Microdissection Genotyping of Mixed Glial and Primitive Neuroectodermal Central Nervous System Neoplasm

Archives of Pathology & Laboratory Medicine,  Oct 2004  by Mohan, Deepak,  Rao, Gutti R,  Swalsky, Patricia A,  Bakker, Anke,  Et al

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A 22-year-old man with previous radiation treatment for childhood astrocytoma underwent resection of a right parietooccipital lesion. Histopathology revealed a malignant neoplasm with areas of astrocytic and primitive neuroectodermal components. To resolve the relationship and cellular origin, representative tissue was microdissected from several targets, obtaining a balanced mixture of each element. Nonneoplastic brain parenchyma was separately microdissected to determine polymorphic marker informativeness and to serve as an internal negative control. Despite the relatively small quantity of tissue removed for each microdissection target, sufficient material was available for reliable, balanced, polymerase chain reaction-format genotyping encompassing a panel of tumor suppressor genes and genetic loci associated with these forms of neoplasia. The findings revealed distinct discordant genotypic profiles for each of the neoplastic components. The efficacy of the approach used for molecular analysis of this complex neoplasm and the implication of the genotypic findings are discussed.

(Arch Pathol Lab Med. 2004;128:1161-1164)

The histopathologic diagnosis of central nervous system neoplasms is often rendered difficult by the relatively small sample size. Reactive astrocytic proliferations can closely resemble a true glial neoplasm, especially in a small biopsy specimen, which may not include adequate amounts of tissue to meet histologie criteria for specific abnormal growth patterns or enough cytologie atypia for the diagnosis of neoplasia.1 The diagnostic difficulty is further compounded by the intrinsic cellular heterogeneity of these neoplasms, which may display varying degrees of anaplasia in topographically distinct regions.2 Ancillary techniques suitable for use on small formalin-fixed tissue specimens and capable of providing objective discriminating information with respect to reactive hyperplasia versus neoplasia would be useful.

Histopathologic classification of brain neoplasms can be controversial and may result in discordant opinions even from expert pathologists concerning the cell of origin for a particular neoplasm.1 It is not uncommon for an individual neoplasm to present considerable variation in neoplastic cell type, particularly in the setting of increased anaplasia, where the cell of origin may be difficult to discern3 By the same token, central nervous system neoplasms often present with biphasic or multiphasic patterns with transitional areas between glial, neural, and other cell types of origin.4 Immunohistochemical stains can be most effective in delineating the differing histogenesis of such neoplasms; however, on many occasions this distinction remains unresolved, despite the use of a broad panel of immunohistochemical markers. Under these circumstances, diagnostic tissue can be exhausted or found to be no longer representative of the cellular patterns observed in initial histology sections.5

Advances in sequencing and increased knowledge of the human genome make it likely that a detailed mapping of cancer-related genes and their alterations will become available for clinical application in the near future. Translation of molecular information concerning the structure of the human genome requires appropriate methods to address critical clinical issues. Given the greater reliance on biopsy techniques to establish an initial histopathologic diagnosis, prognostication will depend on maximal extraction of information from small biopsy samples.

Molecular genotyping based on initial nucleic acid amplification by PCR affords a convenient means to generate information on the status of multiple different genes from a very small amount of starting tissue. This tissue, however, must be sampled in a highly representative manner and must be optimally pure with respect to the cellular elements it contains.6 Light microscopic methods have traditionally achieved this goal, enabling pathologists to directly link morphologic and/or staining features with corresponding biologic characteristics. Microdissected material then serves as the basis for genetic analysis, allowing a variety of specific genes and corresponding gene alterations to be identified.7-10

Using techniques developed in our laboratory that were designed especially for use with small amounts of fixed tissue, we analyzed a complex central nervous system neoplasm notable for the presence of a varying admixture of astrocytic and neuronal growth patterns.11,12 Our specific objective was to address the issue of whether the variations in morphology were based on clonal differences in neoplasm cell populations or whether they were independent of cell type. This distinction is considered important, since in the former case, differences in biological behavior and treatment responsiveness might be anticipated, whereas in the latter a uniform clinical course can be expected, because the histopathologic variability might be considered unrelated to underlying changes directing neoplasm biology.