IMMUNOHISTOCHEMISTRY AND CANCER PROGNOSIS ASSESSMENT USING CYTOLOGIC SPECIMENS
Hormone Receptor Assays
The use of immunocytochemical techniques to determine the presence of estrogen and progesterone receptors in
breast carcinoma fine needle aspirates has been reviewed by numerous investigators.54-63 The same
technical factors influencing IHC in general impact on the interpretation of nuclear immunoreactivity as an
indicator of steroid hormone receptor status. False negative and false positive IHC reactions may result
from technical factors involving specimen fixation and preservation that will lead to false interpretation
of the staining results. Generally, there is good to excellent correlation between IHC whether a
qualitative scoring system or quantitative image analysis based interpretation is used and the traditional
competitive binding dextran coated charcoal biochemical assays.54 Estrogen receptor immunostaining has
been successfully performed on archived Papanicolaou-stained imprints slides.55 Although the IHC method
allows confirmation that tumoral tissue is expressing the receptor protein antigens and the biochemical
methods, on occasion, produce erroneous results when insufficient tumoral nuclei are included in the
submitted cytosol, the biochemical technique is generally preferred in that it is a true functional assay of
estrogen or progesterone binding to the tumor tissue. Current breast cancer screening programs result in
small breast carcinoma tissue samples with limited cellular material available for hormone receptor
measurements. Thus, the IHC method has recently become the predominant technique used to determine hormone
receptor status. Among the errors and pitfalls in the IHC hormone receptor assay, of major importance are
rare cases of mutated hormone receptor genes which produce receptor proteins that stain with available
commercial antibodies but do not physiologically function to bind estrogen. In clinical practice,
approximately 10% of patients with positive IHC hormone receptor assays fail to show response to hormonal
therapy. This observation may, in part, be the result of a mutated receptor and a biologic false positive
IHC reaction. Hormone receptor assays will, on occasion, result in negative staining due to binding of the
receptor in patients receiving estrogen therapy. False negative reactions are also described in
premenopausal patients and patients receiving radiation therapy.54-57 Finally, immunoreactivity for
estrogen receptor protein is not specific to breast cancer nuclei, and carcinomas of other primary sites
including ovary, endometrium, lung and others may be immunoreactive. Thus, IHC for hormone receptors cannot
be used as a method of confirming breast origin in a cytologic sample obtained with a differential diagnosis
of carcinoma of unknown primary site.
Cell Proliferation Markers
A list of the techniques that can be utilized for cytologic specimens to determine the proliferative
compartment or cell proliferation index is provided in Table 5. Deregulation of the cell cycle is considered
to be a major factor for the development and progression of most malignancies.63-65 The differing
methods vary in their applicability to cytologic specimens and may be too tedious and cumbersome for most
laboratories. There is a general lack of standardization of the techniques including defined ranges for
low, medium and high proliferation for various neoplasms. Further testing in the clinical arena appears
warranted before they can fully become a part of the standard of practice.63-65
Table 5. Techniques for Measurement of Cell Proliferation
| Technique | Cell Cycle Phase(s) Detected |
| Mitosis counting | M |
| 3H Thymidine Uptake | S |
| BrdU Labelling | S |
| Flow Cytometry | S (calculated) |
| Image Analysis | S (estimated) |
| Ki-67 (Fresh, Frozen Tissue) | G1, S, G2M (part) |
| Ki-67 MIB1 (Paraffin) | G1, S, G2, M (part) |
| PCNA (Paraffin) | G1, S, G2 |
| p105 (Paraffin) | G1, S, G2M |
| AgNOR (Paraffin) | S, G2M (part) |
| Cyclin A | S |
| Cyclin B | S (part), G2, M (part) |
| Cyclin D | G1 (part) |
| Cyclin E | G1 (part), S (part) |
Mitosis counting, tritiated thymidine uptake and brdu labeling are generally not convenient and difficult to
perform on most cytologic preparations. Although the "S" or synthesis phase of the cell cycle can be
calculated by flow cytometry using mathematic modeling of the cell cycle distribution, this method is
considered imprecise (see below). Image analysis estimates of the S-phase are generally inaccurate when
compared with standardized labeling methods that directly measure the proliferative cell compartment.
Recently, cell proliferation has been measured on cytology specimens using a variety of antibodies employing
standard and modified IHC techniques.62, 65 The use of silver nucleolar organizer region counts as
proliferation markers has been applied mostly to surgical pathology specimens although occasional reports
describe use of the technique in cytologic specimens.66-67 Pitfalls in interpretation of AgNOR counts
are related to the silver impregnation technique and have not been extensively considered.
Immunohistochemical analysis of proliferation markers in cytology has focused on the antibodies Ki-67, the
Ki-67 clone MIB-1, and the proliferating cell nuclear antigen (PCNA).62, 68-70 Standard technical issues
resulting in false negative and false positive staining significantly impact on these techniques that
require quantitative assessment. Significant cell volume is required for the determination of accurate cell
proliferation indices. The cell cycle dependent antibodies are very sensitive to technical errors and
require considerable understanding by the interpreter of the cell cycle phase or phases being detected by
the antibody.65 Measurements of PCNA by IHC have been particularly impacted by variations in staining in
the commercially available antibodies which may detect different epitopes of the proliferation associated
antigen and not be directly comparable. Moreover, as previously mentioned, the use of aggressive antigen
retrieval procedures may influence PNCA immunoreactivity and result in virtually 100% staining of microwaved
cells including cells that are not cycling.65 The MIB-1 antibody has become a favorite cell
proliferation marker that can equally be applied to cytology specimens and formalin-fixed paraffin-embedded
surgical pathology tissues.71 Ki67 staining has recently been used to enhance the identification of
malignancy in serous effusion specimens.72 Suggested cut-off points for proliferation compartments are
included in Table 6. Until standardization of IHC techniques for these cell proliferation markers is
achieved and consensus groups recommend cut-off limits for tumor specific low and high proliferation rates,
extreme caution should be taken in the utilization and clinical decision making of IHC determined cell cycle
analysis.
Table 6. Suggested Cut-offs for Cell Proliferation
| Low | Medium | High |
| S PHASE FLOW CYTOMETRY | <5-7% | <7-12% | >12% |
| Ki-67 IMMUNOHISTOCHEMISTRY | <10-14% | <14-20% | >20% |
Molecular Markers Measured by Immunohistochemistry
A wide variety of antibodies have been developed during the past decade designed to measure in human tumors
the presence of oncoproteins, tumor suppressor proteins, growth factors, growth factor receptors, and
invasion and metastasis markers. The standard pitfalls and errors in IHC technique apply to these molecular
marker antibodies whether they are used as an adjunct to diagnosis for cytology specimens or in the
prediction of disease outcome for established cases of malignancy. Although IHC procedures have the
advantage of allowing simultaneous morphologic assessment of the cellular material potentially expressing a
molecular marker, IHC techniques have generally been less sensitive than methods using molecular biology
techniques to directly detect gene amplification, mutation or dysfunction. The p53 protein has been
evaluated by IHC in cytologic specimens both as a method to detect the presence of malignant cells and to
predict prognosis in malignant breast tumors and other cancers.21,70,73,74 The major pitfall in this
approach is the fact that among the commercially available antibodies, a significant percentage
(approximately 5-10%) of immunoreactive nuclei for p53 protein are positive for increased wild-type protein
and are not specific for p53 gene mutation when subsequent sequence analysis of extracted DNA from the same
tumor is analyzed.75 P53 staining has recently been employed to improve the detection rate
ofadenocarcinoma in serous effusion samples.76
Another molecular marker frequently evaluated by IHC and applied to cytology specimens especially in breast
fine needle aspirates is the HER-2/neu oncoprotein, a cell surface receptor protein similar to epidermal
growth factor receptor.62,77 Although a relatively cancer-specific gene and protein when overexpressed,
IHC procedures to detect HER-2/neu gene amplification have been influenced by varying sensitivity in
formalin-fixed, paraffin-embedded tissues of commercially available antibodies to the HER-2/neu protein.78
Thus, false negative reports indicating no amplification by IHC may occur in cases in which molecular
analysis by either Southern blotting or in-situ hybridization show gene amplification. In a recent study,
HER-2/neu protein overexpression was seen by IHC to be significantly greater in alcohol-fixed FNA specimens
than in the corresponding paraffin blocks from the resected breast cancer tissues.79
Ras gene mutations are frequent in a variety of human malignancies and have been detected by molecular
methods in cytologic specimens as an ancillary method of confirming the presence of malignancy.80-82
Another example of an error in the application of IHC techniques to cytology is the false positive
immunoreactivity for ras gene associated proteins that mistakenly conclude that a ras gene is mutated or
that the wild type gene is amplified.83 Cell adhesion molecule staining has been used to identify
malignant cells in urothelial cytology samples84 and in the classification of thyroid aspirates.85-86
Heterogeneity of Immunohistochemical Staining
Intermediate filament staining may vary from area to area in a solid tumor; heterogeneous expression of
lymphocyte surface markers is well documented; and tumor specific glycoprotein expression may vary
considerably in a single neoplasm. These examples of IHC staining heterogeneity highlight potential errors
which may occur when the technique is applied to cytologic specimens which often feature limited cellularity
that may not be representative of the overall IHC pattern of the entire neoplasm.
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