ANTICANCER RESEARCH 32
: 3363-3370 (2012)
Mesenchymal Characterization: Alternative to Simple
CTC Detection in Two Clinical Trials
GUISLAINE BARRIERE1, ALAIN RIOUALLON2, JOËL RENAUDIE2,
1Astralab Clinical Laboratory, Limoges, France;
2Department of Gynecology and Surgery. Clinique du Colombier, Limoges, France
Abstract. Background: Breast cancer is one of the most
enriched for CTCs by EpCAM-coated immunomagnetic
common malignancies in women. Approximately 25% of
beads. CTCs are then stained for cytokeratins (CK8, CK18
patients with early-stage disease will develop metastatic
and CK19), for CD45 to eliminate white cells, and with 4’,6-
recurrence. Two clinical trials were undertaken in order to
Diamidino-2-Phenylindole (DAPI) nuclear counterstain for
detect circulating tumor cells (CTCs) in primary breast cancer.
essessment of their viability. Finally, they are enumerated by
Patients and Methods: Four-hundred patients with early breast
at least two pathologists. This method was cleared by the US
cancer were enrolled in the trial. After enrichment from their
Food and Drug Administration (FDA) but not endorsed by the
peripheral blood, their CTCs were characterized by gene
American Society of Clinical Oncology (ASCO). Due to the
expression of cancer cell markers. Results: CTCs had a
use of a sole antibody, CTCs can escape the enrichment and
predominant epithelial phenotype in 8.75% of patients and de-
detection phases, as EpCAM expression is decreased in
differentiated characteristics (mesenchymal, stem phenotypes
mesenchymal cells (3). In one-third of patients, CTCs are
alone or both) in 37.6%. Conclusion: Tumor epithelial cells
undetected by conventional methods (4). This impairment
undergoing epithelial–mesenchymal transition give rise to
prompted the development of methods where CTCs are
cells with mesenchymal aggressive phenotype. Detection of
qualified by molecular biology techniques, revealing
mesenchymal and cancer stem cells, which are tumor-
expression of specific genes (5). AdnaGen technology is one
initiating cells, is more relevant than simple counting of CTCs
of those techniques (6). It is based on a combined mixture of
to assess their presence in the blood of patients with breast
antibodies (EpCAM and two epitopes of MUC1) to trap
cancer. This study will be the basis for future evaluation of the
CTCs which are defined by expression of genes involved in
outcome of the disease and the prognostic value of early-
epithelial–mesenchymal transition (EMT). Most CTC
detection trials were dedicated to metastatic breast cancer (7).
CTCs were detected, depending on the stage of the disease
Detection of circulating tumor cells (CTCs) has been recently
and the undertaken methodology, in 8% to 95% breast cancer
developed and can be considered as a prognostic tool (1). A
patients (8-12). Due to the range of results, conclusive
limited number of markers are currently used for the isolation
relevance is still pending. However, these studies have shown
(cell surface antigens) or detection (antigens or mRNA) of
the prognostic potential of CTCs in primary and metastatic
CTCs. One such marker is the epithelial cell adhesion
breast cancer (13). Our preliminary results on primary breast
molecule (EpCAM) (2); others are more cancer-specific, such
cancer and CTCs arising from EMT were presented in Prague
as human epidural growth factor receptor 2 (HER2) and
(June 13-17, 2011) at the TATAA Biocenter qPCR
mucin 1 (MUC1) for breast carcinoma. CTCs are generally
symposium (14). These results were confirmed by Kasimir-
counted by using the CellSearch system (Veridex, Warren,
Bauer et al.
(15). Today, the term CTCs encompasses all
New Jersey, USA). In this technique, blood sample is
types of cells which are considered as foreign entities in theblood, exhibiting cancerous characteristics. This term doesnot describe the diversity of CTC subpopulations. Amongthem, cancer stem and mesenchymal cells have to be taken
Guislaine Barrière, Astralab Clinical
into account. Their features are invasiveness linked to
Laboratory, 7-11 Avenue de Lattre de Tassigny, 87000 Limoges,
increased cell motility and their capability to avoid apoptosis,
France. Tel: +33 555302930, e-mail: [email protected]
anoikis, and general immune defense (16-19). Upon hypoxia,
Circulating tumor cells, de-differentiated circulating
epithelial cancer cells can modify their phenotype. This
tumor cells, epithelial–mesenchymal transition, breast cancer.
phenomenon depends on pleitropic cytokines such as
ANTICANCER RESEARCH 32
: 3363-3370 (2012)
transforming growth factor β (TGF-β) (20). The latter is
were used: AdnaTest Breast Select and Detect, AdnaTest EMT-
induced by hypoxia-inducible factor 1α (HIF1α) (21). The
1/Stem Cell Select and Detect, according to the suppliers
signaling pathway induces loss of the epithelial polarity of
instructions (AdnaGen AG, Langenhagen, Germany). Steps arebriefly described. For semi-quantitative reverse transcription and
cells, which undergo cytoskeletal remodeling. Expression of
polymerase chain reaction (RT-PCR), Sensiscript and HotStarTaq
some proteins which are involved in adhesion structures are
from Qiagen GmbH (Hilden, Germany) were used and the
inhibited. Numerous experiments on this transition have
housekeeping gene was β actin. Thermal profiles are those
demonstrated a decrease of E-cadherin and an increase of N-
recommended by the supplier. Visualization of PCR fragments was
cadherin and vimentin (22-24). Thus, cells acquire a
carried out with a bioanalyser (2100 Bioanalyser; DNA 1000
migratory phenotype and exhibit mesenchymal features.
LabChip; Agilent Technologies; Santa Clara; CA). Peaks were
Epithelial cells and mesenchymal cells can exist
considered to be positive when concentrations were ≥0.15 ng/μl(according to AdnaGen indications).
simultaneously, both types being CTCs. Hence, a clinical trial
Tumor cell enrichment was realized by using
was developed to establish the percentage of patients carrying
AdnaTest Breast Select
. The immunomagnetic beads were coated
these cells at the time of early diagnosis of breast cancer and
with three antibodies, one to EpCAM and two others to MUC1.
before and after surgical eradication of the primary tumor. In
Then, tumor-associated mRNA expressions were analyzed by
this protocol, 400 patients were included and CTCs were
AdnaTest Breast Detect
kit. The following transcripts were separated
evidenced by three markers: HER2, MUC1, epithelial
by capillary electrophoresis: HER2
glycoprotein 40 (GA733-2). Moreover, we analyzed 130
pairs of size 270, 293 and 395 bp, respectively.
Protocol 2: AdnaTest EMT-1/Stem Cell Select
avoided an excess
clinical samples belonging to this cohort to detect
contaminating leukocytes by a special washing buffer procedure.
mesenchymal and stem cells by using more specific EMT
Expressions of tumor-associated mRNA were depicted by AdnaTest
markers: transcription factor TWIST1, protein kinase B
EMT-1/Stem Cell Detect
kit. The following transcripts: TWIST1,
(AKT2), phosphatidylinositol 3-kinase A (PI3KA) and
(203 bp, 306 bp, 595 bp, 165 bp,
respectively) were revealed by capillary electrophoresis. To thesupplied method, we added CD44
and the polycomb group protein
Materials and Methods
) as subsidiary markers of stemness. For each one, theRT sample, previously obtained, was amplified by singleplex PCR.
Sequences of primers were CD44
Four-hundred patients (T1/T3, N–/N+, M0) were enrolled
TTGATGGACC and reverse: GCAGGGATTCTGTCTGTGCTG; and
between 2009-2011. Patients were selected provided they fulfilled
forward: CATTGTCTTTTCCGCCCGC and reverse: CAAAG
the following criteria: age 40-75 years; breast cancer diagnosis
CACACACATCAGGTGGG. The thermal profile used for CD44
confirmed by a pathologist’s analysis of the primary tumor; absence
was as follows: after 15 min denaturation at 95˚C, 33 cycles of PCR
of bone, visceral, cerebral metastasis (controlateral breast
were carried out by denaturation at 94˚C for 30 s, annealing/extension
mammography, liver ultrasonography and entire body-bone
at 59˚C for 30 s, and elongation for 72˚C for 30 s. Termination of the
scanning). Axillary lymph node invasion was assessed. These
PCR reaction was subsequently carried out at 72˚C for 5 min
patients were treated by tumorectomy or mammectomy; axillary
followed by storage of the sample at 10˚C. The procedure for BMI1
clearance was added when required. Characteristics of the primary
was as follows: after 15 min denaturation at 95˚C, 36 cycles of PCR
tumor at the time of diagnosis are shown in Table I. All blood
were carried out by denaturation at 94˚C for 30 s, annealing/extension
samples were obtained after informed consent. The protocol was
at 59.7˚C for 30 s, and elongation for 72˚C for 30 s. Termination of
conducted at the Department of Gynecological Surgery (Private
the PCR reaction was subsequently carried out at 72˚C for 5 min
Hospital Clinique le Colombier) and Astralab Laboratory
followed by storage of the sample at 10˚C. The primers generate
(Department of Specialized Clinical Analyses) in Limoges France.
fragments of the following sizes: (CD44
, 257 bp and BMI1
, 132 bp).
The study was performed with approval of an appropriate Local
expressions were considered positive when the
Ethics Committee (Comité de protection des personnes Sud-Ouest et
transcript concentration was above 0.50 ng/μl. Blood collected from
Outre-mer IV. France) and was in compliance with the Helsinki
20 healthy donors was investigated to determine this cut-off value.
Declaration. This cohort enabled us to realize two trials: the totalityof patients (400) was incorporated in a first protocol and the final
Statistical analyses were performed using
130 patients were also included in a second protocol.
XLStat2011 software (Addinsoft; Paris; France). The Chi-squaretest was used to establish a relationship between CTC detection and
Sampling of biological material.
Blood samples of 7 ml were collected
tumor characteristics and lymphatic invasion.
for cellular enrichment with AdnaCollect tubes (AdnaGen AG,Langenhagen, Germany) before surgery and at least 3 weeks aftersurgery. Blood collections were performed before any drug therapy.
Two tubes of 7 ml were necessary when patients were enrolled intothe second trial. Samples were stored, shipped in the dark at 4-8˚C and
Protocol 1: Patients’ characteristics.
A total of 400 patients
were analyzed within 24 h. Blood collection tubes contain EDTA and
(aged from 40 to 75 years, average 66 years of age) were
a chemical agent to prevent illegitimate RNA expression.
included in the trial. They were studied at the time of newly-
CTC selection and detection.
Selection and detection of CTCs were
diagnosed breast cancer and patients were enrolled after
performed as described elsewhere (5). The following AdnaGen kits
elimination of bone visceral and cerebral metastasis, whether
Table I. Tumor characteristics for 400 patients included in protocol 1. CTC-positivity is indicated for each category of breast cancer and before orafter surgery.
they had axillary lymphatic node invasion or not. Among the
patients failed to clear cancer cells from their blood despite
cohort, 87% of patients had T1 or T2 tumors. Most of the
suppression of the primary tumor. Out of the 20 patients
patients had no positive axillary lymph nodes (67%). The most
found to be CTC-positive after surgery, 12 were cases of new
common molecular characteristic phenotype, assessed on
detection. These data are presented in Table I. There was a
estrogen receptors (ER), progesterone receptors (PR) and
discrepancy between the HER2 phenotype of CTCs and that
HER2 expression of the primary tumor, was hormone receptor
of the primary tumor. Effectively, out of four patients with
positivity. Luminal A type breast cancer was observed in 77%,
HER2-positive primary tumor, only two had CTCs bearing
luminal B in 7%, triple negative (TN) in 8% and HER2 was
the HER2 receptor. In seven cases of HER2-negative tumor,
overexpressed in 4% of breast cancer cases (Table I).
the corresponding CTCs were positive for HER2. This resultshowed a high incidence of conversion for the EGF receptor.
Incidence of CTCs.
In 35 patients, CTCs were detected at
CTC positivity was distributed as follows: 25 luminal A, 3
least once (either before and/or after surgery). Thus 8.75% of
luminal B, 1 HER2-overexpressing and 2 TN. Four breast
the total cohort expressed at least one marker. Out of the 400
tumors were not molecularly characterized. CTCs are often
patients, 359 were submitted to the two series of analyses
detected in luminal phenotypes. However, the imbalance
before and after surgery. The analysis of markers for the 35
between the different types of tumors in the studied cohort is
CTC-positive patients gave the following positivity rates:
not sufficient to support this assertion. Statistical studies
GA733-2, 42%; MUC1, 65%; HER2, 26%. In eight patients,
indicated that CTC detection is independent of the primary
CTCs were detected both pre- and post-operatively. Out of
tumor characteristics. This non-correlation between CTCs and
the 23 patients found to be CTC-positive before surgery, eight
parameters of the tumor was demonstrated by p-
remained positive. Thus, 23% of the initially CTC-positive
ANTICANCER RESEARCH 32
: 3363-3370 (2012)
Table II. Tumor characteristics for 130 patients included in protocol 2. Positivity of mesenchymal or/and stem cells is indicated for each categoryof breast cancer before surgery.
EMT: Epithelial–mesenchymal transition; ddCTC: de-differentiated circulating tumor cell; ND: not determined.
Blood samples from 130 patients, submitted to
applied a transcript cut-off value of 0.50 ng/μl (specificity of
the CTC analyses described above, were tested for CTC
the cut-off is more than 90%, as confirmed in 20 healthy
presence by using EMT and stemness markers. All patients
donor samples). Thus, ddCTCs detected by mesenchymal
were studied at the time of newly-diagnosed breast cancer.
markers and/or ALDHI
were 63% and 40% positive for BMI1
Table II describes clinical and pathological characteristics of
, respectively. Among 84 N– patients, 32 had
the tumors. We distinguish these cells by calling them de-
ddCTCs, and of 37 N+ patients, only 11 had ddCTCs.
differentiated CTCs (ddCTCs), as markers used for their
Statistical studies indicated that ddCTC detection is an
detection are implicated in their mesenchymal or stemness
independent factor of the other pronostic factors based on the
pathologist’s analysis. No p
-value was less than 0.05. In aprevious report, we noticed a correlation between the
. Among 130 patients, 49 had
presence of ddCTCs and lymph node positivity with p<
ddCTCs expressing at least one EMT marker or ALDH1
(5). This value is at the limit of significance, indicating a
mRNA. Thus 37.6% of samples were positive for ddCTCs
trend, and this correlation disappeared as the number of
(Table II). Moreover among this population, 36.7%, 34.6%
and 28.6% exhibited EMT cell, stem cell or both EMT andstem cell markers respectively. The analysis of each marker
for the 49 ddCTC-positive patients gave the followingpositivity rates: TWIST1
, 8.2%; PI3KΑ
, 61.2%; AKT2
In the first protocol, detection of CTCs led to discovery of a
10.2%; and ALDH1
, 63.2%. When analyses detected at least
small rate of CTC positivity at the time of early-breast
one EMT marker or ALDH1
, the sample was further
cancer diagnosis, when cancer cells were detected on the
examined for BMI1
. For these two markers, we
basis of GA733-2, HER2, MUC1 marker expression
(8.75%). These results seem low when compared to those
implicated in regulating the EMT status, such as TWIST1.
previously published, which could be due to the use of
Thus these markers are relevant for the detection of
different technologies. However, Molloy et al.
and Banys et
mesenchymal cells. By using these, we detected 49 patients
who conducted trials comparable to ours, described CTC
out of 130 who had ddCTCs in the blood at the time of early
positivity with 7% and 12% rates, respectively for their
diagnosis of primary breast cancer. These ddCTCs can be
patients (8, 9). To explain such a low percentage of patients
distinguished into three phenotypes: mesenchymal, stem, or
with CTCs, one must bear in mind that CTC isolation is
mixed status (EMT and stemness). Even patients with node-
based on epithelial characterized antibodies of cells (25).
negative invasion had ddCTCs: 32 out of 84 cases. This
Moreover, at the detection step, the markers are limited to
positivity is independant of tumor clinicopathological
some of the total CTC populations. It can be expected that
features. It would be of interest to test blood of these patients
detected cells are epithelial cancer cells and that those
after surgery and during progression-free survival to
loosing the epithelial marker (EpCAM) are missed. Cancer
determine whether these CTCs are implicated in the
cells in the blood arise from the primary tumor and two types
recurrence or metastases occurring in 25% of women at 5
of processes could be implicated: passive or active
years. The stemness characteristics of these cells, particularly
delamination. In the first phenomenon, escape of cancer cells
dormancy, would be at the origin of the “waking-up” of the
occurs as clusters and cells retain their epithelial
disease. We cannot emphasize enough that the selected
characteristics. Collective epithelial cell migration is an
method is crucial in order to truly detect cancer cells in the
integral part of development and cancer progression.
blood. Most of the conventional methods are not able to
Apicobasal polarity is maintained when cell strands or sheets
delineate the subpopulations issued from EMT. In the cohort
leave the primary tumor (26, 27). The lifespan of such cells
of 400 patients, we only found positivity for 8.75% of
is limited by anoikis and solely their presence in the blood is
samples, whereas 37.6% were positive when we examined
an indicator of the existence of a primary tumor. Their
EMT characteristics. Thus it seems evident that the second
evidence does not necessarily indicate that they will survive
method is more appropriate to analyse CTCs in breast cancer
and grow but they can testify to the existence of hidden cell
at the time of early diagnosis. Thus an EMT signature of
subpopulations not detected by the method, coming from an
CTCs may help to stratify early-stage breast cancer.
active delamination process during EMT (28). The data
However, additional studies are required to determine the
reported here also demonstrated that out of the 23 CTC-
prognostic potential of EMT associated with CTCs.
positive patients, eight had persistence of cancer cells in the
Consequently, a follow-up of at least five years may be
blood even three weeks after removal of the primary tumor.
requiered to include mature outcome data.
Moreover, regarding CTC-negative patients before surgery,12 became CTC-positive four weeks after removal of the
primary tumor. This positivity, demonstrating that cancercells are not always cleared by surgery, can be explained by
The aim of the study was to assess CTCs expressing
the presence of a secondary cancer site which releases CTCs
mesenchymal markers in patients with primary breast cancer.
into the blood. Many questions still remain about this
Due to frequent loss of epithelial antigens by CTCs, the most
positive recurrence. The major concern is
invasive cell populations are hidden. Detection has to be
micrometastases exist a long time before they are revealed
improved to identify CTCs which have undergone EMT. The
by their growth. 12-37% of small breast tumors (<1 cm),
two protocols demonstrated that initiating tumor cells which
have already metastasized at diagnosis (29, 30) and some
are subpopulations of ddCTCs are often missed. Such a result
data suggest that systemic dissemination of tumor cells
is of importance because it is these cells which support
occurs at early stages of tumor development (31). Based on
invasion and metastases, and potentially support the genesis
these considerations, we started the second protocol to detect
of residual disease. It has been demonstrated that ddCTCs are
cells arising from EMT and which are not evidenced by the
resistant to chemotherapy and radiotherapy. Neoadjuvent
therapy to reduce the volume of tumor before surgery could
An oncological type of EMT is able to convert
concentrate these cells in the primary tumor. Thus it might be
differentiated epithelial cancer cells into migratory
necessary to establish a follow-up of ddCTCs to monitor how
mesenchymal cancer cells. EMT is associated with poor
they are cleared from the blood. Methodologies to identify
clinical outcome in breast cancer (32, 33). PI3K signaling
ddCTCs should allow for an accurate screening of new
plays a key role in inducing and maintaining EMT. The most
molecules which would be able to target or reverse EMT.
important downstream effector of PI3KΑ is AKT. Moreover,migratory mesenchymal cells acquire some stem cell
characteristics and can be detected by using ALDH1
. Numerous transcription factors have been
The Authors declare that they have no competing interests.
ANTICANCER RESEARCH 32
: 3363-3370 (2012)
11 Giuliano M, Giordano A, Jackson S, Hess KR, De Giorgi U,
Mego M, Handy BC, Ueno NT, Alvarez RH, De Laurentiis M, De
All contributors participated equally to this study. All authors read
Placido S, Valero V, Hortobagyi GN, Reuben JM and Cristofanilli
M: Circulating tumor cells as prognostic and predictive markersin metastatic breast cancer patients receiving first-line systemictreatment. Breast Cancer Res 13
: R67-75, 2011.
12 Krag DN, Ashikaga T, Moss TJ, Kusminsky RE, Feldman S,
Carp NZ, Moffat FL, Beitsch PD, Frazier TG, Gaskin TA, Shook
Our work was financially supported by URCAM- Limousin (Union
JW, Harlow SP and Weaver DL: Breast Cancer Cells in the
Régionale des Caisses d’Assurance Maladie).
Blood: A Pilot Study. Breast J 5
: 354-358, 1999.
13 Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller
MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LW and HayesDF: Circulating tumor cells, disease progression, and survival in
1 Liu MC, Shields PG, Warren RD, Cohen P, Wilkinson M,
metastatic breast cancer. N Engl J Med 351
: 781-791, 2004.
Ottaviano YL, Rao SB, Eng-Wong J, Seillier-Moiseiwitsch F,
14 Barrière G, Riouallon A, Renaudie J, Tartary M, Pelpel M and
Noone AM and Isaacs C: Circulating tumor cells: a useful
Rigaud M: Breast cancer, what do CTCs mean? EMT–Stemness
predictor of treatment efficacy in metastatic breast cancer. J Clin
invasivenes, metastasis and circulating tumor cells. Symposium
Praga (13 June 2011): “Developments in Real-time PCR – From
2 Maetzel D, Denzel S, Mack B, Canis M, Went P, Benk M, Kieu
Preanalytics to Molecular Diagnostics” Circulating Tumor Cells
C, Papior P, Baeuerle PA, Munz M and Gires O: Nuclear
signalling by tumour-associated antigen EpCAM. Nat Cell Biol
15 Kasimir-Bauer S, Hoffmann O, Wallwiener D, Kimmig R and
Fehm T: Expression of stem cell and epithelial–mesenchymal
3 Sieuwerts AM, Kraan J, Bolt J, van der Spoel P, Elstrodt F,
transition markers in primary breast cancer patients with
Schutte M, Martens JW, Gratama JW, Sleijfer S and Foekens JA:
circulating tumor cells. Breast Cancer Res 14
: R15-23, 2012.
Anti-epithelial cell adhesion molecule antibodies and the
16 Morel AP, Lièvre M, Thomas C, Hinkal G, Ansieau S and
detection of circulating normal-like breast tumor cells. J Natl
Puisieux A: Generation of breast cancer stem cells through
Cancer Inst 101
: 61-66, 2009.
epithelial–mesenchymal transition. PLoS One 3
: e2888, 2008.
4 Gradilone A, Raimondi C, Nicolazzo C, Petracca A, Gandini O,
17 Takebe N, Warren RQ and Ivy SP: Breast cancer growth and
Vincenzi B, Naso G, Aglianò AM, Cortesi E and Gazzaniga P:
metastasis: interplay between cancer stem cells, embryonic
Circulating tumour cells lacking cytokeratin in breast cancer:
signaling pathways and epithelial-to-mesenchymal transition.
The importance of being mesenchymal. J Cell Mol Med 15
Breast Cancer Res 13
: 211-221, 2011.
18 Huber MA, Kraut N, and Beug H: Molecular requirements for
5 Barriere G, Riouallon A, Renaudie J, Tartary M and Rigaud M:
epithelial–mesenchymal transition during tumor progression.
Mesenchymal and stemness circulating tumor cells in early
Curr Opin Cell Biol 17
: 548-558, 2005.
breast cancer diagnosis. BMC Cancer 12
: 114, 2012.
19 Oliveras-Ferraros C, Cufí S, Vazquez-Martin A, Menendez OJ,
6 Barriere G, Tartary M and Rigaud M: Epithelial Mesenchymal
Bosch-Barrera J, Martin-Castillo B, Joven J and Menendez JA:
Transition: A New Insight into the Detection of Circulating
Metformin rescues cell surface major histocompatibility complex
Tumor Cells. IRSN Oncology (in press), 2012.
class I (MHC-I) deficiency caused by oncogenic transformation.
7 Fehm T, Hoffmann O, Aktas B, Becker S, Solomayer EF,
Cell Cycle 11
: 865-870, 2012.
Wallwiener D, Kimmig R and Kasimir-Bauer S: Detection and
20 Bi WR, Yang CQ and Shi Q: Transforming Growth Factor-β1
characterization of circulating tumor cells in blood of primary
Induced Epithelial – Mesenchymal Transition in Hepatic
breast cancer patients by RT-PCR and comparison to status of
Fibrosis. Hepatogastroenterology 59, in press, 2012.
bone marrow disseminated cells. Breast Cancer Res 11
21 Zhou G, Dada LA, Wu M, Kelly A, Trejo H, Zhou Q, Varga J and
Sznajder JI: Hypoxia-induced alveolar epithelial–mesenchymal
8 Molloy TJ, Bosma AJ, Baumbusch LO, Synnestvedt M, Borgen
transition requires mitochondrial ROS and hypoxia-inducible
E, Russnes HG, Schlichting E, van’t Veer LJ and Naume B: The
factor 1. Am J Physiol Lung Cell Mol Physiol 297
prognostic significance of tumour cell detection in the peripheral
the bone marrow in 733 early-stage breast cancer
22 Thiery JP and Sleeman JP: Complex networks orchestrate
patients. Breast Cancer Res 13
: R61-71, 2011.
epithelial–mesenchymal transitions. Nat Rev Mol Cell Biol 7
9 Banys M, Krawczyk N, Becker S, Jakubowska J, Staebler A,
Wallwiener D, Fehm T and Rothmund R: The influence of
23 Yang J and Weinberg RA: Epithelial–mesenchymal transition: at
removal of primary tumor on incidence and phenotype of
the crossroads of development and tumor metastasis. Dev Cell
circulating tumor cells in primary breast cancer. Breast Cancer
24 Kang Y and Massagué J: Epithelial-mesenchymal transitions:
10 Bidard FC, Hajage D, Bachelot T, Delaloge S, Brain E,
twist in development and metastasis. Cell 118
: 277-279, 2004.
Campone M, Cottu P, Beuzeboc P, Rolland E, Mathiot C and
25 Criscitiello C, Sotiriou C and Ignatiadis M: Circulating tumor
Pierga JY: Assessment of circulating tumor cells and serum
cells and emerging blood biomarkers in breast cancer. Curr Opin
markers for progression-free survival prediction in metastatic
breast cancer: A prospective observational study. Breast Cancer
26 Wood JM and Olson MF: Collective migration: spatial tension
relief. Curr Biol 22
: R125-127, 2012.
27 Scott RW, Crighton D and Olson MF: Modeling and imaging 3-
32 Liu R, Wang X, Chen GY, Dalerba P, Gurney A, Hoey T,
dimensional collective cell invasion. J Vis Exp 58
Sherlock G, Lewicki J, Shedden K and Clarke MF: The
prognostic role of a gene signature from tumorigenic breast
28 Wicha MS and Hayes DF: Circulating tumor cells: not all
cancer cells. N Engl J Med 356
: 217-226, 2007.
detected cells are bad and not all bad cells are detected. J Clin
33 Sheridan C, Kishimoto H, Fuchs RK, Mehrotra S, Bhat-
Nakshatri P, Turner CH, Goulet R JR, Badve S and Nakshatri H:
29 Wilhelm MC, Edge SB, Cole DD, deParedes E and Frierson HF
CD44+/CD24– breast cancer cells exhibit enhanced invasive
JR: Nonpalpable invasive breast cancer. Ann Surg 213
properties: an early step necessary for metastasis. Breast Cancer
30 Chadha M, Chabon AB, Friedmann P and Vikram B: Predictors
of axillary lymph node metastases in patients with T1 breastcancer. A multivariate analysis. Cancer 73
: 350-353, 1994.
31 Hüsemann Y, Geigl JB, Schubert F, Musiani P, Meyer M,
Burghart E, Forni G, Eils R, Fehm T, Riethmüller G and Klein
CA: Systemic spread is an early step in breast cancer. Cancer
1 Chapter 0: Preliminaries 1.1 Contents of This Chapter • Analysis Versus Numerical Analysis • Describes how numerical analysis differs from analytical analysis and shows where each has special • It briefly lists the topics that will be covered in later chapters • Explains why computers and numerical analysis are intimately related • It describes several ways by which a comput
Universitätsmedizin Göttingen Publikationen und Hochschulschriften 2012 Gastroenterologie und Endokrinologie Journalbeiträge 1. Alekseev D, Goralczyk A, Lorf T, Ramadori G, Obed A (2012) Ten years survival with excellent outcome after living donor liver transplantation from 70 years old donor for primary hepatic neuroendocrine carcinoma: Case report. Int J Surg Case Rep, 3(1): 34-6.