by Michael Emmert-Buck, M.D., Ph.D.; NCI, Lance Liotta, M.D., Ph.D., NCI; and Zhengping Zhuang, M.D., Ph.D, NCI

Our laboratory recently developed a simple, precise system for isolating and genetically microanalyzing extremely small tissue samples. The method allows for the reproducible amplification of DNA or mRNA from individual cells selected microscopically -- from a patient biopsy, for ex-ample.The dissection can be carried out on very small samples that are easy to obtain, prepare, and store, such as single 10-u sections of frozen or paraffin-embedded, formalin-fixed archival material. We believe that this could be a fundamental gateway technique allowing basic and clinical biomedical researchers in a wide range of subfields to dramatically increase the precision of localizing normal and pathological entities, processes and changes.

In our lab, the application of this technique ranges from the discovery of new tumor-suppressor loci involved in cancer susceptibility and progression to genetic diagnosis of cancer and infectious diseases at the microscopic level. This approach could potentially be extended to other genetic analyses in which localization to minute cell clusters is desirable, such as the examination of tissue obtained from transgenic animals or from patients undergoing experimental gene therapy. Other possible applications include pinpointing latent viral infection or tracking the biochemical features of specific cells within heterogeneous tissues.

Figure 1. Microdissection of prostate carcinoma.

Histologic field of invasive prostate carcinoma (panel A). Invasive tumor (T), normal prostate epithelium (N), and a focus of chronic inflammation (CI) are present. The large structure in the middle of the section is a focus of prostatic intraepithelial neoplasia (PIN), which appears to be progressing to invasive cancer. Two separate papillary proliferations are procured for analysis (panels B and C, arrows). Recent studies in collaboration with Paul Duray of the Laboratory of Pathology, and Marston Linehan's group (Rudy Pozzatti, Cathy Vocke, Scott Jennings, and Charles Florence) of the NCI surgical branch indicate the putative in situ tumors are genetically similar to invasive prostate cancer, suggesting that they are indeed precursor lesions.

The Method and How It Works
In the past, tissue heterogeneity has posed a significant problem for investigators conducting genetic analysis of pathologic lesions surgically removed from patients or experimental animals. Tissue sections typically contain multiple cell types. For example, a breast cancer biopsy contains normal epithelial cells, myoepithelial cells, stromal cells, endothelial cells, inflammatory cells and fat cells, as well as cells from muscle and nerve. The actual cancer cells may constitute significantly less than 50 percent of the cells in the tissue sample. Consequently, if the tissue is homogenized, the recovered DNA or mRNA will reflect an average from many cell types and not the specific DNA or mRNA of interest. This problem is compounded further in the genetic analysis of the progressive stages of cancer, in which the cells of interest can only be located with high-power microscopic visualization. Normal and possibly premalignant contaminating host cells pose a significant research impediment to the analysis of chromosome loss of heterozygosity (LOH) because the contaminating cells, with two copies of the allele, will mask the loss of an allele in the tumor cells. This is particularly problematic in assays that use the polymerase chain reaction (PCR), in which an allele from contaminating normal cells can become significantly amplified. Studies using mRNA from microdissected cells, particularly studies screening for mRNA differences between two or more cell populations, e.g., differential display patterns in normal epithelium vs. dysplastic epithelium or in situ carcinoma vs. invasive carcinoma, absolutely require finely microdissected samples that select only the particular cells of interest. Any high-copy mRNA from contaminating cells will interfere with these experiments.

Our method of tissue micro-dissection differs from previously published methods for tissue microdissection used to study human tumors. Earlier microdissection methods required the procurement of a large piece of tissue from a histologic slide of a tumor with an abundance of malignant cells. Selection of an optimal quadrant enriches the malignant cell content in the specimen but does not provide the sample purity necessary for many PCR-based assays, particularly those using mRNA.

To overcome the drawbacks of these previous approaches, our microdissection method amplifies DNA or mRNA from much smaller, purer samples, that is, cells that are singled out and removed from histologic tissue sections under high-power microscopy. The individual cells or groups of cells, which have been stained with eosin, are excised by electrostatic attraction with a 30-gauge needle and placed in a single-step extraction buffer, which provides the starting point for PCR amplification.

In our laboratory's research on genes associated with breast cancer, this microdissection method enables investigators to sample the DNA from pure populations of normal epithelium, in situ carcinoma, and invasive carcinoma, all in the same 10-u section of a patient's biopsy. Studies in collaboration with Maria Merino and Rodrigo Chuaqui, also of the Laboratory of Pathology, NCI, show a novel allelic loss on chromosome 11q13 in 70% of the cases of human breast carcinoma studied (n = 70). We observed the allelic loss in both in situ and invasive components of the tumors. In all cases, the identical allele was lost, providing the first molecular evidence to support the long-held hypothesis that in situ breast cancer is a precursor to invasive cancer.

Figure 2. Detection of allelic loss of chromosome 11q13 in in situ and invasive breast lesions.

Analysis of chromosome 11 loss of heterozygosity by PCR amplification using specific primers for PYGM, a polymorphic DNA marker on 11q13. Microdissection of these two biopsies of breast cancer show normal epithelium (N), in situ tumor (IS), and invasive tumor (IV). PYGM analysis revealed loss of heterozygosity in the in situ and invasive lesions.

The polymorphic DNA marker used in this study was PYGM, located on chromosome 11q13. Reactions were cycled in a Perkin Elmer Cetus thermal cycler as follows: 94deg.C for 1.5 m, 55deg.C for 1 m, 72deg.C for 1 m, for a total of 35 cycles. PCR was performed in 10 uL volumes and contained 1 uL 10X PCR buffer (100 mM Tris-HCl, pH 8.3; 500 mM KCl; 15 mM MgCl2; 0.1% (weight-to-volume) gelatin; 2 uL of DNA extraction buffer; 50 pM of each primer; 20 nM each of dCTP, dGTP, dTTP, and dATP; 0.2 ul [32P] dCTP (6000 Ci/mM); and 0.1 unit Taq DNA polymerase. Labeled, amplified DNA was mixed with an equal volume of formamide loading dye (95% formamide; 20 mM EDTA; 0.05% bromophenol blue; and 0.05% xylene cyanol). The samples were denatured for 5 m at 95deg.C and loaded onto a gel consisting of 6% acrylamide (49:1 acrylamide:bis). Samples underwent electrophoresis at 1800 volts for 2 to 4 hours. Gels were transferred to 3mM Whatman paper, dried, and autoradiographed using Kodak X-OMAT film. The criterion for loss of heterozygosity from the microdissected in situ and invasive breast samples was complete absence of an allele.

DNA Analysis from Tissue Samples
1) 10-u sections of formalin-fixed, paraffin-embedded tissue, or of frozen tissue are prepared on a glass slide per normal surgical pathology protocol (4). mRNA is recovered more efficiently from frozen tissue.
2) Slides with paraffin sections are de-paraffinized by two 5-minute baths in xylene, followed by similar pairs of 2-minute baths in 95% ethanol, 50% ethanol, and distilled water. The slides are then air dried.
3) Slides with frozen or de-paraffinized sections are stained briefly in eosin (1% eosin in 80% ethanol) and air dried.
4) An adjacent hematoxylin- and eosin-stained section is used to scout out the tissue section for optimally clean sites for microdissection--for example, areas in which specific small cell populations of interest are comparatively isolated and free of significant inflammation or other contaminating cells.
5) Microdissection of selected populations of cells is performed under direct light-microscope visualization using an inverted microscope and a sterile, 30-gauge needle. Experienced microdissectors can reliably recover pure cell populations of five or fewer cells, as well as cells arranged as a single layer, such as normal epithelium or the epithelial lining of cystic lesions.

6) Cells of interest are detached from the slide by gentle scraping and will adhere to the tip of the needle via electrostatic attraction.
7) Those cells are immediately suspended in a pH 8.0 solution containing 0.01 M Tris-HCl, 0.1 M ethylenediamine tetraacetic acid (EDTA), 1% Tween 20, 0.1 mg/mL proteinase K, and then incubated overnight at 37deg.C. For optimal PCR amplification we recommend procuring a minimum of 20 cells per uL; however, we have detected loss of heterozygosity (LOH) starting with as few as five cells.

8) The mixture is heated at 95 deg. C for 5 minutes to inactivate the proteinase K, and 2 uL are then used for standard PCR analysis (5). A sample of the PCR protocol is described in the legend to Figure 2.

Tissue-Microdissection Contact
Michael Emmert-Buck, M.D., Ph.D, NCI
phone: 402-2986; fax: 480-9488
e-mail: mbuck@helix.nih.gov

1. M.R. Emmert-Buck, M.J. Roth, Z. Zhuang, E. Campo, J. Rozhin, B.F. Sloane, et al. "Increased gelatinase A and cathepsin B activity in invasive tumor regions of human colon cancer samples." Am. J. Pathol. 145, 1285 - 90 (1994).
2. Z. Zhuang, P. Bertheau, M.R. Emmert-Buck, L.A. Liotta, J. Gnara, W.M. Linehan, et al. "A new microdissection technique for archival DNA analysis of specific cell populations in lesions less than one millimeter in size." Am. J. Pathol. (in press).
3. Z. Zhuang, M.J. Merino, R. Chuaqui, L.A. Liotta, and M.R. Emmert-Buck. "Identical allelic loss on chromosome 11q13 in microdissected in-situ and invasive human breast cancer." Cancer Res. (in press).
4. B.J. Coolidge and R.M. Howard. Animal Histology Procedures. NIH Publication 80-275, Washington, D.C.: Public Health Service (1979).
5. K.B. Mullis and F.A. Faloona, Methods in Enzymology, Volume 155, R. Wu, ed. San Diego: Academic Press, pp. 335 - 50 (1987).

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