Kathleen D. DanenbergResponse Genetics, Inc.

Predictive and Prognostic Markers for

Gastric Cancer

ERCC1

Why would a predictive test for

platinum efficacy be desirable?

Dank et al. Randomized phase III study comparing irinotecan combined with 5-fluorouracil and folinic acid to cisplatin combined with 5-fluorouracil in chemotherapy naive patients with advanced adenocarcinoma of the stomach or esophagogastric junction. Annals of Oncology. 2008; 19:1450-1457.

IF (n=170): irinotecan, folinic acid 5-fluorouracil;CF (n=163): cisplatin and 5-fluorouracil

Median TTPIF: 5.0 monthsCF: 4.2 months

Median OS:IF: 9.0 monthsCF: 8.7 months

Non-platin and platin therapy have similar outcomes

Irinotecan-5-FU Cisplatin-5-FU

Toxic deaths 0.6% 3%

Discontinuation for toxicity

10% 21%

Neutropenia gr. 3-4 25% 52%

Fever or infection 4.8% 10.2%

Thrombocytopenia 1.8% 11.7% (p=0.0003)

Diarrhea 21.6% 7.2%

Stomatitis 2.4% 16.9%

Neurotoxicity 5.4% 22.9%

Cardiovascular toxicity

2.4% 6.0%

…but non-platin treatment is less toxic: Comparative toxicity profiles

of CF and IF

Dank et al. Annals of Oncology. 2008; 19:1450-1457.

• “… Irinotecan/5-fluorouracil (IF) is a platinum-free regimen that has similar efficacy to cisplatin/5-fluorouracil (CF) but with improved tolerance.

• As such, IF could represent a potential platinum-free alternative backbone to be combined with new targeted agents to be explored for the treatment of metastatic gastric cancer.

Dank et al. Annals of Oncology. 2008; 19:1450-1457.

Conclusions

The platins react with DNA to form inter- and intra-strand

crosslinks

ERCC1 is part of the nucleotide excision repair complex that repairs platin crosslinks in DNA

30

40

50

60

70

80

90

% cell viability

cisplatin (4uM)

oxali (2 uM) carbo (20 uM)

control

siRNA

Pre-clinical studies show ERCC1 to be a direct determinant of

cisplatin efficacyERCC1 small interfering RNA expression reduces ERCC1 expression and sensitizes the cells to platinum-containing chemotherapeutic agents.

Youn et al. Oncogenic H-Ras Up-Regulates Expression of ERCC1 to Protect Cells from Platinum-Based Anticancer Agents Cancer Res 2004:64, 4849-4857.

ERCC1 gene expression in gastric cancer cells negatively correlates with

sensitivity to cisplatin.

ERCC1 mRNA expression levels and sensitivity to cisplatin in cells from malignant effusions collected from untreated gastric cancer patients (P= 0.014, r = 0.685).

Wang et al. ERCC1 and BRCA1 mRNA expression levels in metastatic malignant effusions is associated with chemosensitivity to cisplatin and/or docetaxel. BMC Cancer 2008;8:97.

Clinical Study

ERCC1 Threshold for Platin Sensitivity: Response Genetics

Scale

Percent Patients with Low

ERCC1 Benefit Ref

NSCLC: GILT (Platin Doublets) ERCC1<1.7 53 RR=53% Cobo et al JCO 2007

NSCLC: MADeIT (Platin Doublets) ERCC1<1.44 50

RR=44%, Increased Survival Sim et al JCO 2007

CRC: FOLFOX ERCC1<1.7 80

Increased Survival

and Response Shirota et al JCO 2001

CRC: FOLFOX Validation ERCC1<1.7 80

Increased Survival Lenz et al ASCO 2008

Gastric: 5-FU/Cis ERCC1<1.46 50Increased Survival Metzger et al JCO 1998

Gastric: FOLFOX ERCC1<1.79 64Increased Survival J Wei et al ASCO 2007

Gastric: FOLFOX ERCC1<2.2 80Increased Survival

J Wei et al British J of Cancer 2008

Gastric: Platin (S-1/Oxaliplatin) ERCC1<1.85 67

Increased RR and Survival

Matsubara et al British J of Cancer 2008

ERCC1 thresholds and benefit of low

ERCC1 from platin therapy

ERCC1 mRNA levels and response in gastric cancer patients receiving FP

Metzger R, et al. J Clin Oncol. 1998;16:309-316.

p=0.004 by Kruskal-Wallis test.

ER

CC

1 E

xp

ressio

n

20

16

12

8

4

0

Response No Response

ERCC1 mRNA levels and survival of advanced gastric cancer patients treated

with a FOLFOX regimen

Conclusion: “In patients with high mRNA levels of ERCC1, alternative chemotherapy regimens should be

considered.”

p<0.0001

Wei J et al. Br J Cancer. 2008;98:1398-402.

ERCC1 mRNA

Hazard ratio

Low (<0.47)

1

High (>0.47)

9.4 (p<0.0001)

Effect of ERCC1 protein expression on survival in FOLFOX chemotherapy of advanced gastric cancer

Overall survival curve according to ERCC1 expression measured by IHC (P

= 0.0396).

ERCC1 was the only significant independent prognostic factor impacted on OS (hazard ratio 1.91, P = 0.037).

Kwon et al. Ann Oncol. 2007;18:504-9.

Low ercc1

High ercc1

P value Hazard

Adjuvant therapy

47 mos. 7 mos. 0.01 2.77

Surgery only

12 mos. 33 mos. 0.038

Low ercc1

High ercc1

P value Hazard

Adjuvant therapy

undefined

13 mos. 0.007 2.68

Surgery only

21 mos. 43 mos. 0.004 0.18

Median RFS

Median OS

Yiu et al. ASCO 2010 abstract 29

Prediction of survival by ERCC1 expression in gastric cancer treated with surgery followed by FOLFOX or receiving surgery alone.

PELF = cisplatin (P), epirubicin (E), leucovorin (L), 5-fluorouracil (F)

Conclusion: “IHC studies for ERCC1 might be useful to predict the clinical outcome in MGC patients treated with PELF regimen.”

Median overall survival

low ERCC1 13 months

high ERCC1 9 months Log rank p=0.018

1-year survival rate

low ERCC1 62%

High ERCC1 21%

Natoli et al. J Clin Oncol 28, 2010 (suppl; abstr e14603)

ERCC1 expression and activity of PELF regimen as first-line treatment of metastatic gastric cancer.

Br J Cancer. 2008; 98: 832–839. Matsubara et al.

ERCC1expression and outcomes of advanced gastric cancer patients treated with cisplatin and S-1.

0.0

0.2

0.4

0.6

0.8

1.0

0 12 24 36 48 60 72 84 96

log-rank P < .001

Months since start of 1st-line chemotherapy

Pro

bab

ilit

y o

f su

rviv

al

Low ERCC1 and low DPD expression: median survival time, 15.5 monthsAny high expression: median survival time, 10.2 months

Impact of low ERCC1 and DPD on the outcomes of advanced gastric cancer.

Br J Cancer. 2008; 98: 832–839. Matsubara et al.

ASSIGNMENT

RANDOM

n=200, Endpoints: feasibility and increase of PFS

Genotypic Arm: ERCC1 Selection

High ERCC1: CPT11/docetaxel

Low ERCC1: FOLFOX

ASSIGNMENT

RANDOM

High ERCC1:FOLFOX

Low ERCC1: CPT11/docetaxel

SWOG proposed prospective trial using ERCC1to select CPT11/docetaxel or FOLFOX

EGFR expression

Chemotherapy with EGFR-targeted agents: KRAS and EGFR mutations are rare in gastric adenocarcinoma• Mammano E etal. Anticancer Res. 2006;26:3547-50.

– in 49 gastric adenocarcinomas, no specific EGFR gene mutations were detected.

• S.W. Han et al. Br J Cancer 2009;100:298-304. – In 38 gastric patients, no EGFR

amplification or K-ras mutations were observed.These findings suggest a priori that:

a)due to lack of EGFR mutations, gastric tumors will not be very sensitive to EGFR-directed TKI’s (e.g., gefitinib and erlotinib)

b) however, due to the lack of KRAS mutations, they may be sensitive to EGFR-directed antibodies

EGFR Tyrosine Kinase Inhibitors: Phase II, Adenocarcinoma

Gastric Number Patients

% Response

Dragovich (Erlotinib)

25 0%

Doi (Gefitinib) 75 1%

GE Junction

Ferry (Gefitinib)

27 11%

Janmaat (Gefitinib)

26 0%

Tew (Erlotinib) 17 0%

Dragovich(Erlotinib)

43Total: 7/113

9%6%

Doi 1036 Proc ASCO 22, 2003; Ferry Clin Can Res 132:5869; 2007 Janmaat JCO 24: 1612; 2006;Tew GI ASCO 2005; Dragovich JCO 24: 4922; 2006

Bouche O et al. J Clin Oncol 2004;22:4319-4328.

PFS and OS with “classical chemotherapy”

Survival LV5FU2 (n=45)

LV5FU2-cisplatin (n=44)

LV5FU2-irinotecan

(n=45)

OS, months 6.8 9.5 11.3

1-yr OS, % 31 43 43

PFS, months 3.2 4.9 6.9

Abbreviations: LV5FU2), leucovorin-5-FU; OS, overall survival; PFS, progression-free survival

Moehler et al.. Ann Oncol. 2010 Nov 30.: Patients with a complete response (CR) or partial response (PR) had significantly longer OS times and PFS times than patients with SD or PD.

Median PFS(months)

Median OS(months)

LV-5-FU-irinotecan

6.9 11.3

+ cetuximab 9.0 16.5

Median PFS(months)

Median OS(months)

CR + PR 10.6 19.1

SD + PD 6.0 12

p value 0.001 0.041

Moehler et al.. Ann Oncol. 2010 Nov 30.

PFS and OS are increased by addition of cetuximab

Bouche et al.. J Clin Oncol 2004;22:4319-4328

Predicting cetuximab activity

• Since KRAS mutations and EGFR mutations are rare in gastric tumors, can EGFR expression levels predict response to cetuximab?

-For EGFR (+) patients, both TTP (median 7.2 vs 5.0 months, P=0.020) and OS (not reached vs 7.6 months, P=0.013) were significantly longer after adjusting for clinical factors.S.W. Han et al Br J Cancer. 2009;100:298-304.

Phase II study and biomarker analysis of cetuximab combined with modified FOLFOX6 in

advancedgastric cancer.

Response

Overall (n=38)

50%

EGFR(+), low serum ligands (n=11)

100%

Remainder (n=27)

37%

-but tumor EGFR expression did not correlate with PFS

(log-rank P = 0.567) or OS (log-rank P = 0.663).

Response rate relationship to EGFR expression

EGFR(+) 19/26 (73%)

EGFR(-) 3/13 (23%)

EGFR (+) frequency

Responding tumors

16/19 (84%)

Non-responding tumors

10/20 (50%) P=0.041

Moehler et al. Ann Oncol. 2010 Nov 30.

Cetuximab with irinotecan, folinic acid and 5-FU as first-line treatment in advanced gastroesophageal cancer: a prospective multi-center biomarker-oriented phase II study.

-EGFR expression did not significantly correlate with ORRPinto et al. Phase II study of cetuximab in combination with FOLFIRI in patients with untreated advanced gastric or gastroesophageal junction adenocarcinoma (FOLCETUX study). Ann Oncol 2007;18:510-517.

Phase II study of cetuximab plus FOLFIRI in patients with untreated advanced gastric or GE junction adenocarcinoma (FOLCETUX study).

Discrepant results for EGFR expression level as a predictive factor for cetuximab therapy

EGFR associated with: Response Survival

Han et al yes yes

Moehler et al yes no

Pinto et al no -

HER2 status

HER2 inhibitors trastuzumab and lapatinib in gastric cancer

• ASCO 2008, Abstr 4526, Bang, et al.– Analysis of 2484 gastric cancer samples from

the Ph III ToGA trial– 21.9% HER2 positivity

• ASCO 2009, Abstr LBA 4509, ToGA Trial– Rand Ph III, HER2+ gastric cancer– 5-FU/capecitabine + cisplatin +/- trastuzumab– RR 47.3 vs. 34.5%, OS 13.5 vs. 11.1 mo (p =

0.0048)– HR 0.74 (0.60-0.91)– Practice changing!!!

• LOGIC Trial– Rand Ph III, HER 2+ gastric cancer– Capecitabine + oxaliplatin +/- lapatinib

The ToGA trial: Primary end point- OS

Time (months)

294290

246223

209185

173143

147117

11390

9064

7147

5632

4324

3016

2114

137

126

65

40

10

00

No. at risk

11.1 13.8

0.00.10.20.30.40.50.60.70.80.91.0

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36

Event

FC + TFC

Events

167182

HR

0.74

95% CI

0.60, 0.91

p value

0.0046

MedianOS

13.811.1

T, trastuzumab

Rüschoff et al. Virchows Arch. 2010 457:299-307.

HER2 assay by IHC for gastric cancer required a different set of guidelines

than for breast

Sources of HER2 Testing Variation with IHC

Pre-analytic    Time to fixation    Method of tissue processing    Time of fixation    Type of fixationAnalytic    Assay validation    Equipment calibration    Use of standardized laboratory

procedures    Training and competency assessment of staff    Type of antigen retrieval    Test reagents    Use of standardized control materials    Use of automated laboratory methods

Post-analytic    Interpretation criteria    Use of image analysis    Reporting elements    Quality assurance procedures        Laboratory accreditation        Proficiency testing        Pathologist competency

assessment

Wolff et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 2007;25:118-45.

Press et al. HER-2 Gene Amplification, HER-2 and Epidermal Growth Factor Receptor mRNA and Protein Expression, and Lapatinib Efficacy in Women with

Metastatic Breast Cancer. Clin Cancer Res 2008; 14: 7861

HER-2 mRNA expression by PCR correlates with HER-2 FISH (r=0.83)

and IHC (r=0.72)

Comparison of HER2 expression and amplification in primary breast tumors (T) and corresponding lymph node metastases (N) determined with IHC, FISH, and

quantitative RT-PCR

Vinatzer et al. Clin Cancer Res 2005;11:8348-8357

IHCHER2 expression scored as 0 and 1+ (=negative) or 2+ and 3+ (=positive) is indicated.

FISH analysis:red, HER2 signals; green, centromere 17 signals. +, specimens harboring a HER2 amplification; −, nonamplified specimens.

Quantitative RT-PCR:red line, the cutoff between high (scored as HER2 positive) and low relative expressions of HER2.

PCR quantitation of HER2 expression gives the same clinical information as IHC and FISH

Vinatzer et al. Clin Cancer Res 2005;11:8348-8357

Quantitative RT-PCR: -simple, cost-effective, -rapidly produces quantitative, numerical, and reproducible results. -easily amenable to standardization, insensitive to inter-observer variability-results are a number, which can be either above or below a predetermined threshold.

IHC-interpretation of IHC results is inherently difficult and time-consuming, requires experienced pathologists-is influenced by use of different antibodies, fixatives, staining protocols, and inter-observer variability.

FISH-is quantitative and reproducible but results are more difficult to interpret than those of quantitative RT-PCR.-time-consuming, and requires specialized expertise and equipment.

Her2 gene expression associated with OS in patients with metastatic gastric cancer treated with lapatinib

Overall Survivalby Her2 Expression Level

0%

20%

40%

60%

80%

100%

0 6 12 18 24Months After Registration

Above MedianBelow Median

N1717

Events1215

Median in months63

P = .005

Chang H et al,Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No. 18S (June 20 Supplement), 2007: 4647

Summary and conclusions

• ERCC1 mRNA expression appears to be a viable predictive marker for platin therapy.

• The jury is still out on EGFR expression as a predictive marker for cetuximab therapy.

• The IHC and FISH-based assay of HER2 has many issues so PCR should be investigated as an additional tool or as an alternative.