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Validation of a Prognostic Gene Signature for Early Stage
Non-Small Cell Lung Cancer in an Independent Cohort
Sandy D.
D Der1,3, Melania Pintilie1, Christine Frantz 1, Chang-Qi
Chang Qi Zhu 1, Dan Strumpf 1, Igor Jurisica1,3, Frances A
A. Shepherd1,2,3, Ming S.
S Tsao1,3
1
2
3
University Health Network, Ontario Cancer Institute , Princess Margaret Hospital , University of Toronto , Toronto, ON, Canada
Introduction
Results
Adjuvant chemotherapy given to patients with early stage non-small cell lung
cancer (NSCLC) results in prolonged survival.
survival An accurate prognostic test
potentially could provide guidance with respect to the need for and benefit from
post-operative treatment. Recently, we developed an mRNA-based classifier
based on 15 genes from molecular analysis of tumor samples from the NCIC CTG
JBR.10 trial that was both prognostic and predictive for survival benefit from
chemotherapy (presented: ASCO, 2008; maunscript in review). The performance
of this classifier was indicated by in silico validation in 4 large, public NSCLC
expression datasets. Here, we further validated the performance of this 15-gene
classifier by analyzing an independent cohort of 183, snap-frozen, early stage
NSCLC samples collected at Princess Margaret Hospital/UHN from 2000-2005.
Methods
Total RNA was isolated from snap frozen samples using Trizol and processed for
profiling using Affymetrix U133 2.0 Plus arrays. Data was normalized using RMA
and one outlier sample was identified and removed. Expression values for the 15
genes were extracted and the signature was tested using a Cox proportional
hazards model while controlling for clinical factors, age, sex, and histology.
Age
Median (range)
<65
>=65
Sex
Women
Men
Stage
1A
1B
2A
2B
3A
Histology
Adenocarcinoma (ADE)
Squamous (SQC)
Adenosquamous (ASQ)
Large cell (LC)
Other
15 gene signature
Low risk
High risk
UHN-2000-05
N=183
N (%)
BR10 ﾐ OBS
N= 62
N (%)
70 {40-88}
60 (33)
123 (67)
61 {35-77}
44 (69)
19 (31)
84 (46)
99 (54)
18 (29)
44 (71)
49 (27)
80 (44)
9 (5)
45 (25)
34 (55)
28* (45)
Gene
S b l
Symbol
Probe Set
Stage 1 and 2 (A + B)
Gene Title
ATP1B1
201243_s_at
ATPase, Na+/K+ transporting, beta 1 polypeptide
TRIM14
203147_s_at
Tripartite motif-containing 14
FAM64A
221591_s_at
A
A
Family with sequence similarity 64, member A
FOSL2
218881_s_at
FOS-like antigen 2
HEXIM1
202814_s_at
Hexamethylene bis-acetamide inducible 1
MB
204179_at
Myoglobin
L1CAM
204584_at
UMPS
202707_at
Uridine monophosphate synthetase
EDN3
208399_s_at
Endothelin 3
L1 cell adhesion molecule
STMN2
203001_s_at
Stathmin-like 2
MYT1L
210016_at
Myelin transcription factor 1-like
IKBKAP
202490_at
Inhibitor of kappa light polypeptide gene enhancer in B-cells
MLANA
206426_at
Melan-A
MDM2
205386_s_at
Mdm2, transformed 3T3 cell double minute 2
ZNF236
219171_s_at
Zinc finger protein 236
B
Table 2. Genes comprising the classifier.
Tumour Type
n
All NSCLC
62
18.00
5.78-56.05
<0.0001
DCC
ADC
96
2.26
1.02-4.97
0.044
NLCI
All NSCLC
133
2.27
1.18-4.35
0.014
Duke
All NSCLC
48
1.96
0.87-4.42
0.11
SQC
79
3.57
1.48-8.58
0.005
JBR.10,
JBR 10
OBS
UM-SQ
Hazard
Ratio
95% CI
Adjusted p-value
Table 3. Validation of the 15-gene signature for
prognosis of Stage 1B-2 from 4 NSCLC datasets.
Director’ss Challenge Consortium adenocarcinoma dataset;
DCC Director
NLCI: Netherlands Cancer Institute; Duke: Duke University; UM-SQ:
University of Michigan, squamous cell carcinoma dataset
B
HR
Age
Sex
Histology
Stage
RS 15 gene
1.491
1.95
1.514
1.718
2.23
95% Conf.
C f Int.
I t
0.839
1.115
0.858
1.007
1.291
-
2.650
3.411
2.670
2.933
3.853
W ld p-value
Wald
l
0.17
0.019
0.15
0.047
0.004
HR
Age
Sex
Histology
RS 15 gene
2.217
1.906
1.061
2.296
95% Conf. Int.
0.971
0.942
0.540
1.149
5.065
3.856
2.085
4.587
Wald p-value
0.059
0.073
0.86
0.019
C
Figure 1. (A) Univariate analysis to test for ability of the 15-gene
signature to segregate patients into high and low risk groups.
(B) Multivariate analysis indicates the 15-gene signature
outperforms other clinical factors as a prognostic indicator.
Conclusions
130 (71)
43 (24)
2 (1)
8 (4)
32 (52)
26 (42)
90 (49)
93 (51)
29 (47)
33 (53)
4 (6)
* Stage 2 or higher
Table 1. Demographics and summary of clinical factors for the
JBR.10 cohort and the UHN validation cohort studied here.
In addition to in silico validation of this 15 gene prognostic classifier in 4 large, public NSCLC
expression datasets, we have determined validation of its performance in this independent cohort.
Additional ongoing validation studies include the development of a qPCR-based assay to be
tested on both frozen tissue and FFPE samples from this UHN cohort.
Acknowledgements
Research funding for this study was provided by Med BioGene Inc., Canadian Institutes of Health
Research (CIHR) Proof of Principle Program (200711PPP), Canada Foundation for Innovation
(#12301 and #203383), and the Canada Research Chair Program.
D
HR
Age
Sex
Histology
RS 15 gene
0.813
1.729
3.34
2.075
95% Conf. Int.
0.339
0.672
1.111
0.832
-
1.952
4.446
10.037
5.174
Wald p-value
0.64
0.26
0.032
0.12
Figure 2. (A, C) Univariate analysis to study the
prognostic performance of the 15-gene classifier in
Stage 1 or 2 patients separately. (B, D) Multivariate
analysis indicates that magnitude of difference in
segregating low and high risk groups are similar for
Stage 1 and 2, although statistical significance was
obtained with Stage 1 but not with Stage 2.