Diapositiva 1

1
ARE CIRCULATING TUMOR CELLS HELPFUL IN TREATMENT
PLANNING AND IN ASSESSMENT OF RESPONSE? Alberto
Amadori Department of Oncology and Surgical
Sciences University of Padova Istituto Oncologico
Veneto - IRCCS Rome, July 4, 2008
2
Rationale I
Mammary carcinoma is the major cause of
cancer-associated death in women. Despite
advances in clinical research, the possibility of
obtaining complete responses in the metastatic
disease is very low, and in most cases partial
clinical responses are transient. Optimal
therapeutic strategies would rely on identifying
prognostic factors, but despite intense search
for biochemical and molecular markers of
prognosis, in many cases it is very hard to
foresee the course of the disease in other
words, patients categorized as low-risk based on
currently accepted parameters often develop
metastasis, while patients who are classified as
high-risk may survive disease-free for long time.

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Rationale II
Recently, a metanalysis by Braun et al (N Engl J
Med. 2005 Aug 25353(8)793-802) examined data
from 9 clinical studies in a total of 4703
patients with stage I, II, III mammary
carcinoma. Bone-marrow micrometastases were found
in 30,6 of the patients, and were associated
with less favorable pTNM stage, histological
grading and hormonal receptor expression
(plt0,001). As far as survival was concerned, the
presence of micrometastases was associated with a
hazard ratio of 2,26 (95 CI 1,72-2,97, plt0.001)
Kaplan-Meier curves demonstrated that both
overall survival and carcinoma-related survival
were significantly lower in patients with
bone-marrow colonization. Despite these data on
the predictive value of bone-marrow
micrometastasis, the search for this parameter is
not a routine approach in patients, probably due
to its invasiveness indeed, repeated
bone-marrow biopsies over time would be
required.
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Braun et al., NEJM, 353 (8) 793, Table
1     August 25, 2005
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KaplanMeier Estimates of Long-Term Survival and
Outcome in the Complete Patient Group According
to the Presence or Absence of Bone Marrow
Micrometastasis (ibidem)
6
Disseminated Tumor Cells (DTC) or Circulating
Tumor Cells (CTC)?

• the ability of tumor cells to migrate from the
  primary tumor site and seed to distant locations
  is a complex property, which relies on both tumor
  cell features and the tissue microenvironment

7
The metastatic process

• Pantel K, Brakenhoff RH.
• Nat Rev Cancer. 2004

8
CTC ? 1 cell/108 leukocytes

• Jean-François Millet, The Gleaners, 1857

9
DTC may be revealed in different
compartments Bone-marrow Peripheral blood
(CTC) Tissues
DTC/CTC may be detected by different
approaches Immunohistochemistry Molecular
analysis Cytofluorimetric techniques
10
Criteria for an optimal tumor-cell detection
method (ISHAGE 1999)

• Specificity
• Sensitivity
• Reproducibility
• Robustness
• Objective read-out
• Potential for automated analysis
• Quantitation of tumor load
• Characterization of tumor cells
• Proven clinical significance

11
Immunochemistry approaches for DTC detection

• Pantel K, Brakenhoff RH.
• Nat Rev Cancer. 2004

12
Markers utilized for DTC detection in
immunohistochemical approaches

• citokeratins
• Cytokeratins are a family of molecules
  differentially expressed in different epithelial
  compartments
• what is mostly specific for breast carcinoma?

13

• What are the properties of an "ideal" neoplastic
  marker?
• It should be expressed
• in tumor cells but not in normal tissue(s)
• evenly during the entire natural history of the
  disease

14
Molecular approaches for DTC detection

• ? relatively poorly invasive approach
• ? relatively high sensitivity (10-3 - 10-6)

15
Markers utilized for DTC detection in molecular
approaches

• cytokeratin 19
• maspin
• mammaglobin
• CEA
• EGFR
• MUC-1
• HER2
• .................

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Molecular analysis od DTC/CTC conclusions

• No consensus on
• the antigen to be used (cytokeratin or what ?)
• the technique to be used for antigen detection
• (RT-PCR, nested PCR or real-time PCR?)
• ? the major limit seems to be the relatively
  poor specificity

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Immunocytometric approaches for CTC detection

• ? high sensitivity (10-6) and specificity
• ? "single cell" analysis
• ? consensus on classification criteria (ISHAGE
  Working group)
• ?the sensitivity may be increased by appropriate
  enrichment procedures before analysis

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CTC enrichment by filtration (ISET)

• periphearl blood is diluted and passed through a
  polycarbonate membrane (8 m pores)
• the membrane is washed and cells are stained with
  anti-cytokeratin(s)

19
Immunomagnetic enrichment of CTC

• may be preceded by a density gradient
• positive selection (anti-EpCam,
  anti-Cytokeratins)
• negative selection (anti-CD45)

20
CellTracks Products for Cellular Analysis
Kits for Cell Counting
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Immunomagnetic labeling and immunofluorescent
identification of cells
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Aspirate plasma. Add anti-EpCAM Ferrofluid.
1
anti-EpCAM Ferrofluid
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Magnetic incubation.
2
24
Aspirate fluid and unlabeled cells.
3
25
Remove magnets. Re-suspend.
4
26
Permeabilize. Add Staining Reagents.
5
anti-CK-PE
DAPI
anti-CD45-APC
27
Transfer to MagNest device.
6
28
Scanning and Analysis with the CellTracks
Analyzer II
CK- DAPI CD45
EpCAM CK DAPI CD45-
CTC
leukocyte
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Analysis of Tumor Cells
1 2 3 4 5 6
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Breast- Single- Round to Oval Cells
Breast- Clusters
Breast- single cells with blebs
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Breast- Single Cells - Elongated
Breast- Single Cells - Odd Shapes
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Cut-off number for the CTC assay (Cristofanilli,
NEJM 2004)

• Training set results from the first 102 patients
  enrolled in the trial were used to select a cut
  off level of CTCs to stratify the patients into
  two groups favorable and unfavorable prognosis.
• Validation set The cut-off level was then
  validated with the use of the 75 subsequently
  enrolled patients.
• Stratification according to levels of CTCs
  thresholds of 1 to 10,000 cells for the baseline
  levels were systematically correlated with PFS
  for the 102 patients in the training set.
• (Cristofanilli, NEJM 2004)

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Measurable Metastatic Breast Cancer
CTCs Predict Outcome
4.6 2.5 weeks after initiation of therapy
.
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Change in CTCs Predict Survival
lt 5 CTCs at First and Last Draw 83 (47) gt5 CTCs
at First and lt5 at Last Draw 38 (21) lt5 CTCs at
First and gt5 Last Draw 17 (10) gt5 CTCs at First
and Last Draw 39 (22)
green vs. red p0.0001 green vs. blue
p0.3188 green vs. orange p0.0014 blue vs. red
p0.0002
Package Insert, October 2005Clinical Cancer
Research, July 2006
35
gt5 CTCs at 1st and lt 5 CTCs at last time point
n41 (23) PFS 9.5 (4.9 11.5) OS 23.8
(14.6-NR)
10
Age 65 ER/PR() HER
-
2(unk)
Age 65 ER/PR() HER
-
2(unk)
Age 65 ER/PR() HER
-
2(unk)
Age 65 ER/PR() HER
-
2(unk)
20
Age 73 ER/PR (
-
) HER
-
2 (
-
)
Age 73 ER/PR (
-
) HER
-
2 (
-
)
Age 73 ER/PR (
-
) HER
-
2 (
-
)
Age 73 ER/PR (
-
) HER
-
2 (
-
)
19
Mets Dx 14.5 yrs Sites Liver, LN, Bone, Pleural
Effusion, Other
9
18
Mets Dx 1.5 yrs Sites Liver, Bone, Skin
Mets Dx 1.5 yrs Sites Liver, Bone, Skin
Mets Dx 1.5 yrs Sites Liver, Bone, Skin
Mets Dx 1.5 yrs Sites Liver, Bone, Skin
17
5
th
Line Therapy
th
Line Therapy
th
Line Therapy
th
Line Therapy
8
16
2
Line Therapy
2
Line Therapy
2
Line Therapy
2
Line Therapy
nd
nd
nd
nd
15
7
14
13
6
12
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Baseline
Baseline
Baseline
Baseline
CTCs Per 7.5mL of Blood
5
10
9
9
9
9
9
9
Visit 1
Visit 1
Visit 1
Visit 1
Visit 1
Visit 1
Visit 1
Visit 1
4
8
8
8
R1 PD
R1 PD
8
8
8
R1 PD
R1 PD
R1 PR
R1 PR
R1 PR
R1 PR
Baseline
Baseline
Baseline
Baseline
7
7
7
R2 PD
R2 PD
7
7
R2 PD
R2 PD
R2 S
R2 S
R2 S
R2 S
Progression
Progression
Progression
3
Progression
6
6
6
6
6
Progression
Progression
Progression
Progression
Death
Death
Death
Death
5
5
5
5
5
Alive
Alive
Alive
Alive
4
4
4
4
4
2
3
3
3
3
2
2
2
1
1
1
0
0
0
0
0
0
0
14
0
1
2
3
4
5
14
0
1
2
3
4
5
17
0
1
2
17
0
1
2
14
0
1
2
3
4
5
14
0
1
2
3
4
5
17
0
1
2
17
0
1
2
Time (months)
Time (months)
Time (months)
Time (months)
Time (months)
Time (months)
Time (months)
Time (months)
Adriamycin
Navelbine
5-FU Adriamycin Cytoxan
Slow decrease n12 PFS 2.2 (1.9 11.5) OS
15.1 (4.8 -NR) Consider Alternatives
Fast decrease n29 PFS 9.6 (6.1 14.4) OS
gt16.2 (16.2 -NR) Stay the Course
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lt5 CTCs at 1st and gt 5 CTCs at last time point
n19 (10) PFS 5.0 (2.5 7.0) OS 20.8
(7.9-23.6)
Iressa
Aromasin
Change Treatment
Change Treatment
Start 0 CTC, n7 PFS 3.7 (0.9 10.7) OS 21.6
(10.5 -NR)
Start 1-4 CTC, n12 PFS 5.0 (2.3 7.5) OS
28.6 (20.4 -NR)
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Patients and Methods

• The Veridex platform is now operating at the
  Istituto Oncologico Veneto-IRCCS in Padova. We
  are addressing the validation of CTC count as a
  predictive marker of metastasis and therapeutic
  response in the follow-up of breast cancer
  patients. To this purpose we enrolled 45 breast
  cancer patients, stratified according to disease
  condition before surgery, during adjuvant
  treatment or MBC patients. Blood was also drawn
  from healthy control subjects who had no known
  illness at the time of bleeding and no history of
  malignant disease.

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CTC Frequency at the first blood draw

• 45 patients (44 women and 1 man)
• Age 22-80 years (median 58 years)
• No CTC were detected in healthy donors

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Table 1. Patient's characteristics according to
CTCs baseline values
CTC presence did not correlate with tumour size,
lymphnode or Her-2 status at diagnosis
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CTC Frequency according to disease condition
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CTC variation during chemotherapy treatment
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MAY A CLOSE SURVEY OF CTC IN PATIENTS HELP IN
MONITORING THE NATURAL HISTORY OF THE DISEASE AND
FORESEE DISEASE PROGRESSION?
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CTC in follow-up 5/7.5 ml

• ?62 yr-old
• 11/2005 sentinel node negative malignant mammary
  nodule
• infiltrating ductal ca. G3
• pT1b N0, ER e PGR lt10, Hercept test ---
• adjuvant chemotherapy (Epirubicin, 4 cycles
  CMF) RT
• 1/2007 chest Rx neg abdominal eco neg
• normal CEA and Ca15.3 levels
• 3/2007 Ribavirin and alphaIFN for HCV (withdrawn
  on July 2007)
• 9/2007 normal CEA and Ca15.3 levels

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POSSIBLE FUTURE DEVELOPMENTS
Extending CTC analysis to tumors other than
mammary carcinoma
Using the Veridex platform as a tool for
investigative purposes on the nature and biology
of CTC
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CTC expression by cancer type
Clinical Cancer Research Vol.10, 6897-6904
October, 2004
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QUESTIONS TO BE ANSWERED
do CTC simply reflect tumor burden? are CTC
viable cells or pre-apoptotic/apoptotic
entities? do CTC mirror the phenotypic and
genotypic profile of the primary tumor mass
(tumor dormancy)? do CTC possess "stemness"
properties (ability to colonize tissues in the
immunodeficient host)?
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Immunomagnetic CTC Selection
Processing by the CellTracks AutoPrep System
Magnetic incubation
48
A
Composite CD45-APC M30-PE CK-F
DAPI
Intact CTCs
Apoptosis Initiated
Fragments
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Anti-M30-PE to Assess Apoptosis
MCF-7 NT
B

• M30 is a marker of caspase-3 cleavage of
  cytokeratin 18. This marker was used in
  conjunction with the CellTracks Epithelial Cell
  Kit to assess percentage of apoptotic CTC (A).
  Breast cancer cell line MCF7 treated with taxolo
  1/60 served as positive control (B). In a small
  breast cancer pts series 45 of CTC was apoptotic
  (C).

C
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Immunomagnetic Labeling and Immunofluorescent
Identification of Cells
HER2Epithelial Cell
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Examples of CTC
this image was obtained by computer analysis
after staining (DAPI CK (8,18,19) CD45-)
the same cells were isolated by the Immunicon
Profile kit, which allows cell recovery and
phenotypic/molecular analysis
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THANK YOU FOR YOUR ATTENTION!