Matrix metalloproteinase 1 and circulating tumor cells in early breast cancer.

Mego M 1, 2, Karaba M 2, Cierna Z 1, Janega P 1, Minarik G 1, Benca J 2, Sedl‡ ckov‡ T 1, Manasova D 1, Gronesova P 3, Pechan J 2,4,

Reuben JM 5, Mardiak J 1, 2

1 Faculty of Medicine, Comenius University, Bratislava, Slovakia, 2National Cancer Institute, Bratislava, Slovakia, 3Cancer Research

Institute, Bratislava, Slovakia, 4 Slovak Medical University, 5 University of Texas MD Anderson Cancer Center, Houston, Texas, USA

National Cancer Institute

Slovakia Translational Research Unit

Slovakia

Matrix metalloproteinase 1 (MMP1)

•! zinc-dependent endopeptidase •! collagen-cleaving MMP

•! cleave extracellular matrix components

•! produced by tumor cells and tumor associated stroma

•! high MMP1 expression in tumor is associated with:

Ð ! tumor evolution Ð ! poor prognosis Ð ! shortened survival Ð ! induction of epithelial to mesenchymal transition

MMPs and EMT

Radisky et al. J Mammary Gland Biol Neoplasia. 2010, 15: 201-12

Mannello F. BMC Med. 2011 Aug 11;9:95.

CTC heterogeneity

Mego M, Mani SA, Cristofanilli M. Nat Rev Clin Oncol, 2010

Different methods detect different CTC subpopulations with different clinical and biological value

All data regarding CTCs, should be interpreted within the context of the detection method used.

Factors affecting CTC count

Mego M, Mani SA, Cristofanilli M. Nat Rev Clin Oncol, 2010

Study hypothesis

We hypothesize that MMP1 is involved in CTC release, and CTC are detected more often in breast cancer patients with high MMP1 expression in primary tumor.

Study aims

•! To assess association between CTC and MMP1 expression in breast cancer cells

•! To assess association between CTC and MMP1 expression in tumor stroma

•! To correlate MMP1 expression and patients` tumor characteristics

CTC detection

CD45 depletion

CD45 depleted

cells

RNA isolation, reverse transcription, quantitative RT-PCR for EMT related genes (TWIST,SNAIL,SLUG, ZEB2)

and CK19)

Quantitative RT-PCR dissociation curve

RossetteSep 5 mL

Mego, et al. Int J Cancer, 2012

CTC detection

MMP1 expression scoring

•! Immunohistochemical staining was quantified using a weighted histoscore.

•! The proportion of cells with nuclear staining was multiplied by the intensity of staining to provide a score of 0–300.

•! Score = (0 x % not stained) + (1 x % weakly stained) + (2 x % moderately stained) + (3 x % strongly stained)

MMP1 expression scoring

staining intensity 0 staining intensity 1

staining intensity 2 staining intensity 3

Patients` characteristics %

All patients (N = 100) 100.0

Invasive ductal carcinoma 86

T1 66 > T1 33

N0 57 N1-3 43

ER/PR positive 87 HER2 positive 16 Triple negative 15 Grade 3 36

CTC detection Gene expression % samples

KRT 19 12 SNAIL1 0 SLUG 13 TWIST 2 ZEB1 0

Circulating tumor cells % patients

All CTC 24 Epithelial CTC 12 EMT CTC 14 Both CTC 2

CTC and MMP1 expression in cancer cells

CTC - CTC +0

50

100

150

MMP1 and EMT_CTC

EMT_CTC

MM

P1 e

xpre

ssio

n

p = 0.02 p = 0.58

p = 0.37

C T C - C T C +0

5 0

1 0 0

1 5 0

M M P 1 a n d E p ith e lia l C T C

E p ith e lia l C T C

MM

P1

ex

pre

ss

ion

C T C - C T C +0

5 0

1 0 0

1 5 0

M M P 1 a n d A n y C T C

A n y C T C

MM

P1

ex

pre

ss

ion

MMP1 expression in cancer cells and tumor characteristics

p = 0.04

N o n -T N T N0

5 0

1 0 0

1 5 0

2 0 0

M M P 1 a n d tr ip le n e g a t iv e (T N ) b r e a s t c a n c e r

MM

P1

ex

pre

ss

ion

K I6 7 lo w K i6 7 h ig h0

5 0

1 0 0

1 5 0

M M P 1 a n d K i6 7

MM

P1

ex

pre

ss

ion

p = 0.05

p = 0.01 p = 0.01

G r a d e 1 a n d 2 G r a d e 30

5 0

1 0 0

1 5 0

M M P 1 e x p r e s s io n a n d tu m o r g r a d e

MM

P1

ex

pre

ss

ion

H R + H R -0

5 0

1 0 0

1 5 0

2 0 0

M M P 1 a n d h o r m o n a l re c e p to r s (H R )

MM

P1

ex

pre

ss

ion

Multivariate analysis

Independent variable Regression coefficient

Standard error p-value

EMT_ CTC (present vs. absent) 30.1 17.1 0.08

Ki 67 (cut-off 14%) (low vs. high) 39.8 11.8 0.001

CTC and MMP1 expression in stromal cells

p = 0.53 p = 0.3

p = 0.66

C T C - C T C +0

5 0

1 0 0

1 5 0

M M P 1 e x p r e s s io n a n d e p ith e lia l C T CM

MP

1 e

xp

res

sio

n

C T C - C T C +0

5 0

1 0 0

1 5 0

M M P 1 e x p r e s s io n a n d E M T _ C T C

MM

P1

ex

pre

ss

ion

C T C - C T C +0

5 0

1 0 0

1 5 0

M M P 1 e x p r e s s io n a n d a n y C T C

MM

P1

ex

pre

ss

ion

MMP1 expression in stromal cells and tumor characteristics

p = 0.0007 p = 0.0004

p = 0.004

K I6 7 lo w K I6 7 h ig h0

5 0

1 0 0

1 5 0

M M P 1 e x p r e s s io n a n d K i6 7

MM

P1

ex

pre

ss

ion

G r a d e 1 a n d 2 G r a d e 30

5 0

1 0 0

1 5 0

M M P 1 e x p r e s s io n a n d tu m o r g r a d e

MM

P1

ex

pre

ss

ion

In v a s iv e d u c ta l c a r c in o m a O th e r0

2 0

4 0

6 0

8 0

1 0 0

M M P 1 e x p re s s io n a n d tu m o r h is to lo g y

MM

P1

ex

pre

ss

ion

Conclusions •! High MMP1 expression in breast cancer cells, but not in cancer

stroma was associated with CTC_EMT in peripheral blood

•! High MMP1 expression is associated with more aggressive biological features including increased proliferation, hormone receptor negativity and high grade.

•! Our data suggest a link between MMP1 and CTCs with EMT phenotype, and further support a role for MMPs and EMT in tumor dissemination.

National Cancer Institute, Slovakia

Department of Oncology Michal Mego Jozef Mardiak Jozef Sufliarsky Zlatica Péková Zuzana Hlavata Eva Oravcova Andrej Jurik Tomas Minarik Iveta Oravcova Michaela Kubi!ková Veronika "vábová Department of Surgery Marian Karaba Juraj Benca Juraj Pechan Translational Research Unit Denisa Ma#asová Daniela Svetlovska Department of Pathology Gabriela Sieberová Ján Macúch Michal Majer!ík Peter Jáni

Comenius University, Faculty of Medicine

Department of Pathology Zuzana $ierna Pavol Janega Ludovit Danihel Pavel Babal Institute of molecular biomedicine Gabriel Minárik Tatiana Sedla!ková Cancer Research Institute, Slovak

Academy of Sciences Paulína Gronesová Ivana Fridrichová MD Anderson Cancer Center James M Reuben Jefferson Medical College, Philadelphia Massimo Cristofanilli

Acknowledgements

Thank you for your attention

http://www.fmed.uniba.sk/index.php?id=2201