molecular diversity of human breast cancers...stability of the target, e.g. fixation artifacts in...

Molecular Diversity of Human

Breast Cancers: Biologic and Therapeutic Implications

BRCA1

Molecular Diversity of Human

Breast Cancers: Biologic and Therapeutic Implications

HER2

HER-2/neu Program at UCLA

Clinical Material

(Tumor Specimens)

Clinical Trials

(Current & Past Studies)(Current & Past Studies)

Therapeutic Model

(Cell Line and Animal Data)

2/neu Program at UCLA

Molecular Studies

(DNA, RNA,

Protein Analyses)Clinical Trials

(Current & Past Studies)(Current & Past Studies)Clinical Data

(Patient Information)

Basic Science

Hypothesis Testing


HER-2/neu Gene is Amplified in Human Breast

Cancers

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Slamon DJ, et al. Science 235: 177-182, 1987

Gene is Amplified in Human Breast

Cancers

QuickTime™ and a










Clinical Material

(Tumor Specimens)

Clinical Trials


Therapeutic Model



Molecular Studies

(DNA Analyses)

Clinical Trials



Basic Science

Hypothesis Testing


The HER2 AlterationThe HER2 AlterationThe HER2 AlterationThe HER2 Alteration

Southern

Northern

IHC

Western

Slamon et al. Science 1989

HER

Amplification

Median Survival from First Diagnosis

HER

HER

Slamon et al, 1987

HER-2 Oncogene

Amplification

HER-2 Oncoprotein

Overexpression

Breast Cancer

Shortened Survival


HER-2 overexpressing 3 yrs

HER-2 normal 6 - 7 yrs


in a Node-Positive Cohort

♦Median survival in the HER2 normal (non

amplified) cohort = 6.8 years

♦Median survival in the HER2 amplified cohort = ♦Median survival in the HER2 amplified cohort =

<3 years

♦HER2 amplification was an independent prognostic

variable in multi-variate analyses using all standard

prognostic variables


Positive Cohort

Median survival in the HER2 normal (non-

amplified) cohort = 6.8 years

Median survival in the HER2 amplified cohort = Median survival in the HER2 amplified cohort =

HER2 amplification was an independent prognostic

variate analyses using all standard

CONTROVERSIES

♦It is NOT amplified at a rate of ~25% but much

less frequently (~10-15%)

♦There is no association between amplification

and clinical outcome

CONTROVERSIES

It is NOT amplified at a rate of ~25% but much

15%)

There is no association between amplification


Clinical Material

(Tumor Specimens)

Clinical Trials


Therapeutic Model



Molecular Studies

(DNA, RNA,




Basic Science

Hypothesis Testing


Breast Cancer Subtypes are associated

with disease outcome

Breast Cancer Subtypes are associated

with disease outcome

Sørlie et. al. PNAS 2003


Clinical Material

(Tumor Specimens)

Clinical Trials


Therapeutic Model



Molecular Studies

(DNA, RNA,




Basic Science

Hypothesis Testing


Target Validation Target Validation - A

Human Breast Cancer Cells

MCF-7

Single copy

Low Expressor

Transfect

HER-2/neu

Human Ovarian Cancer CellsHuman Ovarian Cancer Cells

CaOv-3

Single copy

Low Expressor

Transfect

HER-2/neu

*Consistent results in 9 additional Breast & Ovarian Cancer Cell Lines

Human Breast Cancer Cells

Transfect

2/neu

MCF-7*

Multiple copy

High Expressor

Human Ovarian Cancer CellsHuman Ovarian Cancer Cells

Transfect

2/neu

CaOv-3*

Multiple copy

High Expressor

*Consistent results in 9 additional Breast & Ovarian Cancer Cell Lines

Immunohistochemistry

MCF 7

CaOV 3

+ Control

Immunohistochemistry

+ HER-2/neu

Engineered HER-2 Over-expression in MCFIncreased Proliferation and Decreased Contact Inhibition

Anchorage-Independent Growth

MCF-7 CN

MCF-7 H2

expression in MCF-7 cellsIncreased Proliferation and Decreased Contact Inhibition

Growth on Plastic

2 4 6 8

200

600

1000

1400

MCF-7 H2

MCF-7 CN

days

Number of cells x 10

3 ••

9

MCFMCF--7 CN7 CN MCFMCF--7 H27 H2

Biologic Effects of HER

Overexpression in Human Breast

Cancer Cells

HER2-

Breast Cancer

HER2+

Breast Cancer HER-2

Breast Cancer

Cell Lines

Breast Cancer

Cell Lines

HER-2

Transfection

Biologic Effects of HER-2/neu

Overexpression in Human Breast

Cancer Cells

↑ DNA Synthesis

HER2+

Breast Cancer

↑ Cell Growth

↑ Growth inBreast Cancer

Cell Lines

↑ Growth in

Soft Agar

↑ Tumorigenicity

↑ Metastatic

Potential

E2 Response,

↑ Tam Resist.


Clinical Material

(Tumor Specimens)

Clinical Trials


Therapeutic Model



Molecular Studies

(DNA, RNA,




Basic Science

Hypothesis Testing


Target Validation Target Validation - B

70

80

90

100

Dose-dependent anti- proliferative

HER2- overexpressing breast carcinoma cells

% Cell Proliferation

642040

50

60

70

4D5 ( ug /ml)

% Cell Proliferation

Pegram M, Hsu S, Lewis G, et al., Oncogene. 1999 Apr 1;18(13):2241

proliferative effects of 4D5 against

breast carcinoma cells in vitro

12108

/ml) Pegram M, Slamon D

Semin Oncol 2000 Oct;27(5 Suppl 9):13-9 Pegram M, Hsu S, Lewis G, et al., Oncogene. 1999 Apr 1;18(13):2241-51.

Preclinical Impact of Trastuzumab

on Tumor GrowthTumor volume (mm

3)

1500

2000

Control

Trastuzumab

Effect of Trastuzumab Treatment on HER2+ Breast Cancer Xenografts

Pietras et al. Oncogene. 1998;17:2235.

Tumor volume (mm

Treatment day

500

1000

0 10 20 300

Trastuzumab

Preclinical Impact of Trastuzumab

on Tumor Growth

Trastuzumab

Effect of Trastuzumab Treatment on HER2+ Breast Cancer Xenografts

Treatment day

30 40 50 60 70

Trastuzumab

Trastuzumab

withdrawn

51 Human

Breast Cell Lines

25

Luminal

Non

10

ER positive

Normal HER-2

9

ER positive

HER-2 amplified

6

ER negative

HER-2 amplified

51 Human

Breast Cell Lines

26

Non-luminal

13

Basal/Progenitor

9

Mesenchymal4

Non-malignant

1 HER-2

Amplified

1 HER-2

Amplified


Clinical Material

(Tumor Specimens)

Clinical Trials


Therapeutic Model



Molecular Studies

(DNA, RNA,




Basic Science

Hypothesis Testing


CALGB 9741

Interim Analyses

Proportion Disease-Free

0.4

0.6

0.8

1.0

By Density

Disease-Free Survival

85 vs 81% (P=0.0072)

Years From Study Entry

Proportion Disease-Free

0 1 2 3 4

0.0

0.2

0.4

q 2 wksq 3 wks

N= 988N= 985

Events= 136Events= 179

85 vs 81% (P=0.0072)

N = 1973; Median F/U = 36 mos

CALGB 9741

Analyses

Proportion Surviving

0.4

0.6

0.8

1.0

By Density

Overall Survival

92 vs 90% (P=0.014)

Years From Study EntryProportion Surviving

0 1 2 3 4

0.0

0.2

0.4

q 2 wksq 3 wks

N= 988N= 985

Events= 75Events= 107

92 vs 90% (P=0.014)

N = 1973; Median F/U = 36 mos

Overall Survival (ITT)1.0

0.8

0.6

Cumulative Probability

0.4

0.2

0.0

0 6 12 18 24 30

Cumulative Probability

N Events HR

Stratified Log-Rank

TAC 745 91 0.70

FAC 746 130

Survival Time (months)

Overall Survival (ITT)

FAC

TAC 87%

81%

30 36 42 48 54 60 66

P-value

Rank

.0080

Survival Time (months)

The HER2 AlterationThe HER2 AlterationThe HER2 AlterationThe HER2 Alteration

Southern

Northern

IHC

Western

Slamon et al. Science 1989

80

90

100 100

Disease-Free Survival

B-31

ACAC��THTH

AC��T

74%

87%85%

0 1 2 3 4 5

50

60

70

AC��TH 864 83AC�T 872 171

N Events

HR=0.45, 2P=1x10-9

74%

66%

Years From Randomization

%

80

90

100

Free Survival

N9831

ACAC��THTH

AC��T

78%

87%86%

0 1 2 3 4 5

50

60

70

AC��T 807 90AC��TH 808 51

N Events

HR=0.55, 2P=0.0005

68%

Years From Randomization

Lessons from the HER2 Story

♦1.) Target Identification

♦2.) Target Validation

♦3.) Preclinical Confirmation♦3.) Preclinical Confirmation

♦4.) Determintion of Potential Usage Preclinically

♦5.) Clinical Translation -

♦6.) Clinical Optimization

Lessons from the HER2 Story

3.) Preclinical Confirmation3.) Preclinical Confirmation

4.) Determintion of Potential Usage Preclinically

- Proof of Concept

6.) Clinical Optimization

Clinical Significance of HER2

Testing of Primary Breast Cancers

HER2 geneHER2 geneamplification (FISH)

Why test for HER2?

• HER2 is recognized as an important predictive and prognostic factor

• HER2 overexpression continues throughout the course of the disease and drives tumor growth4

• HER2 positivity is required for consideration of HER2Herceptin® (trastuzumab) and Lapatinib (Tykerb) therapy

1. Witton et al. J Pathol. 2003;200:290; 2. Ross et al. Oncologist. 2003;8:307; 3. Konecny et al. Clin Cancer Res. 2004;10:1706; 4. Simon et al. J Natl Cancer Inst5. Herceptin® (trastuzumab) PI, February 2005.

HER2 protein

Clinical Significance of HER2

Testing of Primary Breast Cancers

HER2 proteinoverexpression (IHC)

Why test for HER2?

HER2 is recognized as an important predictive and prognostic factor1-3

HER2 overexpression continues throughout the course of the disease

HER2 positivity is required for consideration of HER2-targeted and Lapatinib (Tykerb) therapy5

. 2003;8:307; J Natl Cancer Inst. 2001;93:1141;

Testing Issues

♦Integrity of the macromolecule being analyzed DNA, RNA, or protein

♦Acurracy of the reagent - variability of the antibodies

♦Stability of the target, e.g. fixation artifacts in proteins antigenic sites and recognition that the preantigenic sites and recognition that the precannot be controlled

♦Accuracy of the testing method

♦Heterogeneity of the sample being tested

Testing Issues

Integrity of the macromolecule being analyzed - degradation of

variability of the antibodies

Stability of the target, e.g. fixation artifacts in proteins - altering antigenic sites and recognition that the pre-analytic phase antigenic sites and recognition that the pre-analytic phase

Heterogeneity of the sample being tested

HERHER--2 Gene Amplification is Responsible for 2 Gene Amplification is Responsible for “Pathologic/Pathogenic”Overexpression“Pathologic/Pathogenic”Overexpression

HER2 Biology

2 Gene Amplification is Responsible for 2 Gene Amplification is Responsible for “Pathologic/Pathogenic”Overexpression“Pathologic/Pathogenic”Overexpression

Molecularly Characterized Cohort

♦A cohort of 189 snap-frozen breast cancer specimens of sufficient size to allow the simultaneous extraction of DNA, RNA and protein same specimen

♦Confirmed intact integrity of the DNA, RNA and protein degradation of the macromolecules PRIOR to commencing analyses

♦Formalin-fixed/paraffin-embedded tissue available from the exact same specimens

♦Serves as the “REFERENCE COHORT”

Molecularly Characterized Cohort

frozen breast cancer specimens of sufficient size to allow the simultaneous extraction of DNA, RNA and protein - all from the

of the DNA, RNA and protein - I.e. no degradation of the macromolecules PRIOR to commencing analyses

embedded tissue available from the exact same

“REFERENCE COHORT” for all of our subsequent studies

HER-2 Molecularly Characterized Samples

COHORT” assembled in Multi

Evaluation of Diagnostic Methods

Amplification Level :> 10

5 - 10

2 - 5

1Southern

Frozen IHC

Northern

Western

2 Molecularly Characterized Samples “REFERENCE

assembled in Multi-Tumor Blocks

Multi-Tumor Paraffin-Embedded

Tissue Block

Testing Issues






Testing Issues





Percent of Breast Cancers in Various Expression Categories Identified by Immunostaining with 28

Different Antibodies.

60

80

100

120

0

20

40

Percent of Breast Cancers in Various Expression Categories Identified by Immunostaining with 28

Different Antibodies.

>5-fold

2 to 5-fold

1High

1Low

Frozen IHC

Amplification Level :

Northern

Western

> 10

5 - 10

2 - 5

1

Southern

Testing Issues






Testing Issues





Fixation and Paraffin Embedding Result in

Decreased Antigenicity2 to 5-fold Amplified with Overexpressed

Immunohistochemistry in Archival Tissue Samples

Slamon et al., Science 244: 707

Fixation and Paraffin Embedding Result in

Decreased Antigenicity2 to 5-fold Amplified and Overexpressed

Immunohistochemistry in Archival Tissue Samples

244: 707-712, 1989

Enter - “Antigen Retrieval”“Antigen Retrieval”

Schematic Summary of HER-2 Assay Results: Concordance

with Known HER2-Positive Status

IHC: HER2 in Frozen Tissue

IHC: HER2 in Paraffin Tissue

FISH: Paraffin Tissue

Estimated Concordance or Accuracy*

0 25% 50% 75%

*Based on Results from Science, 1989; Cancer Res

Oncology, 1997; Journal of Clinical Oncology, 2002;

IHC: HER2 in Paraffin Tissue

2 Assay Results: Concordance

Positive Status “REFERENCE COHORT”

IHC: HER2 in Frozen Tissue 99%

84%

With Ag Retrieval

Estimated Concordance or Accuracy*

75% 100% 125%

Cancer Res., 1993; Cancer Res., 1994; Journal of Clinical

, 2002; Clinical Cancer Res., 2005.

97%

Testing Issues






Testing Issues





Comparison of Six Different HER

Molecularly Characterized

Breast Cancers Specimens

Press et al., Journal of Clinical Oncology 20: 3095

Comparison of Six Different HER-2 Assays in

Molecularly Characterized “REFERENCE COHORT”

Breast Cancers Specimens


Northern

> 10

5 - 10

2 - 5

1

Southern

20: 3095-3105, 2002.

Frozen IHC

Western

Comparison of FISH vs. IHCComparison of FISH vs. IHC

Results

1:1 population

-

FISH

0

207

Concordance Study: Two things to note

+ 7

3%Amplification rateAmplification rate

Breast Cancer Research and Treatment 93: 3-11, 2005.

Overall concordance between FISH and IHC results was 82% (95% CI; 78

3.7%

67

CTA-IHC

3+

21

2+1+

28

Concordance Study: Two things to note

67

176

89%

21

24%

2

7%

11, 2005.

Overall concordance between FISH and IHC results was 82% (95% CI; 78–85%) ( p < 0.0004).

3.7%

Results

-

0

538

BCIRG Central Laboratory Concordance Study

-

+

Central FISH

538

20

4%Amplification rateAmplification rate

Press et al., Clinical Cancer Research, 11: 6598-6607, 2005.

Overall concordance between FISH and IHC results was 79% (95% CI; 77

4.3%

67

Local IHC

3+

90

2+1+

230

BCIRG Central Laboratory Concordance Study

73%67

316

90

78%

33

17%

15

230

6%4%

73%

23%

N = 1407

Overall concordance between FISH and IHC results was 79% (95% CI; 77–81%).

4.3%

Arguments Against Screening with IHC

and Reflex Testing with FISH

♦Between 9 - 17% of women with HER

are IHC-negative (0/1+) : definite false negatives

♦Between 8 and 22% of women with IHC 3+ do not ♦Between 8 and 22% of women with IHC 3+ do not

have the HER-2/neu alteration (gene amplification by

FISH) : ? false positives.

♦Trastuzumab (Herceptin) and lapatinib are expensive

therapeutics; errors in testing are costly.

♦Women deserve the most accurate testing methods.

Arguments Against Screening with IHC

and Reflex Testing with FISH

17% of women with HER-2/neu alteration

definite false negatives.

Between 8 and 22% of women with IHC 3+ do not Between 8 and 22% of women with IHC 3+ do not

alteration (gene amplification by

Trastuzumab (Herceptin) and lapatinib are expensive

therapeutics; errors in testing are costly.

Women deserve the most accurate testing methods.

Response Rates in the Genentech

H0649 Pivotal Clinical Trial of

Trastuzumab______________________________________________________

FISH Ratio Non-Resp (n) Responder

<2.0 36 0 0%* 0%, 10%

2.0 - 6.0 75 11 13%*2.0 - 6.0 75 11 13%*

>6.0 65 22 25%

______________________________________________________

FISH results obtained for 209 of the 222 (94%) women entered in trial.

Fisher’s exact test, overall p=0.0005; *p= 0.033, #

Response Rates in the Genentech

H0649 Pivotal Clinical Trial of

Trastuzumab______________________________________________________

Responder (n) Rate (%) 95% CI

<2.0 36 0 0%* 0%, 10%

6.0 75 11 13%*# 7%, 22%6.0 75 11 13%*# 7%, 22%

>6.0 65 22 25% # 17%, 36%

______________________________________________________

FISH results obtained for 209 of the 222 (94%) women entered in trial.

# p= 0.052.

Response Rates in the Genentech H0650

Clinical Trial of Trastuzumab

_______________________________________________________

FISH Ratio Non-Resp (n) Responder

<2.0 28 1 3%* 0.1%, 18%

2.0 - 6.0 24 10 29%*

>6.0 31 18 37%

_______________________________________________________FISH results obtained for 112 of the 114 (98%) women entered in trial.

Fisher’s exact test: overall p-value = 0.002; *p=0.008,

Response Rates in the Genentech H0650

Clinical Trial of Trastuzumab

_______________________________________________________

Responder (n) Rate (%) 95% CI

<2.0 28 1 3%* 0.1%, 18%

6.0 24 10 29%*# 15%, 47%

>6.0 31 18 37% # 23%, 52%

_______________________________________________________FISH results obtained for 112 of the 114 (98%) women entered in trial.

value = 0.002; *p=0.008, #p=0.64.

Testing Issues






Testing Issues





Correlation of HER-2 Gene Amplification with Overexpression


> 10

5 -10

2 -5

1

HER2 Biology

4.4 kb -

12.5 kb -

27% 63%

4.4 kb -

p185 -

Slamon et al., Science 244: 707-712, 1989

2 Gene Amplification with Overexpression

Northern

1

Southern

Frozen

IHC

Northern

Western

10%63% % Women

“Single Copy” Overexpression

HER2 Biology

H & E

Slamon et al., Science 244:707-712, 1989; Pauletti et al., Oncogene 13:63

“Single Copy” Overexpression

IHC

FISHFISH

712, 1989; Pauletti et al., Oncogene 13:63-72, 1996

HER-2 Gene Assessment by FISH

FISH

< 2.0 Not Amplified(FISH-)

2 Gene Assessment by FISH

≥ 2.0 Amplified(FISH+)

Results

Pos (+)

Gene Amplification bySouthern or Dot blot Hybridization

Ampl

49

Comparison: Solid Matrix Blotting Methods (frozen tissues)

with Fluorescence In Situ Hybridization (FISH) (paraffin

Pos (+)

Neg (-)

FISH

49

1

Press et al., Journal of Clinical Oncology 15:2894

Sensitivity = 98%, Specificity = 100%.

ResultsGene Amplification by

Southern or Dot blot Hybridization

Not Ampl

0

Comparison: Solid Matrix Blotting Methods (frozen tissues) REFERENCE COHORT

with Fluorescence In Situ Hybridization (FISH) (paraffin-embedded tissues)

90

0

15:2894-2904, 1997.

N = 140

ASCO/CAP Guidelines

♦New guidelines - A case is indeterminate i.e. may be called amplified, normal or equivocal if the ratio is between 1.8 instead of the FDA approved definition of >2.0 = amplified.

♦HAVE THEY DISCOVERED NEW FUNDEMENTAL BIOLOGY SINCE WATSON & CRICK OR THE KNOWN FIDELITY OF SINCE WATSON & CRICK OR THE KNOWN FIDELITY OF DNA REPLICATION DURING THE CELL CYCLE ???

♦The consequence of this change has easier. Instead, non-amplifed cases are now sometimes called amplified. Conversely amplified cases may now be called nonamplified and hence either not receive the drug or incorrectly be classified as negative cases which benefit from trastuzumab or lapatinib

ASCO/CAP Guidelines

A case is indeterminate i.e. may be called amplified, normal or equivocal if the ratio is between 1.8 -2.2 instead of the FDA approved definition of >2.0 = amplified.

HAVE THEY DISCOVERED NEW FUNDEMENTAL BIOLOGY SINCE WATSON & CRICK OR THE KNOWN FIDELITY OF SINCE WATSON & CRICK OR THE KNOWN FIDELITY OF DNA REPLICATION DURING THE CELL CYCLE ???

The consequence of this change has not been to make things amplifed cases are now sometimes called

amplified. Conversely amplified cases may now be called non-amplified and hence either not receive the drug or incorrectly be classified as negative cases which benefit from trastuzumab or

FISH Ratios Plotted from Lowest to Highest in the BCIRG TrialsFISH Ratios Plotted from Lowest to Highest in the BCIRG Trials

Use of Fixed/Paraffin Embedded

Tissues for m-RNA Expression LevelsTissues for m-RNA Expression Levels

Use of Fixed/Paraffin Embedded

RNA Expression LevelsRNA Expression Levels

Testing Issues






Testing Issues





Chen, J et al. Diagn Mol Path 2007

Acknowledgements (con’t)

♦Genentech:Axel Ullrich H. Michael Shepard, Hank Fuchs, Bob Mass, Mark Sliwkowski

♦Amgen:Frank Calzone Elena CajulisElena Cajulis

♦Nat. Br. Ca. Coalition

♦USC:Michael Press

Acknowledgements (con’t)

Axel Ullrich H. Michael Shepard, Hank Fuchs, Bob Mass,

Frank Calzone

♦Revlon Foundation:Ronald Perlman James Conroy Lilly Tartikoff

♦Herceptin Clinical Investigators Network & BCIRGNetwork & BCIRG

♦Community-based/UCLA Clinical Research Network

♦The Group of 20

molecular diversity of human breast cancers...stability of the target, e.g. fixation artifacts in...

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