrhoeae
M. tuberculosis
Cytomegalovirus
Herpes Simplex virus
Human papilloma virus
Hepatitis B virus – quantitation
Hepatitis C virus: qualitative (ultrasensitive)
quantitative
genotype
VZV
HIV: quantitative standard
quantitative ultrasensitive
genotype
Enterovirus
Infectious Diseases
Respiratory Virus Panel
Influenza A
„ Influenza B
„ Parainfluenza Types 1 – 3
„ Adenovirus
„ Respiratory Syncytial Virus Types A and B
„ Rhinovirus
„ Metapneumovirus
„
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Molecular Hematology Tests
BCR major breakpoint (p210) - qualitative
„ BCR minor breakpoint (p190) - qualitative
„ BCR-ABL quantitation
„ FLT 3 gene mutations (ITD and D835)
„ JAK2 gene mutation - qualitative
„ JAK2 gene mutation – quantitative
„ CLL gene expression analysis and IgVH
mutational status
„
Molecular Pathology Laboratory Test
Volumes
70000
60000
50000
40000
30000
20000
10000
07
06
20
05
20
20
03
04
20
02
20
01
20
00
20
99
20
98
19
97
19
96
19
95
19
94
19
93
19
19
19
92
0
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Molecular Pathology Laboratory Test Volumes
Year
# tests performed
1992
307
% increase from previous year
1993
550
79%
1994
1747
218%
1995
3604
106%
1996
4472
24%
1997
9376
110%
1998
10821
15.4%
1999
13292
22.8%
2000
20,953
57.6%
2001
23,392
11.6%
2002
28,531
22%
2003
38,792
36%
2004
43,461
12%
2005
47,450
9%
2006
53,552
12.9%
2007
64,346
20.2%
Molecular Pathology Laboratory
Test
BCR gene rearrangement
B/T cell gene rearrangement
BclBcl-2 gene rearrangement
DNA Bank
Fragile X Syndrome
HerHer-2/neu gene amplification
HCV qualitative
Chlamydia trachomatis
PCR
LCR
PCR
Year Offered
1991
1992
1992
1992
1992
1993
1994
1994
1996
2001
18
Molecular Pathology Laboratory
Test
Factor V Leiden
Neisseria gonorrhea
LCR
Year Offered
1995
1996
PCR
HCV quantitative
HLA α class II typing
Hereditary Hemochromatosis
MTHFR mutation (homocysteine)
Cytomegalovirus
Herpes Simplex virus
HIV quantitation (standard and ultrasensitive)
Prothrombin mutation
Angiotensin converting enzyme (ACE)
2001
1996
1996
1997
1998
1998
1998
1998
1998
1999
Molecular Pathology Laboratory
Test
Glycoprotein Pl A1/A2
HCV genotyping
Human papillomavirus
Angiotensinogen mutation
Angiotensin receptor type 1
CYP2C9 genotype (Coumadin hyperhyperresponsiveness)
HBV quantitation
Apo E genotype
HIV genotyping
Herpes Simplex Virus
Cystic Fibrosis
Year Offered
2000
2001
2001
2002
2002
2002
2002
2002
2002
2003
2003
19
Molecular Pathology Laboratory
Test
Year Offered
Varicella Zoster Virus
2004
FLT 3 gene mutations
2004
BCR qualitative
2004
Enterovirus
Ashkenazi Jewish Panel
JAK2 mutation - qualitative
2005
2005
2006
BCR quantitative
2007
JAK2 mutation – quantitative
2007
Molecular Pathology Laboratory
Test
Year Offered
Chronic lymphocytic
leukemia (CLL) gene
expression and IgVH
mutational status
2007
Respiratory Virus Panel
(Influenza A and B,
Parainfluenza types 1 -3,
Adenovirus, Rhinovirus,
Respiratory Syncytial Virus
Type A and B, Metapneumovirus)
Metapneumovirus)
2008
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Current and Near Future
Molecular Breakthroughs
Respiratory Viral Panel
„ Circulating tumor cell detection
„ Specific prostate cancer molecular markers
„ Pharmacogenomics
„ Nutrigenomics
„ Microarray applications
„ Epigenetics
„
Respiratory Viral Panel
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Luminex xTAGTM RVP: FDA approved
January 3, 2008
First multiplexed molecular test FDA-cleared for
respiratory infections
First test cleared for detection of human
metapneumovirus
First test cleared for Flu A subtyping
First test cleared for adenovirus
Detects >85% of respiratory virus infections
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RVP Targets
Viral Family
Respiratory Syncytial
Virus (RSV)
Influenza A
Viral Subtype
A
B
NonNon-specific Influenza A
H1
H3
Influenza B
Parainfluenza 1
Parainfluenza 2
Parainfluenza 3
Metapneumovirus
Rhinovirus
Adenovirus
Sample Collection
NA Amplification
Bead Hybridization
Luminex xMAP
Bead Analysis
Target Specific Primer
Extension
xTAG RVP
Data Analysis
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RVP Testing Sites
North Shore - LIJ Health Systems
Laboratories, Long Island, NY
„ William Beaumont Hospital, Royal Oak, MI
„ St. Joseph’s Healthcare, McMaster
University, Hamilton, ON, Canada
„ Mount Sinai Hospital, Toronto, ON, Canada
„
Respiratory Virus Statistics
7th leading cause of death in the US.
More than 60,000 deaths annually
„ 80% of respiratory infections are viral
„ Viral respiratory infections responsible for
75-80% of all doctor visits
„ 11 million adults diagnosed annually with
lower respiratory viral infections and
208,000 hospitalized
„
23
Respiratory Virus Statistics
(cont’d)
5.3 million children diagnosed with lower
respiratory viral infections (2.8 million <5
years old) and 430,000 hospitalized (90%
under the age of 5)
„ Annual cost to society $10 billion
„ Approx 60% of viral infections are
misdiagnosed or diagnosed late and
inaccurately treated with antibiotics
„
RVP Viruses - Statistics
Influenza
• can affect up to 20% of US population each
year
• hospitalizations >200,000 annually
• deaths: 36,000 annually
RSV A and B
• most common cause of bronchiolitis and
pneumonia in infants and children
• deaths: 17,000 annually
Metapneumovirus • recently discovered virus causing fluflu-like
symptoms in all age groups
• second most frequent cause of lower
respiratory infections in children
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RVP Viruses - Statistics (cont’d)
Adenovirus
• generally causing mild infections
Rhinovirus
• CDC recently reported multiple deaths
(NY, OR, TX, WA)
• common cold
• 66 million in US annually
Parainfluenza 1,2,3 • lower respiratory infections in adults and
children
• cause 4040-50% of croup cases
• cause 1010-15% of bronchiolitis and bronchitis
Advantages of Multiplex RVP
Testing
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Collection: noninvasive NP swab
One patient sample- 12 virus detection
High sensitivity and specificity (important for
reference lab with long distance shipping and
decline in infectivity)
Automated
Rapid TAT 6-8 hours vs traditional testing for this
number of viruses, requiring multiple individual
diagnostic tests and up to several days to provide a
complete diagnosis
25
Sensitivity and Specificity
of xTAG RVP
Virus
Influenza A
Flu A Subtype H1
Flu A Subtype H3
Influenza B
RSV A
RSV B
Parainfluenza 1
Parainfluenza 2
Parainfluenza 3
Rhinovirus
Adenovirus
Metapneumovirus
Sensitivity
96.4%
100%
91.7%
91.5%
100%
100%
100%
100%
84.2%
100%
78.3%
96%
Specificity
95.9%
100%
98.7%
96.7%
98.4%
97.4%
99.8%
99.8%
99.6%
91.3%
100%
98.8%
Source: Luminex package insert for xTAG RVP kit reagents. Figures are based on testing of 544 prospectively
prospectively
collected specimens at four North American clinical laboratories during the 20052005-6 flu season in a trial that compared
xTAG with DFA and/or culture.
Advantages of Multiplex RVP
Testing (cont’d)
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Allows starting appropriate treatment quickly
Promote appropriate antiviral use
Avoid inappropriate use of antibiotics
Cohorting: guide accurate isolation procedures
Reduce unnecessary diagnostic studies
Surveillance: identify types of virus circulating in
the community; assist MDs and public health
officials in preventing outbreaks
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Cost and Reimbursement
Cost to perform: $80-90
„ CPT code 87798: Infectious agent detection
by nucleic acid; amplified probe technique,
each organism
„ Reimbursement: • hospital-based clinical
labs vs reference labs
• $49.04 (Medicare
Michigan) x multiple
„
Limitations
„
„
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„
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Cost: development, validation, equipment
Personnel training
Need for automation of NA extraction
Not rapid enough for ER patients (Rapid
immunoassays and DFA have shorter TAT, but
low sensitivity)
A positive molecular test may be analytically true,
but of unclear clinical significance (detect viable
and non-viable viruses, viruses shed for daysweeks after recovery from clinical illness, or after
live attenuated vaccine)
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Circulating Tumor Cell Detection
FDA approved tests CellSearchTM (Veridex)
ƒ
ƒ
ƒ
Metastatic breast cancer - January 2004
Metastatic colorectal cancer - November 2007
Metastatic prostate cancer - February 2008
CellSearch detection
Detection and quantitation of circulating
tumor cells (CTC) of epithelial origin that
detach from solid tumor and enter the blood
stream
„ Principle: identification of CTCs CD45-,
EpCAM + cytokeratins + in whole blood
„ Presence and number of CTCs correlate
with progression-free survival (PFS) and
overall survival (OS) in all 3 types of cancer
„
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Clinical Trial
Metastatic Breast Cancer (MBC)
Multi-institutional - 20 centers
„ 177 MBC patients
„ 145 healthy individuals
„ 200 patients with benign diseases
„ Clinical outcomes monitored up to 50
months
„
Predictive Value: OS of MBC
Patients with <5 or >5 CTC at
Baseline (N=177)
100%
CTC / 7.5mL
at Baseline
<5 CTC
>5 CTC
%Probability of Survival
90%
80%
70%
Logrank
p < 0.0001
60%
N (%)
89 (50%)
88 (50%)
Median OS in
Months (95% C.I.)
21.9 (20.1 to 28.6)
10.9 ( 7.0 to 15.2)
Cox Hazards Ratio = 2.4
chi-square = 19.54
(p-value < 0.0001)
21.9 Months
50%
10.9
Months
40%
30%
20%
10%
0%
0
5
10
15
20
25
30
35
Time from Baseline (Months)
40
45
50
29
%Probability of Progression Free Survival
Predictive Value: PFS of MBC
Patients with <5 or ≥5 CTC at
Baseline (N=177)
100%
90%
CTC / 7.5mL
at Baseline
<5 CTC
>5 CTC
80%
N (%)
89 (50%)
88 (50%)
Median PFS in
Months (95% C.I.)
7.0 (5.6 to 8.9)
2.7 (2.1 to 4.4)
70%
60%
50%
40%
7.0 Months
Cox Hazards Ratio = 1.9
chi-square = 14.44
(p-value = 0.0001)
2.7
Months
30%
20%
Logrank p = 0.0001
10%
0%
0
5
10
15
20
25
30
35
Time from Baseline (Months)
40
45
50
Clinical Trial
Metastatic Colorectal Cancer
(MCRC)
Multi-institutional
„ 413 MCRC patients
„ 158 healthy individuals
„ 55 patients with benign diseases
„ Clinical outcomes monitored up to 36
months
„
30
Predictive Value: OS of MCRC
Patients with <3 or >3 CTC at
Baseline (N=413)
CTC / 7.5mL
at Baseline
N (%)
<3 CTC
305 (74%)
108 (26%)
>3 CTC
100%
90%
%Probability of Survival
80%
Median OS in
Months (95% C.I.)
18.5 (15.5 to 21.2)
9.4 ( 7.5 to 11.6)
Cox Hazard Ratio = 2.5
chi-square = 31.48
(p-value < 0.0001)
70%
60%
18.5 Months
50%
9.4
Months
40%
Logrank
p < 0.0001
30%
20%
10%
0%
0
2
6
4
8
10 12 14 16 18 20 22 24
Time from Baseline Blood Draw (Months)
26
28
30
%Probability of Progression Free Survival
Predictive Value: PFS of MCRC
Patients with <3 or ≥3 CTC at
Baseline (N=413)
100%
CTC / 7.5mL
at Baseline
N (%)
<3 CTC
305 (74%)
108 (26%)
>3 CTC
90%
80%
Median PFS in
Months (95% C.I.)
7.9 (7.0 to 8.6)
4.5 (3.7 to 6.3)
Cox Hazard Ratio = 1.6
chi-square = 12.19
(p-value = 0.0002)
70%
60%
7.9 Months
50%
4.5
Months
40%
Logrank p = 0.0002
30%
20%
10%
0%
0
2
4
6
8
10 12 14 16 18 20 22 24
Time from Baseline Blood Draw (Months)
26
28
30
31
Predictive Value: OS of MPC
Patients with <5 or >5 CTC at
Baseline (N=219)
CTC / 7.5mL
at Baseline
N (%)
<5 CTC
94 (43%)
125 (57%)
>5 CTC
100%
%Probability of Survival
90%
Median OS in
Months (95% C.I.)
21.7 (21.3 to ------)
11.5 ( 9.3 to 13.7)
80%
70%
Logrank
p < 0.0001
60%
21.7 Months
50%
11.5
Months
40%
30%
20%
Cox Hazard Ratio = 3.3
chi-square = 34.48
(p-value < 0.0001)
10%
0%
0
2
4
6
8
10 12 14 16 18 20 22 24
Time from Baseline Blood Draw (Months)
26
28
30
A Reduction in CTC Below 5 After the Initiation of
Therapy Predicts Longer OS whereas an Increase in
CTC Count to 5 or above Predicts a Shorter OS in
MPC Patients
Group
Description
1
<5 CTC at All Draws
2
>5 CTC at BL & <5 CTC at Last Draw
3
<5 CTC at Early Draw & >5 CTC at Last Draw
4
>5 CTC at All Draws
N (%)
88 (38%)
45 (20%)
26 (11%)
71 (31%)
Median OS in
Months (95% C.I.)
>26 (21.4 to ------)
21.3 (18.4 to ------)
9.3 ( 8.2 to 11.3)
6.8 ( 5.8 to 10.3)
100%
%Probability of Survival
90%
80%
70%
60%
1
50%
40%
30%
20%
10%
Curve
Comparison
1 vs. 2
1 vs. 3
1 vs. 4
2 vs. 3
2 vs. 4
3 vs. 4
Logrank
p-Value*
0.1528
<0.0001
<0.0001
<0.0001
<0.0001
0.5013
*p-values not adjusted for
multiple hypothesis tests
0%
0
2
4
6
4
2
3
8 10 12 14 16 18 20 22 24
Time from Baseline Blood Draw (Months)
26
28
30
32
MBC, MCRC & MPC
Overall Survival Comparison
100%
90%
80%
70%
60%
50%
40%
1
30%
20%
10%
0%
0 5 10 15 20 25 30 35 40 45 50
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
4
0%
0 5 10 15 20 25 30 35 40 45 50
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
0
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
0
MBC
MCRC
MPC
<cut-off at all time points
22.6
18.6
>26
≥cut-off at all time points
4.1
3.9
6.8
≥cut-off at baseline and
<cut-off at final draw
19.8
11.7
21.3
<cut-off at early draw
and ≥cut-off at final draw
10.6
7.1
9.3
2
5 10 15 20 25 30 35 40 45 50
3
5 10 15 20 25 30 35 40 45 50
Molecular Pathology Staff, 2008
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