Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

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Chapter | 25 Tumor Markers
B. Cell Surface Determinants
With the use of specific monoclonal antibodies, the flow
cytometer can be used to characterize cell populations
based on their cell surface determinants. Although particularly
well suited for the study of leukemias, this technique
may also be adapted to the study of solid tumors. Aberrant
expression of cluster differentiation (CD) molecules has
been identified as a common, and distinguishing, feature
of canine lymphomas ( Wilkerson et al. , 2005 ). The flow
cytometer also allows rapid immunophenotyping of lymphomas
using an aspirate specimen ( Culmsee et al. , 2001 ).
Further work is needed to determine how these findings
may correlate with prognosis.
IV. PROLIFERATION MARKERS/
APOPTOSIS
A. Mitotic Counts
Determining the frequency of mitotic figures visible in histological
sections of biopsy specimens is a long-standing
method for assessing cell proliferation. Cells in the mitotic
phase are easily recognizable on routinely prepared specimens.
There are various methods for reporting the mitotic
activity, including number of mitoses visible per certain
number of high, dry microscopic fields or the number of
mitoses present in a certain number of cells (mitotic index).
It has been shown that the mitotic index correlates better
to other indices of proliferation and tumor grade compared
to mitoses per area ( Sarli et al. , 1999 ). Although determining
mitotic counts is technically simple, it is limited in its
usefulness because of lack of reproducibility, and errors in
interpretation may be introduced by delays in tissue fixation,
variation in section thickness or size of microscopic
field of view, and in difficulty recognizing mitoses.
High mitotic index has been reported as a negative prognostic
indicator of various tumors, including canine ocular
melanoma ( Wilcock and Peiffer, 1986 ), feline fibrosarcoma
( Bostock and Dye, 1979 ), and canine soft tissue sarcomas
( Kuntz et al. , 1997 ). However, the mitotic index may
have limited value for predicting the behavior of canine
melanoma ( Spangler and Kass, 2006 ), canine skin cancers
( Martin De Las Mulas et al. , 1999 ), or canine lymphoma
(Kiupel et al. , 1999 ).
B. Thymidine-Labeling Index and
BrdU Incorporation
The synthesis of DNA occurring in the S phase of the cell
cycle can be measured by labeled DNA precursor incorporation.
Although there are radiometric methods using tritiated
thymidine, bromodeoxyuridine (BrdU), a halogenated
thymidine analogue, is more commonly used for direct
estimation of DNA synthesis. The use of specific monoclonal
antibodies to BrdU allows immunohistochemical
detection in paraffin-embedded sections with simultaneous
morphological examination of the tissues. The thymidine
labeling index is defined as the ratio of the number
of positively stained cells to the total number of cells.
The S-phase fraction of the cell cycle can be determined
by counting nuclei labeled with BrdU providing an accurate
assessment of the proliferative capacity of the tumor.
One drawback to this technique is that it requires infusing
patients with BrdU before surgery to permit its incorporation
into tumor DNA.
BrdU incorporation and calculation of tumor potential
doubling time has been associated with biological behavior
and prognosis in canine chondrosarcoma, osteosarcoma,
epulides, and lymphoma ( Ohta et al. , 2004 ; Schwyn et al. ,
1998 ; Vail et al. , 1996 ; Yoshida et al. , 1999 ).
C. Nucleolar Organizing Regions
Quantifying the proteins associated with nucleolar organizing
regions (NORs) of interphase chromosomes is another
method to assess the proliferation rates of tumors. The
NORs are visualized microscopically with a simple silverstaining
method that is effective because of the argyrophilia
of their nonhistone acidic proteins. The staining procedure
can be done on both histological and cytological specimens.
These argyrophilic nucleolar organizing regions (AgNORs)
are representative of actual or potential transcriptional activity
of ribosomal DNA and may be used as a marker for cell
proliferation. AgNOR counts may be expressed as the mean
number of AgNORs per nuclei or as the percentage of tumor
cells with 5 AgNORs per nucleus. It is believed that the
mean AgNOR count reflects DNA ploidy, whereas the percentage
of tumor cells with 5 AgNORs per nucleus represents
the proliferative activity.
Increasing AgNOR scores have been shown to be predictive
of poor outcome in canine soft tissue sarcoma ( Ettinger
et al. , 2006 ), canine mast cell tumors ( Bostock et al. , 1989 ;
Scase et al. , 2006 ; Simoes et al. , 1994 ), canine mammary
gland tumors ( Sarli et al. , 2002 ), canine lymphoma ( Kiupel
et al. , 1998, 1999 ; Vail et al. , 1996 ), transmissible venereal
tumors ( Harmelin et al. , 1995 ), and feline mammary carcinoma
( Preziosi et al. , 2002 ). However, AgNOR scores do
not seem to be predictive of outcome in feline lymphoma
(Rassnick et al. , 1999 ; Vail et al. , 1998 ), and other studies
suggest that AgNOR scores do not predict outcome in
canine mammary gland tumors ( Lohr et al. , 1997 ) .
D. Proliferation Markers
Methods for detection of cell cycle-related antigens are
described in Section V.D.