A PROGNOSTIC AND PREDICTIVE 15-GENE SIGNATURE FOR NSCLC IDENTIFIED BY MARSHA ALGORITHM

C.Q. Zhu, K. Ding, and M. S. Tsao Ontario Cancer Institute-Princess Margaret Hospital, University of Toronto

One in 3 women and one in 2 men in the United States will develop cancer in his or her lifetime

Estimated death

Estimated new cases

Cancer statistics 2009, CA Cancer J. 2009

Classification of lung cancer

Small cell lung cancer (SCLC) ~21%

Non-small cell lung cancer (NSCLC)

Adenocarcinoma (ADC) 40%

Squamous cell carcinoma (SQCC) 30%

Large cell carcinoma (LCUC) 9%

5-year survival rate:15%

Early stages Advanced Cancer

statistics, CA

Cancer J 2009

Survival differs even Within individual stage

30-40% of stage I NSCLC will relapse

The 5-year survival advantage of

adjuvant chemotherapy is minimal,

from 4%-15% Trial Stage No. of

Patients

Agents Hazard

ratio

P Value

IALT I-III 1867 Cisplatin/

Vinca or VP16 0.86 <0.03

JBR.10 IB-II 482 Cisplatin/

vinorelbine 0.7 0.01

ANITA IB-IIIA 840 Cisplatin/

vinorelbine 0.79 0.013

LACE

(meta-

analysis)

I-III 4584 Platinum

doublets 0.89 0.0004

JLCRG I 978 Uracil-Tegafur 0.71 0.047

Adapted from Wakelee HA, et al. Clin Lung

Cancer July 2006

No Benefit for Adjuvant Chemotherapy

in Stage I Patients

Trial Stage Hazard ratio 95% CI

ALPI I 0.97 0.71-1.33

IALT I 0.97 Not available

ANITA IB 1.10 0.76-1.57

JBR10 IB 0.94 Not available

CALGB 9633

IB 0.80 0.60-1.07

Prognostic Gene Signatures in NSCLC by

Microarray or RT-QPCR

Tumor Type Gene Set

Raponi/Beer (2006) SQCC/ADC 50/47

Potti (2006) NSCLC Metagene sets

Lu (2006) NSCLC 64

Larsen (2007) ADC 54

Larsen (2007) SQCC 111

Chen (2007) NSCLC 5

Bianchi (2007) NSCLC 10

Lau (2007) NSCLC 3

Director’s Challenge 08 ADC Multiple sets

None of these was tested as

predictive marker

JBR.10 - Study Design

R

A

N

D

O

M

I

S

E

Stratified by

Nodal

* N0

* N1

Ras

* Neg

* Pos

* UNK

T

I

S

S

U

E

R

E

G

I

S

T

E

R

Observation

Only

Cisplatin

Vinorelbine

BR.10 Tumor Bank

Consort diagram of the JBR. 10 patients used for this microarray study

Microarray study is representative of the

entire JBR.10 cohort

Winton T, et al NEJM 2005,

352 2589-97

• To establish a prognostic gene expression signature that can classify surgery only patients into risk groups with significantly different survival outcomes

• To test the predictive value of this signature for benefit from adjuvant chemotherapy

Study Goals

Methods Expression Profiling by Affymetrix U133A microarrays

Data was pre-processed using RMA (Robustic Multi-array average)

Batch difference was adjusted by the distance-weighted discrimination algorithm (DWD)

Candidate genes were pre-selected by univariate survival analysis at p<0.005 (n=172)

Expression level was standardized (z-score) and a single risk score was introduced in the Cox model

Risk score was the product of z score weighted by its association estimate in the univariate analysis

Goodness-of-fit (R-square), p value in the model and p value in the univariate analysis model was the selection criteria

Software: SAS v9.1

Effect of batch difference

DWD (distance-weighted discrimination) was used to homogenize the batch difference

Prognostic Gene Set Selection

Signature is Prognostic in Observation but Not in

Chemotherapy Treated Patients JBR.10, chemotherapy (n=71)

High risk Low risk

Perc

en

tag

e

0

20

40

60

80

100

0 3 6 Time (Years)

9

35 36

28 25

19 15

3 1

HR 1.15 (95% CI 0.56-2.37)

p=0.6942

JBR.10, observation (n=62)

Low Risk High Risk

Pe

rce

nta

ge

0

20

40

60

80

100

0

31 31

3

28 9

6 Time (Years)

20 3

9

1 0

High risk Low risk

HR 15.02 (95% CI 5.12- 44.04)

p<0.0001

# at Risk

15-gene Signature is Prognostic in Stage I

and Stage II Patients

HR 13.32 (95% CI 2.86-62.11)

p<0.0001

Perc

en

tag

e

Stage II (n=28)

0

20

40

60

80

100

0

11 17

3

9 3

6 Time (Years)

7 1

9

0 0

High risk Low risk

HR 13.47 (95% CI 3.00-60.43)

p<0.0001

Low Risk

High Risk

Stage IB (n=34)

Perc

en

tag

e

0

20

40

60

80

100

0

20 14

3

19 6

6 Time (Years)

13 2

9

1 0

High risk Low risk

No. at Risk

Validation of the signature*

Zhu CQ et al, 2010, JCO, 28:4417-24

*adjusted for stage,histology (NSCLC), sex and age

Validation of 15-gene Signature in Director’s

Challenge Stage I-II Patients

HR 3.21 (95% CI 1.69-6.11)

p=0.0002

Low Risk High Risk

Pe

rce

nta

ge

0

20

40

60

80

100

0

87

82

20

81

69

40

Time (Months) 65

48

60

49

28

High risk Low risk

No. at Risk

DCC, no adjuvant (n=169)

HR 1.10 (95% CI 0.47-2.53)

p=0.8294

Perc

en

tag

e

0

20

40

60

80

100

0

21 20

20

18 18

40

Time (Months) 13 10

60

8 6

High risk Low risk

DCC, adjuvant chemo (n=41)

Interaction p = 0.0001

HR 0.33 (95% CI 0.17-0.63)

p=0.0005

Observ. Chemo

31

36

9

25

3

15

0

1

Perc

en

tag

e

0

20

40

60

80

100

0 3 6

Time (Years) 9

Chemo Observation

No. at Risk

JBR.10, high risk (n=67)

Chemotherapy Benefits High Risk but Not

Low Risk Patients

HR 3.67 (95% CI 1.22-11.06)

p=0.0133

Pe

rce

nta

ge

0

20

40

60

80

100

0

31 35

3

28 28

6 Time (Years)

20 19

9

1 3

Chemo Observation

JBR.10, low risk (n=66)

15 Genes in the Signature

Probe Set Gene Symbol

Gene Name

201243_s_at ATP1B1 ATPase, Na+/K+ transporting, beta 1 polypeptide

203147_s_at TRIM14 Tripartite motif-containing 14

221591_s_at FAM64A Family with sequence similarity 64, member A

218881_s_at FOSL2 FOS-like antigen 2

202814_s_at HEXIM1 Hexamethylene bis-acetamide inducible 1

204179_at MB Myoglobin

204584_at L1CAM L1 cell adhesion molecule

202707_at UMPS Uridine monophosphate synthetase

208399_s_at EDN3 Endothelin 3

203001_s_at STMN2 Stathmin-like 2

210016_at MYT1L Myelin transcription factor 1-like

202490_at IKBKAP Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein

206426_at MLANA Melan-A

205386_s_at MDM2 Mdm2, transformed 3T3 cell double minute 2

219171_s_at ZNF236 Zinc finger protein 236

Further validation of the signature in UHN183 (Medbiogene Inc)

Clinical characteristics UHN183

n=183 (%)

Age Median 70 (40-88)

<65y 63 (33)

>=65y 123 (67)

Sex Women 84 (46)

Men 99 (54)

Stage 1A 49 (27)

1B 80 (44)

2A 9 (5)

2B 45 (25)

Histology ADC 130 (70)

SQCC 43 (24)

ADSQ 2 (1)

LCUC 8 (4)

Signature is prognostic

Univariate analysis in separate

Stage 1 and 2

Conclusions

A novel 15-gene prognostic signature may be

used to substaging NSCLC

The signature may identify early stage non-small

cell lung cancer patients who are most likely to

benefit from chemotherapy after complete

surgical resection