biomarkers needed for diagnosis of lung cancer, a brief review of them is at times needed

1. Predictive Biomarkers for Lung Cancer
Current Status / Perspectives:
Although curative resection of patients with
early-stage lung CA are performed, the risk
of relapse remains substantial
Indicates that there may be micro-
invasion/metastasis have not been
detected by general imaging and/or
pathological examinations
Predictive biomarkers will allow the selection
of lung cancer patients who may need more
aggressive screening and treatment

2. Predictive Biomarkers for Lung Cancer
Intended Goals:
 Defining categories or tumor subsets that may
improve the diagnostic classification of lung
tumors
 Identifying specific genes, proteins, or accessory
cells that could serve as targets for improved
diagnosis and/or therapy
 Associating biomarkers with clinical outcomes

3. Predictive Biomarkers for Lung Cancer
Hurdles:
There are no biomarkers universally
recommended to help in the clinical
management of lung cancer today.
 Probable valid biomarkers
 Candidate biomarkers
 General trends
 Poor study design / analysis
 Assay variability
 Lack of standardization protocols

4. Predictive Biomarkers for Lung Cancer
Challenges:
 Single biomarker approach has not been proven to
have strong predictive potential in lung cancer
 Use of molecular and nano-IVD technologies bring
a key promise for identification of clinically
meaningful biomarkers
 Clinical validation of candidate biomarkers
remains a major challenge

5. Predictive Biomarkers for Lung Cancer
Challenges:
 Use of biomarkers for early detection of
lung cancer is promising but still methodologically
challenging
 Clinical management of lung cancer will most
probably first benefit from use of biomarkers
 Development of new therapeutic options for lung
cancer will stimulate identification and clinical
validation of new biomarkers

6. Predictive or diagnostic modelling
• Tissue based.
• Serum or urinary based
• Cellular based
Use of one or more biomarkers to determine prognosis
or response to treatment beyond usual clinical criteria

7. Overview of Genomic Approach
 DNA / RNA microarray
 MicroRNA microarray
 Single nucleotide polymorphism (SNPs)
 Epigenetic (e.g. methylation) profiling

8. Metagene Analysis in NSCLA
Potti et al,
NEJM, 2006

9. Metagene Analysis in NSCLA
Application of the lung metagene model to refine
the assessment of risk and guide the use of
adjuvant chemotherapy in Stage 1A NSCLC Potti et al,
NEJM, 2006

10. Unique Micro RNA Profile in Lung
Cancer Diagnosis and Prognosis
• miRNAs are small non-coding RNAs which
play key roles in regulating the translation
and degradation of mRNAs
• Genetic and epigenetic alteration may
affect miRNA expression, thereby
leading to aberrant target gene(s)
expression in cancers
• Yanaihara et al, Cancer Cell, 2006:
- miRNA profiles of 104 pairs of primary
lung cancers and corresponding non-
cancerous lung tissues were analyzed by
miRNA microarrays
- 43 miRNAs showed statistical differences

11. Unique Micro RNA Profile in Lung
Cancer Diagnosis and Prognosis
• A univariate Cox proportional hazard
regression model with a global permutation
test indicated that expression of the miRNAs
has-mir-155 and has-let-7a-2 was related to
adenocarcinoma patient outcome
• Yanaihara et al, Cancer Cell, 2006:
- miRNA profiles of 104 pairs of primary
lung cancers and corresponding non-
cancerous lung tissues were analyzed by
miRNA microarrays
- 43 miRNAs showed statistical differences
• Lung adenocarcinoma patients with
either high has-mir-155 or reduced
has-let-7a-2 expression had poor survival

12. Overview of Proteomic Approach

13. (Mass/Charge)
3000 5500 8000 10500 13000
RelativeIntensity
NL
LC
Spectra from human normal lung and NSCLC tissues
* *
*
*
*

14. Cluster analysis between Tumor and Normal lung
(82 signals)

15. 0 10 20 30 40
50
Poor Prognosis Group
P < 0.0001
Good Prognosis Group
1.0
0.8
0.6
0.4
0.2
0
Kaplan-Meier survival curves
based on 15 MS peaks
Time in Months
Survival

16. Grand Serology: Pedigreed database

17. Clinical Correlations in NSCLC (interim data)
Clinical Correlations in Esophageal Cancer (interim data)

18. Cellular Biomarkers
 Circulating cancer cells (EpCAM+ cells)
 Endothelial progenitor cells (CD133+VEGFR2+ cells)
 Hemangiocytes (CXCR4+VEGFR1+ myelomonocytic
precursor cells; pro-angiogenic; pre-metastatic niche)
 Stromal cells (pericytes, myofibroblasts)

19. Pro-angiogeic
Bone marrow
Endothelial
progenitorshematopoietic
stem/progenitor cells
Pro-angiogeic
Bone marrow
Endothelial
progenitorshematopoietic
stem/progenitor cells
Inflammation
Tumor, Ischemia
Regenerating Tissue
Hypoxia
Wound Healing
CXCR4+
VEGFR1+
CD133+
VEGFR2+
Neo-angiogenic Niche
Chemokine
(SDF-1)
Mobilization
Recruitment
Differentiation
Incorporation
Assembly
Niche Migration
(endosteal  vascular)

20. Hypothesis
“NSCLC is associated with an elevated
hemangiogenic profile, therefore, surgical
removal of primary tumor may normalize
this dysregulation in hemangiogenesis”

21. Plasma
Functional angiogenic activity
(HUVEC-based angiogenic scale)
ELISA
Flow
cytometry CEP (CD133+
VEGFR2+
)
Cells
Lysate
Hematopoietic colony-
forming assay
VEGF, SDF-1
Figure 5. Schema
Plasma
Functional angiogenic activity
(HUVEC-based angiogenic scale)
ELISA
Flow
cytometry CEP (CD133+
VEGFR2+
)
Cells
Lysate
Hematopoietic colony-
forming assay
VEGF, SDF-1
Figure 5. Schema
Assessment of Hemangiogenic Biomarkers in
NSCLC
Schema:
EPCs

22. Angiogenic Activity
0: Well separated HUVECs
1: Cells begin to migrate and align
2: Visible capillary tubes; no sprouting
3: Sprouting of new capillary tubes
4: Polygonal structures begin to form
5: Presence of complex mesh-like structures
HUVEC-Based Functional Angiogenic
Scale
0
1
2
3
4 5

23. 0
1
2
3
4
5
Pre-op baseline
2 weeks post-op
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
FunctionalAngiogenicScale
0
1
2
3
4 *p<0.05
Figure 4
70000
80000
R2+
lood)
Figure 5 Pre-op baseline
2 weeks post-op
0
1
2
3
4
5
Pre-op baseline
2 weeks post-op
Pre-op baseline
2 weeks post-op
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
FunctionalAngiogenicScale
0
1
2
3
4 *p<0.05
Figure 4
70000
80000
R2+
lood)
Figure 5 Pre-op baseline
2 weeks post-op
Functional Angiogenic Scale

24. 0
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0
10000
20000
30000
40000
50000
60000
70000
80000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingCD133+VEFR2+
EPCs(permlperipheralblood)
0
20000
40000
60000
80000
*p<0.05
Figure 5 Pre-op baseline
2 weeks post-op
Ctrl Pre-
op
Post-
op
80
100
120
140
160
180
200
latingHPCs
eripheralblood)
Figure 6
Pre-op baseline
2 weeks post-op
100
150
200 p<0.05*
0
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0
10000
20000
30000
40000
50000
60000
70000
80000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 251 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingCD133+VEFR2+
EPCs(permlperipheralblood)
0
20000
40000
60000
80000
*p<0.05
Figure 5 Pre-op baseline
2 weeks post-op
Ctrl Pre-
op
Post-
op
80
100
120
140
160
180
200
latingHPCs
eripheralblood)
Figure 6
Pre-op baseline
2 weeks post-op
100
150
200 p<0.05*
80
100
120
140
160
180
200
latingHPCs
eripheralblood)
80
100
120
140
160
180
200
latingHPCs
eripheralblood)
Figure 6
Pre-op baseline
2 weeks post-op
Pre-op baseline
2 weeks post-op
100
150
200 p<0.05*
0
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0
10000
20000
30000
40000
50000
60000
70000
80000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingCD133+VEFR2+
EPCs(permlperipheralblood)
0
20000
40000
60000
80000
*p<0.05
Figure 5 Pre-op baseline
2 weeks post-op
Ctrl Pre-
op
Post-
op
80
100
120
140
160
180
200
latingHPCs
eripheralblood)
Figure 6
Pre-op baseline
2 weeks post-op
100
150
200 p<0.05*
0
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0
10000
20000
30000
40000
50000
60000
70000
80000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 251 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingCD133+VEFR2+
EPCs(permlperipheralblood)
0
20000
40000
60000
80000
*p<0.05
Figure 5 Pre-op baseline
2 weeks post-op
Ctrl Pre-
op
Post-
op
80
100
120
140
160
180
200
latingHPCs
eripheralblood)
Figure 6
Pre-op baseline
2 weeks post-op
100
150
200 p<0.05*
80
100
120
140
160
180
200
latingHPCs
eripheralblood)
80
100
120
140
160
180
200
latingHPCs
eripheralblood)
Figure 6
Pre-op baseline
2 weeks post-op
Pre-op baseline
2 weeks post-op
100
150
200 p<0.05*
Circulating CD133
+
VEGFR2
+
Endothelial
Progenitor Cells
0
1
2
3
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
FunctionalAngioge
0
1
2
3
4 * p<0.05
0
10000
20000
30000
40000
50000
60000
70000
80000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingCD133+VEFR2+
EPCs(permlperipheralblood)
0
20000
40000
60000
80000
*p<0.05
Figure 5 Pre-op baseline
2 weeks post-op
Ctrl Pre-
op
Post-
op
160
180
200
Cs
lblood)
Figure 6
Pre-op baseline
2 weeks post-op
0
1
2
3
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
FunctionalAngioge
0
1
2
3
4 * p<0.05
0
10000
20000
30000
40000
50000
60000
70000
80000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 251 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingCD133+VEFR2+
EPCs(permlperipheralblood)
0
20000
40000
60000
80000
*p<0.05
Figure 5 Pre-op baseline
2 weeks post-op
Ctrl Pre-
op
Post-
op
160
180
200
Cs
lblood)
Figure 6
Pre-op baseline
2 weeks post-op
160
180
200
Cs
lblood)
160
180
200
Cs
lblood)
Figure 6
Pre-op baseline
2 weeks post-op
Pre-op baseline
2 weeks post-op
0
1
2
3
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
FunctionalAngioge
0
1
2
3
4 * p<0.05
0
10000
20000
30000
40000
50000
60000
70000
80000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingCD133+VEFR2+
EPCs(permlperipheralblood)
0
20000
40000
60000
80000
*p<0.05
Figure 5 Pre-op baseline
2 weeks post-op
Ctrl Pre-
op
Post-
op
160
180
200
Cs
lblood)
Figure 6
Pre-op baseline
2 weeks post-op
0
1
2
3
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
FunctionalAngioge
0
1
2
3
4 * p<0.05
0
10000
20000
30000
40000
50000
60000
70000
80000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 251 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingCD133+VEFR2+
EPCs(permlperipheralblood)
0
20000
40000
60000
80000
*p<0.05
Figure 5 Pre-op baseline
2 weeks post-op
Ctrl Pre-
op
Post-
op
160
180
200
Cs
lblood)
Figure 6
Pre-op baseline
2 weeks post-op
160
180
200
Cs
lblood)
160
180
200
Cs
lblood)
Figure 6
Pre-op baseline
2 weeks post-op
Pre-op baseline
2 weeks post-op

25. Plasma SDF-1 Levels
0
200
400
600
800
1000
1200
1400
1600
1800
2000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
PlasmaLevelsofSDF-1(pg/ml)
Figure 8
Pre-op baseline
2 weeks post-op
0
600
1200
1800 *p<0.05
0
200
400
600
800
1000
1200
1400
1600
1800
2000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
PlasmaLevelsofSDF-1(pg/ml)
Figure 8
Pre-op baseline
2 weeks post-op
0
200
400
600
800
1000
1200
1400
1600
1800
2000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 251 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
PlasmaLevelsofSDF-1(pg/ml)
Figure 8
Pre-op baseline
2 weeks post-op
Pre-op baseline
2 weeks post-op
0
600
1200
1800 *p<0.05
0
200
400
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
I
V
0
200
400
600
0
200
400
600
800
1000
1200
1400
1600
1800
2000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
PlasmaLevelsofSDF-1(pg/ml)
Figure 8
Pre-op baseline
2 weeks post-op
0
600
1200
1800 *p<0.05
0
200
400
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 251 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
I
V
0
200
400
600
0
200
400
600
800
1000
1200
1400
1600
1800
2000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
PlasmaLevelsofSDF-1(pg/ml)
Figure 8
Pre-op baseline
2 weeks post-op
0
200
400
600
800
1000
1200
1400
1600
1800
2000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 251 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
PlasmaLevelsofSDF-1(pg/ml)
Figure 8
Pre-op baseline
2 weeks post-op
Pre-op baseline
2 weeks post-op
0
600
1200
1800 *p<0.05

26. Predictive Modelling
Permit risk stratification.
Customize treatment
Less extensive surgery
Rational drug selection
Monitoring response to therapy.

27. 0
10000
20000
30000
40000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingC
EPCs(permlp
0
20000
40000
60000
*
Ctrl Pre-
op
Post-
op
0
20
40
60
80
100
120
140
160
180
200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingHPCs
(permlperipheralblood)
Figure 6
Pre-op baseline
2 weeks post-op
0
50
100
150
200 p<0.05*
Ctrl Pre-
op
Post-
op
0
10000
20000
30000
40000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 251 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingC
EPCs(permlp
0
20000
40000
60000
*
Ctrl Pre-
op
Post-
op
0
20
40
60
80
100
120
140
160
180
200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingHPCs
(permlperipheralblood)
Figure 6
Pre-op baseline
2 weeks post-op
0
50
100
150
200 p<0.05*
Ctrl Pre-
op
Post-
op
0
20
40
60
80
100
120
140
160
180
200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingHPCs
(permlperipheralblood)
0
20
40
60
80
100
120
140
160
180
200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 251 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CirculatingHPCs
(permlperipheralblood)
Figure 6
Pre-op baseline
2 weeks post-op
Pre-op baseline
2 weeks post-op
0
50
100
150
200 p<0.05*
Ctrl Pre-
op
Post-
op
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0
Ctrl Pre-
op
Post-
op
0
20
40
60
80
100
120
140
160
180
200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
(permlperipheralblood)
Figure 6
Pre-op baseline
2 weeks post-op
0
50
100
150
200 p<0.05*
Ctrl Pre-
op
Post-
op
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 251 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0
Ctrl Pre-
op
Post-
op
0
20
40
60
80
100
120
140
160
180
200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
(permlperipheralblood)
Figure 6
Pre-op baseline
2 weeks post-op
0
50
100
150
200 p<0.05*
Ctrl Pre-
op
Post-
op
0
20
40
60
80
100
120
140
160
180
200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
(permlperipheralblood)
0
20
40
60
80
100
120
140
160
180
200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 251 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
(permlperipheralblood)
Figure 6
Pre-op baseline
2 weeks post-op
Pre-op baseline
2 weeks post-op
0
50
100
150
200 p<0.05*
Ctrl Pre-
op
Post-
op
Circulating Hematopoietic Progenitor Cells

28. 0
200
400
600
800
1000
1200
1400
1600
1800
2000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
VEGF-A(pg/ugprotein)
0
200
400
600
800
1000
1200
1400
1600 *p<0.05
Figure 7
Pre-op baseline
2 weeks post-op
2000
ml)
Figure 8
Pre-op baseline
2 weeks post-op
0
200
400
600
800
1000
1200
1400
1600
1800
2000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 251 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
VEGF-A(pg/ugprotein)
0
200
400
600
800
1000
1200
1400
1600 *p<0.05
Figure 7
Pre-op baseline
2 weeks post-op
Pre-op baseline
2 weeks post-op
2000
ml)
Figure 8
Pre-op baseline
2 weeks post-op
2000
ml)
Figure 8
Pre-op baseline
2 weeks post-op
Pre-op baseline
2 weeks post-op
0
200
400
600
800
1000
1200
1400
1600
1800
2000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
IntraplateletLevelsof
VEGF-A(pg/ugprotein)
0
200
400
600
800
1000
1200
1400
1600 *p<0.05
Figure 7
Pre-op baseline
2 weeks post-op
0
200
400
600
800
1000
1200
1400
1600
1800
2000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 251 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
IntraplateletLevelsof
VEGF-A(pg/ugprotein)
0
200
400
600
800
1000
1200
1400
1600 *p<0.05
Figure 7
Pre-op baseline
2 weeks post-op
Pre-op baseline
2 weeks post-op
Intraplatelet VEGF-A Levels

29. .
Cancer-Testis Genes are expressed and are
markers of poor outcome in pulmonary
adenocarcinoma
Ali O. Gure,CCR 2005