HER2 and adjuvant trastuzumab Soonmyung Paik, MD NSABP Foundation.

HER2 and adjuvant trastuzumab

Soonmyung Paik, MD

NSABP Foundation

Target = Degree of Benefit

NSABP B-14

N-, ER+ (over 10 fmole)

Placebo Tamoxifen

When positive, more ER the better response to

tamoxifen?

Median risk

High risk

Low risk

Amount of target (ER)

Expected prognosis of tamoxifen treated patients

ERpercent

Log Hazard Ratio (DRFI)

0 20 40 60 80 100

-2

-1

0

1

Test for non-linearity: p-value= 0.018681 Overall p-value= 0.00265

ER by IHC is not a linear prognosticator for tam pts

ER percent staining

HR

EstR1.1

Log Hazard Ratio (DRFI)

8 9 10 11 12 13

-2

0

2

4

Test for non-linearity: p-value= 0.676325 Overall p-value= 1e-006

ER by QRT-PCR is linear

HR

ER by QRT-PCR

EstR1.1

10-Year Distant Disease Recurrence Rate

6 8 10 12 14

0.0

0.1

0.2

0.3

0.4

0.5

0.6

B14 PlaceboB14 TAM

ER*tam interaction: p-value=0.0006

Degree of benefit

ER Expression by RT-PCR (relative to ref genes; log2)

Association of Quantitative ER Expression by QRT-PCR and Tamoxifen Benefit

Cut-off based on 10 fmole LBA

0 1 2 3 4 5

50

60

70

80

90

100

0 1 2 3 4 5

50

60

70

80

90

100

Disease-Free Survival

B-31 N9831

Years Years

AC->T+H 864 83AC->T 872 171 AC->T 807 90

AC->T+H 808 51

N Events N Events

HR=0.45, 2P=1x10-9 HR=0.55, 2P=0.0005

AC->T+H AC->T+H

AC->T AC->T

RR of ACTH/ACT for DFS (NSABP B-31)

0.00 0.25 0.50 0.75 1.00 1.25 1.50

FISH- & IHC <3 (174)

IHC <3 (299)

IHC 3+ (1488)

FISH- (207)

FISH+ (1588)

RR

Categories (N)

Interaction p=0.60 for FISHInteraction p=0.26 for IHC

Note: RR adjusted for ER and nodal status

CP1270832-10

Clinical Significance of Clinical Significance of Polysomy 17 in the HER2+ Polysomy 17 in the HER2+ NCCTGNCCTG N9831 Intergroup N9831 Intergroup

Adjuvant Trastuzumab TrialAdjuvant Trastuzumab Trial

Reinholz MM, Jenkins RB, Hillman D, Lingle WL, Davidson N, Martino P, Kaufman P, Kutteh L, and

Perez EA.

NCCTG, ECOG, SWOG, CALGBReinholz et al: SABCS 2007 (abstract #36)

CP1270832-11

Adjuvant Trastuzumab May Benefit Pts with Normal HER2 Breast Tumors (n=103)

40

50

60

70

80

90

100

0 1 2 3 4 5

Time (years)

Percent

AC_ T+H

HER2 FISH ratio < 2.0

AC_ T N Events DFS 3 yr 5 yrAC_ T 74 19 82.0 63.7AC_ T+H 82 11 91.0 80.8

40

50

60

70

80

90

100

0 1 2 3 4 5

Time (years)

Percent

AC_ T+H

AC_ T

IHC 0,1,2+ and HER2 FISH ratio <2.0

N Events DFS 3 yr 5 yrAC_ T 44 14 82.6 60.9AC_ T+H 59 9 90.2 81.2

40

50

60

70

80

90

100

0 1 2 3 4 5

Time (years)

Percent

AC_ T+H

AC_ T

IHC 0,1,2+

N Events DFS 3 yr 5 yrAC_ T 142 20 88.2 67.6AC_ T+H 191 19 89.1 82.3

p = 0.12p = 0.26

p = 0.14

CP1270832-12

Hazard Ratio of Benefit to Trastuzumab by HER2 FISH Ratio

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

11.0-15.0 (328)

< 2.0 (156)

2.0-5.0 (253)

5.0-8.0 (515)

8.0-11.0 (473)

≥ 15.0 (70)

p = 0.12

p = 0.05

p = 0.03

p = 0. 04

p = 0.96

p = 0.004

Ratio (N) p-value

Hazard ratioHazard ratio

CP1270832-13

DFS Based on Chromosome 17 Status and Treatment Arm

40

50

60

70

80

90

100

0 1 2 3 4 5

Time (years)

Percent

n17, AC_ T

n17, AC_ T+H

p17, AC_ T

p17, AC_ T+H

N Events DFS 3 yr 5 yrp17: AC_ T+H 442 29 89.5 88.9 n17: AC_ T+H 262 18 89.2 88.0 n17: AC_ T 282 51 74.9 67.9p17: AC_ T 423 48 83.2 77.6

p = 0.04

All had HER2 amplification

DFS for HER2 Normal (by IHC and FISH) Patients by Chromosome 17 Status

and Treatment Arm

40

50

60

70

80

90

100

0 1 2 3 4 5

Time (years)

Percent

n17, AC_ T+H

p17, AC_ T+H

p17, AC_ T

n17, AC_ T

N Events DFS 3 yr 5 yr

n17: AC_ T+H 34 4 93.7 84.1 p17: AC_ T+H 24 6 78.6 65.5

p17: AC_ T 13 3 83.3 75.0n17: AC_ T 33 13 76.7 54.5

B-31 FISH neg without polysomy 17

Years After Surgery

% D

isea

se-f

ree

0 1 2 3 4 5

02

04

06

08

01

00

Trt N EventsACT 80 19ACTH 69 7 RR=0.38 p=0.02667

FISH negative with Polysomy 17

Years After Surgery

% D

isea

se-f

ree

0 1 2 3 4 5

02

04

06

08

01

00

Trt N EventsACT 34 4ACTH 24 1 RR=0.32 p=0.30719

Disease-free-Survival According to Local Immunohistochemistry for HER2 and Central FISH for Patients Treated with Adjuvant Chemotherapy with/without Trastuzumab in the HERA Trial

_____________________McCaskill-Stevens W, Procter M, Azambuja E, Dafni U,

Leyland-Jones B, Ruschoff J, Dowsett M, Jordan B, Dolci S,Abramovitz M, Stoss O, Viale G, Gelber RD, Piccart-Gebhart

M,for the HERA Study Team

Prospective testing for

eligibility before randomization

Local IHC 3+ → central IHC 3+

Local IHC 2+ → central FISH+

Local FISH + → central FISH+

HER2 status testing

central FISH+ = FISH Ratio ≥ 2.0

Central FISH results are available for:

1131 pts. prospectively (eligibility screening)

940 pts. retrospectively (assay banked specimens)

2071 (61%) total out of the 3401 patients

p-value for interaction = 0.29 (FISH ratio ≥ 2 only)

•p-value for interaction = 0.38 (HER2 copy ≥ 4 only)

RR of ACTH/ACT for DFS in B31

0.00 0.25 0.50 0.75 1.00 1.25 1.50

FISH- IHC 0 (25) FISH- IHC 1+ 2+ (149)

FISH- IHC<3 (174)FISH- IHC 3+ (31)

FISH+ IHC<3 (125)FISH+ IHC 3+ (1380)

IHC 1+ (119)IHC 2+ (146)

IHC 1+2+ (265)IHC<3 (299)

IHC 3+ (1488)FISH- (207)

FISH+ (1588)

RR

Interaction p=0.60 for FISHInteraction p=0.26 for IHC

Interaction p=0.60 for FISHInteraction p=0.26 for IHC

Questions

• Testing issue??– Non-linearity of current tests

Questions

• Testing issue??– Non-linearity of current tests

• Artifact of subset analyses??

Questions

• Testing issue??– Non-linearity of current tests

• Artifact of subset analyses??

• Differences in mechanism of action of trastuzumab in metastatic versus adjuvant setting??

Chromosome 17q HER2 locus is not amplified in HER2 negative tumors in B-31

P-value cut off at 0.0001 for blue colorHeight indicates fold difference between HER2 pos and negative

Chr 17

Chr 1

Chromosome 17q HER2 locus is not amplified in HER2 negative tumors in B-31

P-value cut off at 0.0001 for blue colorHeight indicates fold difference between HER2 pos and negative

Chr 17

Chr 1

Adjusted P=0

HER2 mRNA (210930_s_at)

4

8

16

32

64

128

256

512

1024

HER2 neg HER2 pos

Adjusted P= 4.62507e-32

C17orf37 mRNA

8

16

32

64

128

256

512

1024

2048

HER2 neg HER2 pos

SKBR3 (1

0)

MCF7

(10)

B30 H

ER neg

(33)

B30 H

ER2 pos

(10)

both n

egat

ive

(154

)

FISH+

IHC- o

r FIS

H- IHC+ (1

21)

both p

ositiv

e (2

99)

1

2

4

8

16

32

64

128

256

512

HE

R2

mR

NA

Central HER2 assay negative cases have similar HER2 mRNA levels as in HER2 negative cases from B-30 trial

SKBR3 (1

0)

MCF7

(10)

B30 H

ER neg

(33)

B30 H

ER2 pos

(10)

both n

egat

ive

(154

)

FISH+

IHC- o

r FIS

H- IHC+ (1

21)

both p

ositiv

e (2

99)

1

2

4

8

16

32

64

128

256

512

HE

R2

mR

NA

Central HER2 assay negative cases have similar HER2 mRNA levels as in HER2 negative cases from B-30 trial

STEPP Analysis

Lower 1/3

Mid 1/3

Upper 1/3A gene on 8q is a predictor of degree of benefit from adjuvant trastuzumab

HER-2 total (H2T) HER-2 homodimerization (H22D)

The Principle of VeraTag Technology - A Novel Proximity-based Assay

• A monoclonal antibody specific for a unique epitope of HER2 is conjugated to a fluorescein VeraTag reporter (Pro11) or a molecular scissors (S) by means of a cleavable tether.

• The molecular scissors liberates singlet O2 upon irradiation with red light.

HER-2 total (H2T) HER-2 homodimerization (H22D)

The Principle of VeraTag Technology - A Novel Proximity-based Assay

• The free radicals cleave all thioether bonds within approximately 200 nM, releasing the “VeraTag reporter.”

• The signal (Pro11) can then be collected and analyzed on a capillary electrophoresis array.

VeraTag HER2 Total protein assay in FFPE

VeraTag H2TAssay

Performance Characteristics

The H2T and H22D assays quantitate HER2 expression and HER2:HER2 dimers over a 3-log dynamic range.

Accuracy, precision, sensitivity, and reproducibility.

CLIA-validated and CAP-certified.

Similar results for H22D.

Accuracy H2T Sensitivity H2T

Precision H2T Reproducibility H2T

ELISA:MDA MB 435: ~2 ng/mg

Replicates: 15 each(105 pairwise comparisons)100% within 1.7-fold>95% within 1.45-fold

55 replicates96% within 2-fold

FFPE breast tumorsFFPE cell lines

FFPE cell linesFFPE cell lines

Correlation of HER2 expression by VeraTagand IHC in 170 breast tumors (FFPE)

0 1+ 2+ 3+0.01

0.1

1

10

100

IHC

IHC FISH

Comparison of HER2 assessments by VeraTag with IHC (N=170) and FISH (N=64) in FFPE breast tumors

Correlation of HER2 expression by VeraTagwith centromere-corrected FISH copy number

in the Bordet cohort

0 500 1000 1500 2000 25000123456789

101112131415161718

r = 0.18p = 0.15

HER2 expression VeraTag

0 10 20 30 40 500

0.2

0.4

0.6

0.8

1.0

≥ median H2T*, n=29< median H2T, n=29

HR=0.48p=0.01

4.0 12.8

Time (months)

Pro

po

rtio

n P

rog

res

sio

n F

ree

Time-to-progression (TTP)

0 10 20 30 40 500

0.2

0.4

0.6

0.8

1.0≥ median H22D, n=29< median H22D, n=29

HR=0.63

p=0.11

4.5 12.0

Time (months)

Pro

po

rtio

n P

rog

res

sio

n F

ree

Time-to-progression (TTP)

0 20 40 60 800

0.2

0.4

0.6

0.8

1.0≥ median H2T, n=29< median H2T, n=29

HR=0.64p=0.17

27.331.8

Time (months)

Ov

era

ll S

urv

iva

l

Overall survival (OS)

0 20 40 60 800

0.2

0.4

0.6

0.8

1.0≥ median H22D, n=29< median H22D, n=29

HR=0.81p=0.49

27.331.8

Time (months)

Ov

era

ll S

urv

iva

l

Overall survival (OS)

H2T H22D

H2T H22D

Figure 3: Univariate Kaplan-Meier (KM) analyses examining the impact of Trastuzumab treatment on time-to-progression (TTP) and overall survival (OS) in patients with high vs. low HER2 expression (H2T, left) and HER2 homodimer (H22D, right) levels.

Summary

• Three large adjuvant studies failed to demonstrate association between the amount of HER2 gene copy or protein expression and degree of benefit from trastuzumab added to chemotherapy

• HER2 testing method is evolving to more quantitative tests with improved linearity of prediction of trastuzumab response