Page 92 - Withrow and MacEwen's Small Animal Clinical Oncology, 6th Edition

CHAPTER 3 The Pathology of Neoplasia 71

This likely reflects the situation in veterinary medicine. In a sub- histochemical staining technique that has demonstrated prognos-
set of cases, and for clinicians seeking further prognostic and/ tic relevance in canine lymphoma, 118 MCTs, 119 and mammary
120
tumors.
or predictive information, ancillary tests, such as histochemical

VetBooks.ir stains, IHC, TEM, PCR, and flow cytometry, may be necessary
(see Chapter 8). As molecular and genomic research in veterinary
oncology continue to evolve, new ancillary tests also will likely Immunohistochemistry
emerge, resulting in greater sophistication in tumor diagnoses, IHC can aid the classification of several tumors in veterinary
prognostication, and theranostics, including more frequent iden- medicine and is a widely used diagnostic technique. IHC is a
tification of novel therapeutic targets. 117 staining procedure that uses commercial antibodies to iden-
tify specific cellular and extracellular molecules ex vivo, such as
Special Histochemical Stains cytoplasmic intermediate filaments, cell surface markers, or even
secretory substances; all “molecules” or “markers” are tissue pro-
Histochemical stains consist of chemical substances that, when teins, also referred to as tissue antigens. IHC can be performed
applied to tissue sections, result in a direct chemical reaction on frozen sections or specimens routinely fixed in formalin and
with the tissue’s constituents. For all intents and purposes, rou- processed into paraffin blocks. Tissue sections are incubated with
tine H&E is a histochemical stain in which hematoxylin reacts primary antibodies to specific cell proteins (antigens). Sections
with nucleic acid and eosin with cytoplasmic protein. Some com- with bound primary antibody are then exposed to secondary
mon histochemical stains that can be used in veterinary oncologic antibodies directed against the primary antibody. The secondary
pathology to assist in the diagnosis of certain poorly differenti- antibodies are linked to peroxidase or avidin-biotin peroxidase
ated tumors are listed in Table 3.6. Stains that identify extracel- complexes. The peroxidase catalyzes a reaction in the presence
lular matrices that may indirectly support tumor histogenesis of dye that precipitates at the site of the complex and is visible
include Masson trichrome (collagen; fibrocytic tumors), Alcian with light microscopy. 121,122 As an alternative, alkaline phospha-
blue (proteoglycans; myxoma, myxosarcoma), and Congo red tase enzyme systems also are available. A list of common diagnos-
(amyloid; plasma cell tumors, amyloid producing odontogenic tic IHC markers used in veterinary oncology, and the respective
tumors). Although histochemical stains often are used for poorly tumor types for which their use is indicated, is provided in
differentiated tumors, the stains themselves do not differentiate Table 3.7. ICC uses similar technical aspects; however, it is per-
between benign and malignant. With the advancement of IHC, formed on cytologic samples obtained from an aspirate or impres-
many histochemical stains have lost popularity; however, they are sion smear; this technique has been increasing in popularity over
still available and can be useful in the appropriate setting. Silver recent years. 123
staining of nucleolar organizer regions (AgNOR) is an additional IHC also may be prognostically useful; for example, it can assist in
evaluating proliferation or the tumor growth fraction through detec-
tion of markers such as Ki67 (MIB-1) and PCNA. 124 Prognosis also
TABLE 3.6 Special Histochemical Stains to Support has been linked to markers of multidrug resistance (e.g., P-glyco-
Tumor Histogenesis protein) 125 and altered proto-oncogenes or tumor suppressor genes
(e.g., p53, c-kit/CD117, p21, Rb, and PTEN). 81,82,126–128 Other
Tumor Type (Reactive immunohistochemically detectable markers linked to the prognosis
Constituent) Histochemical Stain Reference
include VEGF and its receptors, 129 cyclooxygenase-2, 68,130–132 and
Granular cell tumor Periodic acid-Schiff (PAS) 160,161 platelet-derived growth factor receptor. 133 Many other potential
(cytoplasmic lysosomes) prognostic markers in a variety of veterinary tumor types detect-
able by IHC have been and continue to be studied, (e.g., epidermal
Liposarcoma and lipid-rich Oil Red-O, Sudan stains 162–165 growth factor receptor, human epidermal growth factor receptor-2,
neoplasms (lipid) (black, III, IV) a
urokinase plasminogen activator, heat shock proteins). 134–136 As the
Mast cell tumor (cytoplas- Toluidine blue, Giemsa, 17,34 realm of IHC in veterinary medicine continues to expand, so, too,
mic granules) PAS b will the discovery of tumor-specific diagnostic markers and markers
Melanoma (melanin) Fontana-Masson c 166,167 for prognostic (biologic aggressiveness) and predictive (therapeutic
responsiveness) utility.
Neuroendocrine tumor Silver stains (Pascual’s, 168,169 Although IHC can be a valuable tool, some complicating fac-
(cytoplasmic granules) Grimelius, Sevier- tors exist. A negative stain does not exclude a certain cell type.
Munger), Churukian- Technical components or tumor cell dedifferentiation, with loss
Schenk
of antigen expression, may result in a negative result. One of the
Plasma cell tumor (amy- Congo red 170–172 more common technical problems that can cause negative stain-
loid), APOT (amyloid) ing is prolonged formalin fixation that results in excessive cross-
Rhabdomyosarcoma (myo- Phosphotungstic acid- 173 linking of the antigenic components or loss of soluble proteins into
cyte cross striations) hematoxylin (PTAH) the fixative. Antibody-specific antigens (epitopes) that have been
masked by protein cross-linking often can be “unmasked” by pre-
a These stains must be performed on nonprocessed tissue because exposure to xylene during treating sections with trypsin or pepsin or by using heat-induced
processing dissolves lipid components. epitope retrieval techniques. 121,122 Decalcification of tissue may
b May be used in feline and ferret mast cell tumors because the granules in these species are also result in alteration of target proteins so that they are no lon-
often better visualized with PAS. ger recognized by the respective antibody. The type and duration
c Reacts with melanin, which is also visible on routine light microscopy and therefore may
be beneficial when trying to confirm melanin versus an alternative cellular pigment (e.g., of decalcifying solution, however, may mitigate these deleterious
hemosiderin, lipofuscin). Melanin bleach may provide similar information. effects. 136,137 Areas of tissue necrosis, autolysis, hemorrhage, sec-
tion drying, and sometimes collagenous matrix components can