Our Methodology: DNA Extraction and TaqMan® Tumor Genotyping
The laboratory isolates DNA from tissue samples containing tumor cells and analyzes specific sites within particular genes for the presence or absence of mutations.
Types of specimen analyzed:
The starting material may be formalin-fixed, paraffin-embedded (FFPE) tissues as used for standard histologic assessment; fine needle aspirate (FNA) or other cytology specimens (eg., pleural fluid); or fresh-frozen tissues. Upon receipt of a completed Tumor Profiling Requisition form, the laboratory will coordinate with the Surgical Pathology or Cytopathology division of the Department of Pathology to obtain the appropriate patient specimen(s), whether from within the medical center or from an outside laboratory. A pathologist (usually Dr. Walther) will review the relevant microscopic sections and designate the tumor tissue to be analyzed.
Routinely, at least 250,000 tumor cells are necessary for the analysis. For example, appropriate numbers of tumor cells are usually contained within 4 to 6 unstained, 1 cm square sections, each 5 microns in thickness, cut from paraffin-embedded tissue blocks. If the only available tumor tissue is less than this amount, special procedures may be employed to amplify the extracted DNA prior to mutational analysis (see below). For all samples, at least 50% of the total cells analyzed should be tumor cells. If the malignant component of the tissue is less than 50%, the laboratory can enrich for neoplastic cells by manual or laser-capture microdissection of histologic sections.
Mutation detection method:
Testing for mutations is performed by TaqMan® Array real-time PCR of the DNA extracted from the tissues. This technique permits completion of the entire test in a single tube that remains sealed throughout procedure, thereby greatly reducing the chance of artifact due to inter-specimen contamination with DNA or PCR products.
Analysis of very small specimens:
In cases in which only a small amount of tumor tissue is available for analysis (less than 250,000 malignant cells, after microdissection), DNA will be extracted and subjected to a “pre-amplification” step before being analyzed by TaqMan® Array. Pre-amplification is a highly multiplexed PCR reaction designed to amplify specifically those regions of DNA in which the presence or absence of mutations will be assessed. Pre-amplification is currently validated in our laboratory for a subset of the full panel of mutation tests, including the majority of tests for mutations in BRAF, EGFR, KRAS, NRAS and PIK3CA.
Most tumor specimens, even after microdissection, contain a mixture of neoplastic and non-neoplastic cells. The sensitivities of the TaqMan® assays in our panel have been tested individually, using well-characterized human tumor cell lines when possible, and all have been shown to reliably detect mutations when present in a 1:1 mixture of heterozygous mutant (tumor) and normal DNA [analytical sensitivity = 50% tumor cells]. Most assays in the panel have considerably greater analytical sensitivity and are able to detect 10 to 30% tumor cells. In the future, it is anticipated that a newer version of the TaqMan® detection method that is currently under development will further improve the sensitivities of individual assays such that they will all reliably detect mutations in specimens containing as little as 1% tumor cells.