Application of On-Treatment Ki67 in ALTERNATE Trial

Cynthia Ma, M.D. Ph.D.

Associate Professor of Medicine

Washington University in St. Louis

Case Study

• A 60-year-old postmenopausal woman presented with a palpable 4cm size tumor of the left breast. Biopsy of this lesion revealed invasive ductal carcinoma that is ER+ (Allred score 8), PR+ (Allred score 8), HER2-, grade 2. She desires lumpectomy rather than mastectomy although she is not a candidate for lumpectomy. What is your recommendation for the management of her cancer?

A. Recommend neoadjuvant dose dense Adriamycin plus cyclophosphamide for 4 cycles followed by taxol for 4 cycles, then surgery

B. Recommend neoadjuvant hormonal therapy with an aromatase inhibitor for 4 months followed by surgery

C. Proceed to surgery with mastectomy

D. Recommend radiation therapy before surgery

Phase 2 randomized trial of primary endocrine therapy versus chemotherapy in postmenopausal patients with estrogen receptor‐positive breast cancer

Cancer Volume 110, Issue 2, pages 244-254, 30 MAY 2007 DOI: 10.1002/cncr.22789

Postmenopausal Women, ER+ and/or PR+, T2N1-2, T3N0-1, T4N0M0, ineligible for breast conservation therapy (N=239)

Neoadjuvant Chemotherapy (N=118)

Neoadjuvant Endocrine Tx (N=121)

Anastrozole (N=61)

Exemestane (N=60)

Surgery

3 months

Phase 2 randomized trial of primary endocrine therapy versus chemotherapy in postmenopausal patients with estrogen receptor‐positive breast cancer

Cancer Volume 110, Issue 2, pages 244-254, 30 MAY 2007 DOI: 10.1002/cncr.22789

Endocrine Therapy (N=121)

Chemotherapy (N=118)

Clinical Respsonse 65% 64%

pCR 3% 6%

Breast conservation 33% 24%

ER Allred > 6 Endocrine Therapy (N=70)

Chemotherapy (N=63)

p

Clinical Response 70% 60% 0.07

Breast conservation 43% 24% 0.05

Neoadjuvant Endocrine Therapy to Improve Breast Conserving Surgery Rate

P024Letrozole

Tamoxifen

SURGERY

ER+ Stage 2/3

Anastrozole

Combination

SURGERY

TamoxifenER+ Stage 2/3

2-week BiopsyIMPACT

L T

AT C

Letrozole

SURGERY

4-week Biopsy

ER+ Stage 2/3

POL

Anastrozole

Tamoxifen

SURGERY

CombinationER+ Stage 2/3

2-week BiopsyIMPACT

Data from Matthew Ellis

2-4 Wk Ki67 10% Cutpoint Predicted RFS in IMPACT and POL

POL 4W Ki67 % PEPI 0 RFS (events), median F/U 5 years>10% 1/19 (5%) 8/21 (38%)≤10% 10/36 (28%) 3/45 (7%)P Value P=0.08 (Fisher) P=0.003

 IMPACT 2W Ki67 % PEPI 0 RFS (events), median F/U 37 months>10% 0/32 (0%) 9/35 (26%)≤10% 21/101 (21%) 13/118 (11%)P Value P=0.004 (Fisher) P=0.008 (log rank)

Pathologic T, N, ER and Ki67 post neoadjuvant endocrine therapy predicted RFS and BCSS

Ellis MJ, Tao Y, Luo J, et al: Outcome prediction for estrogen receptor-positive breast cancer based on postneoadjuvant endocrine therapy tumor characteristics. J Natl Cancer Inst 100:1380-8, 2008

P024Letrozole

Tamoxifen

SURGERY

ER+ Stage 2/3

Pathology, Biomarkers Factors

RFS BCS

HR Points HR Points

Tumor size T1/2 T3/4

-2.8

03

-4.4

03

Node status No Yes

-3.2

03

-3.9

03

Ln Ki67 level 0 -1 1+ -2 2+ -3 3+ -4 4+

-1.31.72.22.9

01123

-1.42.02.73.8

01233

ER Allred 0-2 3-8

2.8-

30

7.0-

30

Ellis et al JNCI 2008: 100, 1380-8

Preoperative Endocrine Prognostic Index (PEPI)

Preoperative Endocrine Prognostic Index (PEPI) Data from P024 and POL and trial

PEPI 0 pT1/2pN0

Ki67 ≤ 2.7% ER Allred 3-8

Data from Matthew Ellis

PEPI 0

PEPI non-0

Months

Dis

eas

e F

ree

Su

rviv

al

1.00

0.75

0.50

0.25

0.00

P024Letrozole

Tamoxifen

SURGERY

ER+ Stage 2/3 Letrozole

SURGERY

4-week Biopsy

ER+ Stage 2/3

POL

0 20 40 60 80 100

Exemestane

Letrozole

Anastrozole

2-4 week biopsy

Ki67 ≤ 10%Continue AI therapy

SURGERY

PEPI score 0 stage 1/0 No Chemo

PEPI > 0Stage > 1MD decision

FOLLOW

Ki67 > 10% Chemotherapy or Immediate

Surgery

SURGERY

FOLLOW

ACOSOG Z1031

http://www.ctsu.org/

Path CR rate?

Eligibility:• Postmenopausal• Clinical Stage II or III • ER+ (Allred 6-8)• HER2-

R

Cohort B

High Risk Baseline Intrinsic Subtype Enrichment in Ki67 > 10% at 2 to 4 weeks

Ellis et al SABCS 2012

Efficacy of Standard Chemotherapy in patients with AI resistance (Ki67>10% at 2-4 week)

• 49 patients (23%) had 2 to 4 week Ki67>10%

• 36 received chemotherapy per protocol (27) anthracycline (A) and taxane (T)-based, 7 T-based, 1 A-based and 1 non-NCCN)

• 2/35 getting HCCN-guideline chemo had a subsequent pCR (5.7%).

Ellis et al SABCS 2012

ALTernate approaches for clinical stage II or III Estrogen Receptor positive breast cancer NeoAdjuvant TrEatment (ALTERNATE) in postmenopausal women:

A Phase III Study (A011106)

Arm ISURGERY

FOLLOW

SURGERY

Arm II

Cycle 1 day 2 optional biopsy(if receiving paclitaxel)

Neoadjuvant Chemotherapy GroupChemotherapy (4-6 cycles)

RANDOMIZE

Arm IA x 4.5 yrs

Arm IIF x 1.5 yrs then A x 3 yrs

FOLLOW

AFTER SURGERYTherapy of Physician Choice

Arm III

BIOPSY

Arm III(A+F) x 1.5 yrs then Ax3 yrs

Modified PEPI 0

Chemo NOT recommended

Modified PEPI > 0

Chemo recommended

Endocrine therapy of Physician’s Choice

FOLLOW

Arm I

Arm II

Arm III

Ki67

<10%

Ki67>10%

4w12 w

BIOPSY

6 cycles (each cycle is 4 weeks)

BEFORE SURGERY AFTER SURGERY

Arm I: Anastrozole (A)Arm II: Fulvestrant (F)Arm III: Anastrozole + Fulvestrant

Red: required tissue collectionBlue: optional tissue collection

ALTERNATE Schema

REGISTRATION

Eligibility

Post-menopausalClinical Stage II or III

ER+ (Allred 6-8) HER2-

Key Inclusion Criteria

• Postmenopausal women• Clinical T2-T4c, any N, M0 invasive breast cancer

– Patients with multifocal invasive breast cancer are not eligible

• Invasive breast cancer is ER+ with an Allred score of 6, 7 or 8 and HER2 negative defined as 0 or 1+ by IHC or with a FISH ratio (HER2 gene copy/chromosome 17) < 2 if IHC 2+.

• ECOG performance status 0-2• Must agree to undergo the required research biopsies at

baseline, week 4 and at surgery.

Key Exclusion Criteria

• An excisional biopsy of this breast cancer • Surgical axillary staging procedure prior to study entry

Note: FNA or core needle biopsy of axillary node is permitted

• Treatment for this cancer including surgery, radiation therapy, chemotherapy, biotherapy, hormonal therapy or investigational agent prior to study entry

• History of invasive breast cancer or contralateral DCIS

Primary Objectives

• To compare the efficacy of three neoadjuvant endocrine therapy regimens in achieving modified PEPI 0 (1st Phase)– Anastrozole alone– Fulvestrant alone– Fulvestrant/Anastrozole combination

• To demonstrate the 5-year RFS rate in patients with a modified PEPI score of 0 is at least 95% (2nd Phase)– Anastrozole alone – Fulvestrant alone (if efficacy superior than anastrozole in the 1st Phase analysis)– Fulvestrant/Anastrozole combination (if efficacy superior than anastrozole in the 1st Phase

analysis)

Primary Objectives

• To compare the efficacy of three neoadjuvant endocrine therapy regimens in achieving modified PEPI 0 (1st Phase)– Anastrozole alone– Fulvestrant alone– Fulvestrant/Anastrozole combination

• To demonstrate the 5-year RFS rate in patients with a modified PEPI score of 0 is at least 95% (2nd Phase)– Anastrozole alone – Fulvestrant alone (if efficacy superior than anastrozole in the 1st Phase analysis)– Fulvestrant/Anastrozole combination (if efficacy superior than anastrozole in the 1st Phase

analysis)

Statistics (First Phase)– Primary endpoint:

• To compare the Modified PEPI 0 rate of the three neoadjuvant treatment.

– N=400 each arm (total n=1200)• one sided alpha=0.025 chi-square test • 82% chance to detect at least 0.10 difference in modified PEPI

0 rate

– Upon completion of the first phase enrollment, the anastrozole arm will continue to enroll patients for the 2nd phase of the trial while waiting for the primary endpoint analysis.

– Only the fulvestrant containing arm (s) which showed a superiority over anastrozole in the primary endpoint will be continued to the second phase.

Statistics (Second Phase)

– Primary endpoint: • RFS for the modified PEPI 0 group in pts treated with neoadjuvant

anastrozole or fulvestrant or anastrozole/fulvestrant if proceeded to the 2nd phase

– A sample size of 317 pts with a modified PEPI score of 0 are needed so that:

• With a sample size of 317 patients, a one-sided alpha=0.025 nonparametric Brookmeyer-Crowley type one sample survival test will have a 90% chance of rejecting that 5 year RFS rate is 95%, when the true 5 year RFS rate is at most 90%. (That is, we will conclude that the 5 year RFS rate is significantly less than 95% if the p-value for this test is less than 0.025).

– A sample size of 940 patients (including the 400 enrolled in the first phase of the trial) in each arm is needed to reach the goal of 317 women with a modified PEPI score of 0 assuming the modified PEPI 0 rate is 33%.

Biopsy/Shipment Kit

cryovials

Formalin cup inside

Blood draw kit OCT and chamber

Biopsy/Shipment Kit

dry ice and frozen samples• OCT frozen tissue block• Serum, plasma cryovials

Room temperature samples• Formalin cup • EDTA whole

blood

Biopsy gun

Central CLIA Ki67 Analysis at Wash U AMP Laboratory

– Standard SOP for Ki67 staining• FDA approved CONFIRM anti-Ki67(clone 30-9) rabbit

monoclonal antibody and reagents• FDA approved Ventana Benchmark XT platform

– Standard SOP for Ki67 scoring• Image analysis-based Ki-67 quantification algorithm with visual

point counting for selected cases• FDA approved Ventana Companion Algorithm Ki67 (30-9)

image analysis with iScan Coreo Au scanner running Virtuoso software

– Ki67 reporting• Available within 2 weeks • Specimen coordinator for sample and Ki67 result tracking.

Development of Image Analysis – Based Ki67 Scoring SOP

• Research Ki67 assay used in prior studies– P024/POL trial

• Ki67 antibody: SP6 antibody (Neomarkers)• Shandon Sequenza® Immunostainer (Ellis Lab)• Scoring: Manual point counting methodology

– IMPACT• Ki67 antibody: MIB-1 (DakoCytomation)

• CLIA Ki67 assay used in ALTERNATE trial– Central testing at Wash U AMP laboratory– Uniform sample collection using biopsy/shipment kits– Standard SOP for Ki67 analysis

• Antibody: FDA approved CONFIRM anti-Ki67 (clone 30-9) rabbit monoclonal antibody (Ventana, Tuscson, AZ)

• Staining: FDA approved Ventana Benchmark XT platform and reagent• Scoring: FDA approved iScan Coreo Au scanner (Ventana) with the

Virtuoso software; Cases with complex histological features will be scored by visual point counting.

Point counting methodology for Ki67 scoring

Photomicrographs of at least 3 randomly selected fields are taken with a background grid and color printed. The observer counts the total tumor cells and the number of Ki67 positive cells that intersect with first grid line. This process is repeated on every third gridline. Cells are counted from three separate, 40X images, or all the cells on the slide are counted if three images cannot be obtained, so that at least 200 total tumor cells will be counted.

Inter observer ICC was 0.96329

when the same photomicrographs

are provided.

Ventana Ki-67 (30-9) Image Analysis

• The VENTANA Virtuoso Ki-67 (30-9) Image Analysis Software assists the pathologist in the semi-quantitative measurement of Ki-67 in tissues stained with Ventana Medical Systems, Inc. CONFIRM anti-Ki-67 (30-9) monoclonal antibody.

• This application generates an Ki-67 score that can be reviewed and accepted by the pathologist, or if necessary, overridden by the pathologist.

• The image analysis application is an assist to the pathologist in the scoring and interpretation of Ki-67 staining on breast cancer tissues.

• The Virtuoso Ki-67 (30-9) Digital Read Application allows the pathologist to view Ki-67 stained slides as images on a computer monitor, similar to what can be viewed under a microscope.

• Training set (to develop Ki67 scoring SOP)– pN+ post neoadjuvant surgical samples from the P024/POL trial

• Validation sample set:1. pT1/2 N0 post neoadjuvant surgical samples from P024/POL trial to

validate the 2.7% cut point

2. 4-week biopsy of POL samples to validate the 10% cutpoint

3. Z1031 surgical samples

Development of Image Analysis – Based Ki67 Scoring SOP

Goncalves R, Ma C, Tao Y, et al: The development of a standardized Ki-67 assay for the ALTERNATE trial: An experience in academic investigational device development, 2013 San Antonio Breast Cancer Symposium, Abstract P3-05-10

Ki67 stained slide

Scanned

Scoreby

Image Analysis

Scoreby

Point counting

Data analysis

Pathologist Evaluation (FOV selection and triage cases for point counting

Triage criteria met for point counting

yesno

 SOP for Ki67 scoring

Goncalves R, 2013 SABCS, Abstract P3-05-10

Criteria to triage for point counting

• Abundant intermixed lymphocytes within the invasive tumor.• Sparse invasive tumor.• Diffusely infiltrative tumor with no clear consolidation (ie,

streaming invasive tumor with a large amount of intervening stroma).

• Any situation leading to abundant marking of stromal elements (ie, non-fascicular “plump” fibroblasts).

• Any situation where an abundant amount of stained cells are not being included in the analysis such as general diffuse and faint nuclear staining.

Goncalves R, 2013 SABCS, Abstract P3-05-10

Validation Data at 2.7% cutpointK-M RFS by Ki67 < 2.7% (modified PEPI 0) vs Ki67 > 2.7% (modified PEPI > 0) for pT1/2 N0 cases in P024/POL trial

Logrank p=0.0440

N=30

Goncalves R, 2013 SABCS, Abstract P3-05-10

Validation Data at 10% cutpointK-M RFS by Ki67 < 10% vs Ki67 > 10% on

4-week biopsy in POL trial

N=46

Goncalves R, 2013 SABCS, Abstract P3-05-10

Validation on reproducibility from 2 pathologists

Scatterplot of Ki67 image analysis using FOVs selected by Ventana pathologist vs Wash U pathologist

R-Square: 0.8928.

The Spearman Correlation

Coefficient : 0.8988 (p<0.0001)

Goncalves R, 2013 SABCS, Abstract P3-05-10

 Wash U Pathologist

>2.7% <2.7% Total

Ventana Pathologist>2.7% 29 3 32

<2.7% 1 23 24

Total 30 26 56

• The agreement was 29/30 (0.9667) (95% exact confidence limit: 0.9025; 1) in predicting Ki67 > 2.7%.

• The agreement was 23/26 (0.8846) (95% exact confidence limit: 0.7618; 1) in predicting Ki67 <2.7%.

• Simple Kappa Coefficient was 0.8557 (95% Confidence limit: 0.7197; 0.9916).

Analysis of concordance at 2.7% cutpoint

Validation on reproducibility from 2 pathologists

Goncalves R, 2013 SABCS, Abstract P3-05-10

 Wash U Pathologist

>10% <10% Total

Ventana Pathologist

>10% 9 1 10

<10% 0 46 46

Total 9 47 56

• The agreement was 100% in predicting Ki67 over 10%.

• The agreement was 46/47 (0.9787) (95% exact confidence limit: 0.9374; 1) in predicting Ki67 <10%.

• Simple Kappa Coefficient was 0.9367 (95% Confidence limit: 0.8139; 1.0).

Analysis of concordance at 10% cutpoint

Validation on reproducibility from 2 pathologists

Goncalves R, 2013 SABCS, Abstract P3-05-10

Image Analysis SOP*

mPEPI-0 N (%)

mPEPI-non-0 N (%)

Total

Visual Point Counting

(VPC)

mPEPI-0 N (%)

36 (17.9) 3 (1.5%) 39

mPEPI-non-0N (%)

8 (4%) 154 (76.6) 162

Total 44 157 201

mPEPI score concordance data by Image analysis SOP vs visual point counting (Z1031 samples)

*6% were triaged to VPC

Sanati S, 2014 SABCS, Abstract P4-11-13

Time to breast cancer recurrence in Z1031 patients with mPEPI-0 (dashed line) versus mPEPI>0 (solid line)

Median F/U: 5 (2-7) years

N=201 (53.3% of the Z1031 population)

Among the 112 patients who did not receive ACT, the 5-year RFS rate was 100% among the 33 mPEPI-0 patients and 87.5% among the 79 mPEPI >0 patients.

Sanati S, 2014 SABCS, Abstract P4-11-13

Summary

• On-treatment Ki67 assessment during and at the completion of neoadjuvant endocrine therapy using established SOP predicted endocrine responsiveness in previous clinical trials.

• High Ki67 (>10%) at 2-4 week biopsy indicates poor response to endocrine therapy.

• Prospective validation of PEPI 0 as a biomarker of endocrine sensitivity is being prospectively validated in the ongoing ALTERNATE trial.– PEPI 0 status after 6 months of endocrine therapy identifies

patients who do not need chemotherapy – PEPI 0 rate could be used as a surrogate endpoint in clinical

trials of endocrine therapy agents.

PEPI 0

Evolving Clinical Approach Managing Early Stage ER+ HER2- Breast Cancer

AI therapyalone?

Endocrine resistantPEPI > 0

2-4 wks

AI AI

3-6 mos

Endocrine resistant(Ki67 High)

Acknowledgement• Washington University Investigators

– Dr. Matthew Ellis– Dr. Souzan Sanati, Dr. Katherine DeSchryver (Pathologists)– Dr. Rodrigo Goncalves– Dr. Erika Crouch (AMP laboratory)– Jeremy Hoog (Sample coordination)– Dr. Yu Tao (Statistical analysis)

• ACOSOG Investigators– Dr. Vera Suman

• NCI– Dr. Lisa McShane

• Ventana – Dr. Michael Barnes

• Funding– R01-CA095614, U24-CA114736, U10-CA076001, and U01-CA114722 from

NCI; the Breast Cancer Research Foundation; Komen St Louis Affiliate Clinical Trials Grant; AVON foundation, and grants to support ACOSOG for the conduct of Z1031 from Pfizer and Novartis.