|
|
|
|
|
|
|
|
|
|
|
|
|
Morphologic Characterization of Circulating Tumor Cells:
The detection of CTCs may have
important prognostic and therapeutic implications but because their numbers can
be very small, these cells are not easily detected.
CTCs have been observed in the peripheral blood of patients with
epithelial-derived cancers at ultra low concentrations of 1 in 106 to 107
peripheral blood mononuclear cells. (Gross,
Verwer et al. 1995;
Kraeft, Ladanyi et al. 2004)
These CTCs can be detected and analyzed by using immunofluorescent microscopy
with immunocytochemical markers such as CK (antibodies against cytokeratins) and
use of nuclear staining with DAPI. Prospective clinical trials have established
that these CK+ cells are malignant (
Fehm, Sagalowsky et al. 2002) and their presence in blood is correlated with
poor outcome and lower survival rate in a number of studies .(
Cristofanilli, Budd at al. 2004;
Cristofanilli, Hyes et al. 2005)
CTC Identification: Fluorescent Microscopy
|
 |
Criteria for CTC identification from fluorescent images consists of CK+
fluorescence in both Alexa 555 and Alexa 488, with an appropriate CK-typical
staining pattern, overlying the DAPI nuclear stain. To investigate the
morphologic detail of CTCs, a subset of identified CK+ individual rare cell
clusters from this population were relocated and further evaluated with the
Wright-Giemsa stain by routine bright-field microscopy. (It yields information
in nuclear detail, nuclear contour, presence/absence of nucleoli, quality of
cytoplasm, amount of cytoplasm, and allows for fine qualitative distinctions
that are not possible on a fluorescent-only image.)
Cell Identification Criteria:
- CD45- (Green)
- CK+ (Red)
- DAPI Nucleus (Blue)
- Appropriate cytoskeleton staining pattern
|
CTC Pleomorphism
Complementing fluorescent analysis with light microscopy analysis
creates detailed nuclear and cytoplasmic information, demonstrating pleomorphism
within the population. There were identified CTCs and CTC clusters with variable
nuclear-to-cytoplasmic (N/C) tumor tissue as well as CTCs that exhibit apoptotic
hallmarks.
CTC pleomorphism in the blood of metastatic colorectal
patients.
|
Cytomorphologic Comparison of CTCs to the Primary Tumor
- Fluorescently labeled CTC (Red: cytokeratin, Green: CD45, Blue: DAPI); 60x,
digitally enlarged.
- Same CTC, restained with Wright-Giemsa; 100X(oil).
- A similar cell from the original biopsy four years prior with arrow pointing
to lobated nucleus, 100X (oil)
Cytologically, the primary and metastatic tumor deposits show pleomorphism
among the individual cells of the tumor tissue. Circulating cells from these
same patients retain this cytomorphology, showing overall pleomorphism with
variations in size, shape , and N/C as well. The CTCs show nuclear contour
irregularity and lobation, with eccentrically located cytoplasm corresponding to
primary tumor type cells.
It supports the premises that CTCs retain primary tumor cytologic
characteristics, and that CTCs represent a random
sampling of many phenotypic cell types present in the primary and metastatic
tumor deposits.
<< Back to top
<< Back / Next >>
|
|
|
|
 |
|
|
|
|