SYMPOSIUM PAPER |
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Summary |
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Immunohistochemistry (IHC) has provided major insights about the classification of brain tumors by identifying cellular markers of phenotype and about tumor growth potential with nuclear markers of proliferation. In situ hybridization (ISH) research shows promise for diagnostic applications in tumor classification. The avidinbiotin conjugate IHC procedure is highlighted for diagnostic use on routinely processed clinical specimens. The immunophenotypes of brain tumors are tabulated in reference to their common IHC markers. Tumors that have been correctly classified by their IHC phenotypes include the giant-cell glioblastoma, primary brain lymphoma, and central neurocytoma. Phenotypes that may be more definitively detected by ISH, such as pituitary hormone, immunoglobulin light chain, and collagen messages are described. IHC of nuclear proliferation markers correlates with grade of malignancy, predicts tumor growth potential, and is prognostic for patient survival. The incorporation of bromodeoxyuridine, the expression of proliferating cell nuclear antigen, and the expression of Ki-67 antigen detected by MIB-1 antibody are compared in regard to their cell cycle activity and labeling index determinations. Fluorescence in situ hybridization (FISH) of brain tumor interphase nuclei and chromosomes is described. Abnormal FISH signals of specific chromosomes are associated with different types of brain tumors, with different grades of malignancy, and with mesenchymal drift of glioma cells in culture. (J Histochem Cytochem 46:585594, 1998)
Key Words: FISH, glioblastoma, glioma, immunohistochemistry, in situ hybridization, lymphoma, malignancy, MIB-1
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Introduction |
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Brain tumors perplex histologists, neurobiologists, and pathologists. The histological features of primary brain tumors, including gliomas, neuronal tumors, and neuroembryonal tumors, elude comparison with visceral and connective tissues. Some barely resemble brain.
The neuropathological interpretation of brain tumors begins with their classification by their histological and cytological features that resemble elements of the nervous system (
Determination of grade of malignancy concludes the neuropathological interpretation of a brain tumor (
In the terminology of "translational research," i.e., the clinical utilization of basic science, IHC has been translated to regular clinical use for brain tumor diagnosis. Concurrently, new IHC phenotypic markers continue to be found, and the best of these will translate into diagnostic use. Nuclear IHC markers of proliferation are presently being translated into the diagnostic arena. Their ultimate diagnostic utility will depend in large part on the extent of their power to predict patient survival, which appears to be substantial for astrocytomas (
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Immunohistochemistry of Phenotypic Markers |
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What cell type does a brain tumor resemble? Phenotypic analyses lay the foundation for classification of brain tumors into specific diagnostic categories (
Figure 1 is an H&E-stained section of a brain tumor with remarkably large malignant cells. Glycosylated collagen between these malignant cells (Figure 2) led people to believe it to be a sarcoma. For decades it was considered to be a "monstrocellular sarcoma" (
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Another tumor originally mistaken for a reticulum cell sarcoma or microglioma is the primary brain lymphoma (
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Until a few years ago, most central neurocytomas were interpreted to be gliomas (
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Table 1 lists alphabetically the IHC stains commonly used for evaluating tumors at the University of Michigan at this time. The majority use monoclonal antibodies, but GFAP and S-100 are detected with polyclonal antibodies. Polyclonal antibodies tend to be quite sensitive, useful in detecting low levels of antigens, but are not as specific as monoclonal antibodies.
The source of many brain tumor antigens to which IHC antibodies were raised was a differentiated cell of specific lineage. Although the neoplastic counterparts of these cell lineages often express the expected antigens, this oncological fidelity is not universal. Neoplasms of a high grade of malignancy are prone to express unusual antigens. IHC of the neuroembryonal tumors has produced startling examples of this (Table 1). Primitive neuroectodermal tumors, including cerebellar medulloblastomas common in childhood, may express intermediate filaments commonly found in carcinoma, muscle tumors, gliomas, and neuronal neoplasms (
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Immunohistochemistry of Nuclear Markers |
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Brain tumors are given a histopathological grade of malignancy (relying mainly on features in H&E-stained slides) to estimate the patient's prognosis. The histopathologically defined grades of gliomas include noninvasive juvenile astrocytomas as the lowest grade and invasive, mitotically active glioblastoma as the highest (
New methods of assessment of cell cycle parameters with proliferation markers are ways of quantitating a larger and more representative portion of proliferating cells than is possible by counting mitoses (Figure 10 and Figure 11). These methods are compared below.
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BUDR is a halopyrimidine analogue of thymidine that is incorporated into DNA during its synthesis in the S-phase of the cell cycle. Cells that have incorporated BUDR are detected with a specific monoclonal antibody after ethanol fixation and heat and/or acid treatment of tissue sections to expose the BUDR epitopes (
Malignancy is associated with high BUDR labeling indices. In 38 patients with various brain tumors resected after a 1-hr pulse of in situ BUDR, high histopathological grades of malignancy were associated with a high BUDR LI (
The answer to this question comes from studying tumors of the same histopathological grade that look similar to the pathologist but nevertheless are associated with variable patient survival. Applied to histopathologically indistinguishable low-grade astrocytomas, BUDR LI distinguished two groups with different prognoses. Patients with astrocytomas having an LI of less than 1% had better survival than the group with LIs greater than 1% (
The proliferation antigens represented by PCNA, Ki-67 antibody, and MIB-1 antibody are attractive alternatives to BUDR. Unlike BUDR, they occur naturally in tissue (
PCNA/cyclin is an auxiliary protein to DNA polymerase. It is synthesized in late G1- and S-phases of the cell cycle (
The prognostic value of PCNA among brain tumors has yet to be agreed upon. One impediment is variation in LI among laboratories and even among IHC batches in the same laboratory (
The MIB-1 and Ki-67 monoclonal antibodies recognize Ki-67 epitopes on a nuclear protein present in a portion of the cells in G1-, S-, G2-, and M-phases of the cell cycle (
The advent of an MIB-1 monoclonal antibody opens a new era in the application of proliferation markers because the MIB-1 antibody detects Ki-67 epitopes in paraffin sections of formalin-fixed tissue (
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In Situ Hybridization of Phenotypic Markers |
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The detection of mRNA codes is theoretically attractive for the diagnosis of neoplasms with little or no expression of the suspected protein antigen or with protein markers that lack sensitive and specific antibodies for their detection. Thus far, ISH is primarily a research method, but it has provided insights into the molecular pathology of pituitary adenoma and glioma. Three different examples follow.
ISH can detect a latent phenotype in specific cases where immunohistochemical markers are only partially effective. The mRNA messages detected by ISH correspond with their encoded peptide hormones localized immunohistochemically in pituitary adenomas. More importantly, ISH detected the messages for two hormones in some adenomas that expressed only a single hormone detectable by IHC (
ISH can detect a latent phenotype where specific immunohistochemical markers are inadequate. The individual detection of - and
-light chains of immunoglobulins provides particular power in the assessment of neoplastic vs inflammatory lymphocyte proliferation. Neoplastic lymphocytes express either
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-light chains, but not both as do lymphocytes in inflammation. This diagnostic discriminator of lymphoma is called light-chain restriction. Unfortunately, immunoglobulin light chains lack sensitive and specific antibodies for their detection in paraffin sections, confounding the interpretation of light-chain reaction. ISH for immunoglobulin mRNA has been proposed as a more specific alternative to these anti-immunoglobulins on paraffin sections (
ISH can detect the cell of origin for extracellular protein markers. Unlike the rest of the body, normal brain contains very little collagen. Nevertheless, certain glioblastomas contain abnormal bands of collagen and spindled cells that resemble sarcoma mixed with more typical malignant glioma cells (
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In Situ Hybridization of Nuclear Markers |
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Fluorescence in situ hybridization (FISH) can be used to localize specific DNA sequences either in nuclei or on chromosomes. FISH can locate repetitive sequences specific for individual chromosomes, whole chromosomes, and specific genes when a long enough gene probe is available (
FISH can reveal chromosomal marker signals in cells cultured from gliomas (Figure 12). A long unanswered question in glioma cell biology has been the source of phenotypic alterations in cultured glioma cells. These alterations have been termed "mesenchymal drift" because the cultured glioma cells tend to lose glial and gain mesenchymal features, including collagen production (
Applied to metaphase spreads of brain tumor chromosomes, FISH provides new insights about cytogenetic observations. For example, elongation of a glioblastoma chromosome 17 by what appears to be the insertion of a single small band on GTG-banded chromosomes was shown by microdissection and FISH to be the insertion of DNA from three different chromosomes (
Comparative genomic hybridization (CGH) is a different type of FISH in which the tumor DNA is a labeled probe rather than DNA fixed on a slide. It utilizes two-color FISH to compare this brain tumor DNA with an internal standard of normal DNA. Tumor DNA is labeled with one molecular tag, such as biotin. Normal DNA is labeled with another tag, such as digoxigenin. The binding of these two probes to metaphase spreads of normal human chromosomes is then compared. The result has been a plethora of observations of losses and gains of chromosomal DNA in gliomas (
The diagnostic applications of FISH and CGH have not been fully explored but will be in the next few years. The repetitive sequences that identify specific chromosomes have been identified in nonmitosing (interphase) nuclei with these ISH techniques. Astrocytic tumor progression to higher grades of malignancy is associated with increased variation in such signals for chromosomes 7 and 17 (
ISH has demonstrated that isochromosome 17q (one chromosome composed of two long arms of chromosome 17) is a constant finding in medulloblastoma (
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Summary |
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Immunohistochemistry has been embraced by diagnosticians seeking improved classification of brain tumors and has become the diagnostic standard for many tumors. IHC of nuclear proliferation markers is beginning to assume an important role in estimating prognosis, and in situ hybridization promises future insights. The conspicuous value of these procedures to the diagnosis and management of brain tumor patients encourages those who struggle to innovate and improve these markers to continue their worthy efforts.
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Acknowledgments |
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Supported in part by National Institutes of Health Grants (P03) CA 46592, CA 68545, and CA 47558 from the National Cancer Institute.
The skilled editorial assistance of Ms Dianna Banka is gratefully acknowledged.
Received for publication August 7, 1997; accepted August 23, 1997.
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