Circulating tumor cells (CTCs) have potential clinical applications as biomarkers in colorectal cancer. Studies have found CTCs correlate with disease stage but not other clinical factors. Detecting CTCs before and during treatment can independently predict progression-free and overall survival. While CTC detection provides prognostic information, methodology challenges remain around isolating, quantifying, and characterizing CTCs reproducibly. Further research could help validate CTCs against standard biomarkers and guide personalized therapy.

1. Circulating Tumor Cells in
colorectal cancer

2. Discussion for today
• What are CTC
• Biology of cancer metastasis
• Facts and hypothesis
• How do we measure CTCs?
• Clinical applications and studies
• Summary of the World CTC Summit
• A SWOT analysis

3. Glossary
• CTC Circulating tumor cells
• DTC Disseminated tumor cells
• EpCAM Epithelial Cell Adhesion Molecule
• CK Cytokeratin
• EMT Epithelial Mesenchymal Transition
• MET Mesenchymal Epithelial Transition

4. What are CTCs?
• Tumor cells in circulation!
• CTCs are biomarkers
(substance/s used as an indicator of a biological
state which can be measured objectively)
• They can be used as a Indicator of
1. Normal or pathogenic biological processes
2. Responses to a therapeutic intervention
3. Prognostic vs predictive biomarkers

5. The biology of cancer metastasis

6. The biology of cancer metastasis

7. Some facts…
• Disseminated tumor cells – MC seen in
Bone Marrow of patients with varied ca.
• Seen in 20-40% pts who are clinical N0 M0
• Seen even in cancers which conventionally
have low rate of bone metastases
• Significant correlation between detection of
DTCs and metastatic relapse
• Not all patients with DTCs relapse (50%)

8. Some hypotheses
• A fraction of DTCs in early stage cancers
represent putative metastatic stem cells
• DTCs can be used to monitor response to
systemic (anti-metastatic) therapy
• The ‘lead interval’ between biomarker
detection and overt metastases provides
an intermediate window between early and
metastatic disease, enabling treatment

9. How do we measure CTCs?

10. How do we measure CTCs?
Enrichment
Identification

11. Methods for CTC detection
• Physical and biological properties
• Capture and enrichment – morphological
and phenotypic characteristics of tumor
cells
– Size, Density and protein expression
• CTC identification
– Immunocytochemistry (protein expression)
– Nucleic acid characteristics (RT-PCR)

12. Enrichment methods
• Isolation by Size of Epithelial Tumor
(ISET) Cells
– Cells > 8 micron
• Density gradient (Tumor cells have lower
density than other cells)
– Ficoll-Hypaque
• Based on immunomagnetic techniques
– Separation based on specific surface markers

13. Immunomagnetic separation
• Most commonly used
• MACS, RARE, AdnaGen, CellSearch
• Differentiate tumor cells from other cells by
specific surface markers
• Positive selection – cytokeratin
• Negative selection – CD-45
• More sensitive than size and density
based methods

14. CTC Identification
• Immunocytochemistry
– Uses monoclonal antibodies against specific
tumor types (CK, Mg, CEA)
– Identifies intact cells (preserves the cell)
• RT-PCR
– Assess DNA/RNA changes specific to tumor
cells
– Amplifies minute quantities of tumor RNA
– More sensitive (false +ve: non-CTC mRNA)

15. The CellSearch
• Only FDA approved method
• 7.5 ml blood centrifuged
• Plasma and buffer coat removed
• CTCs captured using EpCAM labelled
ferrofluid
• Differentiated from WBCs by panel of
monoclonal antibodies
– Pan-CK, anti CD-45, nucleic acid dye

16. The CellSearch
• Labelled sample loaded into a cassette
and analysed (CellTracks) – an automated
fluorescence detection system
• CTCs detected as
– Nucleated cells
– CK +ve
– CD-45 -ve
• Highly reproducible

19. Different markers
• K- ras and p53
• Expression of CEA mRNA.
• Expression of CK20 mRNA.
• Expression of CK19 mRNA.

20. Clinical evaluation of CTCs in
CRC

21. • Circulating Tumor Cell Clusters in the
Peripheral Blood of Colorectal Cancer
Patients
Bela Molnar,1 Andras Ladanyi, Lenke Tanko, Lydia Sre´ter, and Zsolt Tulassay II.
Department of Medicine, Semmelweis University, Budapest 1088, Hungary
• Results: Of 20 healthy samples, 2 contained one cytok- eratin-
positive cell. Of 32 single samples from malignant cases, 24 showed
cytokeratin-positive cells. Tumor cell clus- ters, mixed-cell doublets
(one cytokeratin-positive and -neg- ative cell), and mixed-cell
clusters were detected in 22 of 24 patients. In six cases, cytokeratin-
positive dendritic-like cells were detected. Follow-up data indicate
that chemotherapy cannot destroy all of the circulating tumor cell
clusters.

22. • Conclusions: Using the methods
presented, we could detect circulating
colon cancer cells and cell clusters in
colon carcinoma patients. Similar cellular
structures were de- scribed previously only
in rats. Present data prove that such
structures are present in human colorectal
cancer, too.

23. Circulating tumor cells in colorectal cancer:
correlation with clinical and pathological
variables
J. Sastre1*, M. L. Maestro2, J. Puente1, S. Veganzones2, R. Alfonso1, S. Rafael2, J. A. Garcı ´a-Saenz1, M. Vidaurreta 2,
M. Martı ´n1, M. Arroyo2, M. T. Sanz-Casla 2 & E. Dı ´az-Rubio1 1Department of Medical Oncology, 2Department of Central
Laboratory, Hospital Clı´nico San Carlos de Madrid, Madrid, Spain
Received 24 September 2007; revised 26 November 2007; accepted 30 November 2007

24. • Results: Positive CTCs were detected in 34 of 94
patients (36.2%).
• No correlation was found between positive CTCs and
location of primary tumor,
increased carcinoembryonic antigen level,
increased lactate dehydrogenase level or
grade of differentiation.
Only stage correlated with positive CTCs (20.7% in stage
II, 24.1% in stage III and 60.7% in stage IV, P = 0.005).

25. • Conclusions: CTCs detection by
CellSearch is a highly reproducible
method that correlates with stage but not
with other clinical and morphological
variables in patients with colorectal
cancer. Colon cancer tumor cells are
detectable in all stages. Further studies
are warranted

26. Relationship of Circulating Tumor Cells to
Tumor Response, Progression-Free
Survival, and Overall Survival in Patients
With Metastatic Colorectal Cancer
Steven J. Cohen, Cornelis J.A. Punt, Nicholas Iannotti, Bruce H. Saidman, Kert D. Sabbath, Nashat Y. Gabrail, Joel Picus, Michael
Morse, Edith Mitchell, M. Craig Miller, Gerald V. Doyle, Henk Tissing, Leon W.M.M. Terstappen, and Neal J. Meropol
• J Clin Oncol 26:3213-3221. © 2008 by American Society of Clinical Oncology
• Patients and Methods In a prospective
multicenter study, CTCs were enumerated in the
peripheral blood of 430 patients with mCRC at
baseline and after starting first-, second-, or
third-line therapy. CTCs were measured using
an immunomagnetic separation technique.

27. • Results Patients were stratified into unfavorable
and favorable prognostic groups based on CTC
levels of three or more or less than three
CTCs/7.5 mL, respectively. Patients with
unfavorable compared with favorable baseline
CTCs had shorter median progression-free
survival (PFS; 4.5 v 7.9 months; P .0002) and
overall survival (OS; 9.4 v 18.5 months; P
.0001). Differences persisted at 1 to 2, 3 to 5, 6
to 12, and 13 to 20 weeks after therapy.

28. • Conversion of baseline unfavorable CTCs to
favorable at 3 to 5 weeks was associated with
significantly longer PFS and OS compared with
patients with unfavorable CTCs at both time
points (PFS, 6.2 v 1.6 months; P .02; OS, 11.0 v
3.7 months; P .0002). Among nonprogressing
patients, favorable compared with unfavorable
CTCs within 1 month of imaging was associated
with longer survival (18.8 v 7.1 months; P
.0001). Baseline and follow-up CTC levels
remained strong predictors of PFS and OS after
adjustment for clinically significant factors.

29. • Conclusion The number of CTCs before
and during treatment is an independent
predictor of PFS and OS in patients with
metastatic colorectal cancer. CTCs
provide prognostic information in addition
to that of imaging studies.

43. advantages
• Can test both the hypotheses in single pt
• Each person acts as his own control – excludes
bias
• No of trial pts will increase
• Easy to perform
• If IMV is clamped at the beginning if surgery the
tumor cells released in portal circulation can be
trapped and if systemic tumor cells are less at
the end of surgery support our hypothesis of
tumor cell release into systemic circulation

44. Strengths – Established
applications of CTCs
• Primary assessment of recurrence risk
– Provides prognostication beyond conventional
prognostic markers
• Monitor response to treatment
• Provide molecular characterisation of cells

45. Weaknesses - Challenges in
CTC detection
• Isolating, quantifying and molecularly
characterising CTCs is extremely
challenging
• Very little standardisation and automation
of processes
– Laborious sample preparation procedures
– High intra and inter lab differences
– Reproducibility

46. Opportunities - the promise of
CTCs
• Prospective validation of CTCs against
conventional biomarkers
• Markers assessment to guide choice of
personalized therapy
• Identify new therapeutic targets
• Mutation analysis of CTCs can enable
targeted therapy
• Alternative to invasive biopsies

47. Threats
• Has to compete with other biomarkers for
reliability, feasibility of performance
• Expensive technology
• Labour intensive

48. Summary
• CTCs are promising biomarkers with
independent prognostic ability
• Clearly prognostic in varied advanced
cancers
• Methodology is challenging and expensive
• Has promise in varied advanced
applications

49. • Not everything that can be counted
counts, and not everything that counts can
be counted