Circulating Tumor Cells (CTC) and pathological Complete Response (pCR) are strong independent prognostic factors in Inflammatory Breast Cancer (IBC) in a pooled analysis of two multicentre phase II trials (BEVERLY 1 & 2) of neoadjuvant chemotherapy combined with bevacizumab

1. Circulating Tumor Cells (CTC)
and pathological Complete Response (pCR)
are strong independent prognostic factors
in Inflammatory Breast Cancer (IBC)
in a pooled analysis of two multicentre phase II trials
(BEVERLY 1 & 2)
of neoadjuvant chemotherapy combined with bevacizumab
Jean-Yves Pierga, François-Clément Bidard, Aurélie Autret, Thierry Petit, Fabrice André, Florence
Dalenc, Christelle Levy, Jean-Marc Ferrero, Gilles Romieu, Jacques Bonneterre, Florence
Lerebours, Thomas Bachelot, Pierre Kerbrat, Emmanuelle Charafe-Jaufret, Jérôme Lemonnier,
Patrice Viens
Institut Curie, Paris and Université Paris Descartes; Institut Paoli Calmettes, Marseille,Paul Strauss Cancer Center and
University of Strasbourg; Gustave Roussy, Villejuif, Institut Claudius Regaud, Toulouse, Centre François Baclesse, Caen,
Centre Antoine Lacassagne, Nice, Institut du Cancer de Montpellier, Montpellier; Centre Oscar Lambret, Lille,
Centre Léon Bérard, Lyon, Centre Eugene Marquis, Rennes; R&D UNICANCER, Paris, Institut Paoli Calmettes, Marseille,
France

2. Introduction
• Inflammatory breast cancer (IBC) is a highly aggressive form of locally
advanced breast cancer
– Representing up to 5% of breast cancers1
– Characterized by high vascularity and increased microvessel
density
• The 5-year survival is # 40% 1,2
• Bevacizumab, targeting VEGF, significantly improves progression-free
survival and response rate in patients with advanced breast cancer but
not overall survival3
• In neoadjuvant setting of non-IBC, bevacizumab increases tumor
response rate4,5
1. Dawood S. Expert Rev Anticancer Ther 2010. 2. Cristofanilli M, et al. Cancer 2007 3. Miles D. Ann Oncol 2013 4. Bear HD NEJM 2012 5. von Minckwitz G NEJM 2012

3. Circulating tumor cells
Comen, E. et al. Nat. Rev. Clin. Oncol. 8, 369–377 (2011)

4. • CTC count is an independent prognostic factor in early breast cancer1–3
- In the REMAGUS 02 study in patients receiving neoadjuvant chemotherapy,
20–25% of patients had ≥1 CTC/7.5 mL3
- A similar rate (22%) was reported in the GeparQuattro trial4
- Higher incidence of 35% to 54% of CTC has been reported in non metastatic
IBC 5,6.
- We have recently reported that CTC detection is an independent prognostic factor
in 52 primary HER2+ IBC7
• The potential of Circulating Endothelial Cells (CEC) to predict the efficacy of
anti-angiogenic therapy remains debated
1. Rack BK, et al. JNCI 2014 . 2. Bidard FC, et al. Ann Oncol 2010;21:729–33. 3. Pierga JY, et al. Clin Cancer Res 2008;14:7004–10. 4. Riethdorf S, et al. Clin
Cancer Res 2010;16:2634–45. 5. Pierga, Lancet Oncol 2012. 6. Mego M Breast Cancer Res. 2015;17(1):2.7. Pierga J.Y Clin Cancer Res 2015;
Introduction

5. • The BEVERLY 1 & 2 studies were evaluating bevacizumab in combination
with chemotherapy in neoadjuvant setting in patients with HER2-negative and
HER2-positive IBC (T4d), respectively.
BEVERLY 2  HER2-positive IBC
BEVERLY 1  HER2-negative IBC
• An ancillary study was evaluating circulating tumor cells (CTCs) and circulating
endothelial cells (CECs) as candidate biomarkers
• We present a pooled analysis of these two prospective trials.
Introduction

6. 1 2 3 4 5 6 7 8
Radiotherapy
(6 weeks)
FEC x 4 cycles
5FU
Epirubicin
Cyclophosphamide
Docetaxel x 4 cycles
Surgery
Endocrine therapy
if ER+
Trastuzumab (52 weeks)
CTC CEC
inclusion
CTC CEC
cycle 5
CTC CEC
pre-surgery
CTC CEC
post-surgery
BEVERLY 2 treatment schedule (N= 52 HER2+ IBC)
Bevacizumab (24 weeks) Bevacizumab (28 weeks)

7. 1 2 3 4 5 6 7 8
Radiotherapy
(6 weeks)
FEC x 4 cycles Docetaxel x 4 cycles
Surgery
Endocrine therapy
if ER+
CTC CEC
inclusion
CTC CEC
cycle 5
CTC CEC
pre-surgery
CTC CEC
post-surgery
BEVERLY 1 treatment schedule (N= 100 HER2- IBC)
Bevacizumab (24 weeks) Bevacizumab (28 weeks)
Trastuzumab (52 weeks)

8. Methods
• CTC and CEC were detected in 7.5 ml and 4 ml of blood respectively
• CellSearch® system
– CTC detection : EpCAM+ immunoselection
followed by cytokeratin, CD45 and HER2 staining
– CEC detection : CD146+ immunoselection
followed by CD105 and CD34 staining

9. HER2 Cytokeratin
Example of CTC detection
• HER2 CTC staining and correlation with primary tumor status in BEVERLY
studies has been performed

10. Objectives
• To describe CTC and CEC counts at baseline and during treatment
• To correlate CTC and CEC counts with pathological response.
• To evaluate the prognostic value of CTC and CEC counts and changes
on disease-free survival (DFS), recurrence-free survival (RFS), and
overall survival (OS)
Endpoints:
- CTC and CEC counts at the five specified timepoints
- Pathological response rate (Sataloff TA, NA/NB)
- DFS, RFS, and OS at 3 years (current analysis) and 5 years

11. Patients characteristics (n=152 patients, all T4d M0)
Value
Median age, years (range)
ECOG performance status, N (%)
0
1
Lymph node involvement, N (%)
N0
N1
N2
N3
Histology: no special type (ductal)
Tumor grade, N (%)
I
II
III
Receptors, N(%)
ER positive
HER2 positive
Triple negative subtype
49 [21-75]
135 (93%)
10 (7%)
40 (26%)
78 (51%)
17 (11%)
9 (6%)
140 (93%)
4 (3%)
53 (35%)
87 (57%)
61 (40%)
52 (34%)
55 (36%)

12. Results: Baseline CTC and CEC counts
• From October 2008 to September 2010, 152 patients were included
• 137 were evaluable for both CTC and CEC at baseline
• At baseline, 55 patients had ≥ 1 detectable CTC (39%)
– Median CTC count for positive cases was 4, range [1-559]
• CECs were detected in all patients except one
• Median CEC count was 15, range [0-696]
• No correlation was observed between baseline CTC and CEC counts

13. Changes in CTC count during neoadjuvant therapy
CTCs /7.5 mL
-20
0
20
40
60
80
100
120
140
160
NumberofCTC
CTC before C1 CTC before C5 CTC before surg CTC after surg
After 4 cycles of chemotherapy, a
dramatic drop of CTC to a rate of
9% was observed (p<0.0001) .

14. Changes in CEC count during therapy
CEC/4 ml
1
10
100
1000
10000
NumberofCEC(log)
CEC before C1 CEC before C5 CEC before surg CEC after surgery CEC at 1 year
p<0.001
Significant
increase during
docetaxel +
bevacizumab
Surgery

15. Correlation for HER2 status between CTC and primary tumor
Each case with HER2 discrepancy had a low CTC count ( < 5 CTC/7.5ml) and/or less than 25% of discordant CTC
1. Ligthart & Bidard, et al. Ann Oncol 2013
HER2
negative
primary
HER2
positive
primary

16. Results: correlation with pCR
• pCR rate was 40% (95% CI 31-48% ) after central review (Sataloff TA, NA/NB)
• At multivariate analysis higher pCR rates was associated with
– Hormone Receptors negative status HR=3.36 [1.33, 8.49] p=0.01
– HER2 positive status HR=6.90 [2.83, 16.7] p<0.0001
– cN0 status at baseline HR=2.77 [1.09, 7.02] p=0.03
• No correlation between pCR and CTC or CEC levels and changes

17. Results: Univariate analysis for survival
• Median follow-up was 43 months.
Disease-Free Survival Overall Survival
p (log rank) HR [95%CI] p (log rank) HR [95%CI]
Age : > 50 years 0.11 0.7 [0.4-1.1] 0.62 0.9 [0.4-1.6]
cN status : cN1, N2, N3 0.02 2.2 [1.1-4.2] 0.03 2.7 [1.1-6.9]
Tumor grade: III 0.90 1.0 [0.6-1.7] 0.37 1.3 [0.7-2.6]
Hormone R. Status: no expression 0.003 2.2 [1.2-3.7] <0.001 3.3 [1.5-6.6]
HER2 status: positive 0.35 1.3 [0.7-2.3] 0.51 1.3 [0.6-2.8]
Triple Negative 0.11 1.5 [0.9-2.4] 0.02 2.1 [1.1-3.8]
pCR (central review): No <0.001 3.3 [1.6-6.5] 0.009 3.7 [1.3-10]
CTC at inclusion: ≥1CTC <0.001 2.8 [1.6-4.7] <0.001 4.3 [2.1-8.7]
CEC at inclusion: <20CEC 0.13 1.5 [0.9-2.7] 0.1 1.8 [0.9-3.6]

18. Disease Free Survival (ITT)
CTC=0 at C1 n=54
CTC ≥ 1 at C1 n=55
CTC=0 at C1 n=85
HR=2.80, 95%CI[1.65-4.76]
3-year DFS: 70% vs. 39%

19. Overall Survival (ITT)
HR=4.28, 95% CI[2.10-8.74]
3-year OS : 92% vs 56%
CTC=0 at C1 n=85
CTC ≥ 1 at C1 n=55

20. Multivariate analysis for Disease-Free Survival (ITT)
p (Cox model) HR [95%CI]
cN status : cN1, N2, N3 0.09 2.1 [0.9-4.9]
Triple Negative 0.01 2.5 [1.2-5.3]
pCR (central review): No 0.003 3.2 [1.5-6.8]
CTC at inclusion: ≥1CTC 0.004 2.6 [1.4-4.8]

21. Multivariate analysis for Overall Survival (ITT)
p (Cox model) HR [95%CI]
cN status : cN1, N2, N3 0.12 2.7 [0.8-10]
Triple Negative 0.04 2.9 [1.1-7.9]
pCR (central review): No 0.03 3.3 [1.1-9.7]
CTC at inclusion: ≥1CTC 0.008 3.1 [1.3-7.2]

22. Early dissemination and tumor response
• pCR reflects tumor sensitivity to treatment and is correlated with
improved overall survival 1
• However not all patients with pCR are cured
• CTCs reflect early tumoral dissemination
• Could CTC detection identify pCR patients still at risk of relapse?
1Cortazar P et al, Lancet 2014

23. Disease-Free Survival (ITT)
According to CTC at baseline & pCR
N=125*
pCR yes /CTC=0 at C1 n=35
pCR yes /CTC≥ 1 at C1 n=15
pCR no /CTC=0 at C1 n=46
pCR no/CTC ≥ 1 at C1 n=29
*143 patients had surgery,
133 had centrally reviewed pCR
137 evaluable for CTC

24. Disease-Free Survival (ITT)
According to CTC at baseline & pCR
N=125*
pCR yes /CTC=0 at C1 n=35
pCR yes /CTC≥ 1 at C1 n=15
pCR no /CTC=0 at C1 n=46
pCR no/CTC ≥ 1 at C1 n=29
*143 patients had surgery,
133 had centrally reviewed pCR
137 evaluable for CTC
3-year DFS (87%)
3-year DFS (30%)

25. Overall survival (ITT)
According to CTC at baseline and pCR
N=125*
pCR yes /CTC=0 at C1 n=35
pCR yes /CTC≥ 1 at C1 n=15
pCR no /CTC=0 at C1 n=46
pCR no/CTC ≥ 1 at C1 n=29
*143 patients had surgery,
133 had centrally reviewed pCR

26. Overall survival (ITT)
According to CTC at baseline and pCR
N=125*
pCR yes /CTC=0 at C1 n=35
pCR yes /CTC≥ 1 at C1 n=15
pCR no /CTC=0 at C1 n=46
pCR no/CTC ≥ 1 at C1 n=29
*143 patients had surgery,
133 had centrally reviewed pCR
3-year OS (93%)
3-year OS (53%)

27. Conclusions
• This is the largest prospective study evaluating CTC with a standardized
method in non-metastatic IBC.
• We observed a high CTC detection rate of 39% at baseline, with a strong
and independent prognostic value for DFS and OS.
• Combination of pCR after neoadjuvant treatment, with CTC at baseline,
isolates a subgroup of IBC (25%) with excellent survival.
• CTC count should be part of IBC stratification in prospective trials.

28. Perspectives
• Proposal of post-neoadjuvant immune-therapy in patients
with absence of pCR and/or presence of CTC at baseline
IMENEO expected data, as of June 2015
• An International MEta-analysis on individual
data in non-metastatic breast cancer (IBC and
non-IBC) treated by NEOadjuvant therapy has
been initiated (IMENEO study)

29. Acknowledgments
Patient
Beverly1 Sponsored by UNICANCER
Beverly2 Sponsored by Roche
With the financial support of
The Ligue Nationale Contre le Cancer
Roche
Chugai Pharma
Technical support
Circulating Tumor Biomarker lab,
SIRIC, Institut Curie
21 French participating centers
Gustave Roussy, Villejuif, France
Centre Paul Strauss, Strasbourg, France
Institut Claudius Regaud, Toulouse, France
Institut Curie, Paris, France
Centre François Baclesse, Caen, France
Centre Val d'Aurelle, Montpellier, France
Centre Oscar Lambret, Lille, France
Centre Antoine Lacassagne, Nice, France
Centre Eugene Marquis, Rennes, France
Centre Jean Perrin, Clermont Ferrand, France
Centre Léon Bérard, Lyon, France
Centre Paul Papin Center, Angers, France
Institut Curie, Saint Cloud, France
Institut Jean Godinot, Reims, France
Centre Alexis Vautrin, Nancy, France
Centre Catherine de Sienne, Nantes, France
Clinique Armoricaine, Saint Brieuc, France
Institut de Cancérologie de l’Ouest, Nantes, France
Hôpital Civil, Strasbourg, France
Institut Bergonié, Bordeaux, France
Institut Paoli Calmettes, Marseille, France