Advances in Assessment of Tumor Margins
Assessment of surgical margins is a challenging task for the histology technician, pathologist, and submitting veterinarian. It is often difficult to communicate results of margin evaluations to a submitting veterinarian in a written report, or even by phone. This task is further complicated by the various techniques applied in different laboratories to assess margins. The purpose of this article is to familiarize the reader with advances we have made in margin evaluation, which include providing annotated images to our clients.
The most commonly used method to evaluate tumor samples is the cross-sectioning method, also known as the radial method or “halves and quarters” (Fig. 1). The specimen is bisected along its shortest axis. Then, each half of the tissue is bisected through its longest axis, creating quarter sections that demonstrate the mass in a different plane.
While we use this method for routine evaluation of submitted biopsies, this method is not favored for complete margin evaluations since it evaluates a very limited portion of the margin area and makes the erroneous assumption of symmetrical expansile growth of the tumor.
In human medicine neoplasms are sectioned like a “bread loaf” or a “pie” to have the most detailed information for each submission. Parallel slicing at regular intervals (complete bread loafing, serial sectioning) increases the percentage of margin area examined (Fig. 2). Since the distance between sections determines the quality of the margin evaluation, the cost of this approach limits its use in veterinary medicine.
A modified technique combines radial and parallel techniques (Fig. 3). This allows for evaluation of tissue immediately adjacent to the bulk of the tumor and evaluation of some distant margins of the tissue sample.
Tangential sections (shaved edge sections, “orange peel”) provide a complete assessment of surgical margins (Fig. 4). Multiple 2- to 3-mm sections are shaved off the edge of the sample and laid into cassettes with the cut surface down. Any tumor present in these sections is interpreted as incomplete excision. The disadvantage is that the distance of tumor to margins cannot be assessed. By combining cross-sectioning with tangential margin evaluation, we deliver the most complete margin assessment. While expensive, due to the large number of slides and time required for this method, it should be requested for all mast cell tumors, melanomas, mammary tumors, and other carcinomas to more accurately determine tumor extent. By inking the tumor margins the clinician can guide evaluation of certain regions and insure examination of the surgical margins.
Regardless of the method used to evaluate margins, the clinician needs to know to which margin the tumor extends in order to better direct additional resection or select advanced therapy, e.g. radiation. We therefore provide online photographs of tumors submitted for full margin evaluation that detail the exact position of each margin slide that was created by our technician (Figs. 2-5) This allows our pathologist to indicate in the biopsy report where neoplastic cells extend to the margin, and the clinician can view the photos and decide exactly where additional surgical resection or radiation may be needed.
Complete margin evaluations are not recommended for spindle cell sarcomas, as these neoplasms often infiltrate adjacent tissue via extension of thin tentacle-like processes. When such a tentacle is examined in cross section it can be very difficult to differentiate from reactive fibroblasts. Thus, histologic margin evaluation is not always definitive for these cases. Advanced imaging techniques, such as CT and MRI, are much more helpful in determining the extent of these neoplasms prior to surgical resection.
While complete margin evaluations are typically requested for cutaneous neoplasms, online digital photographs are also very useful for assessing margins of neoplasms in other organs and tissues, especially amputated toes, mandibles/maxillas, tails, lung lobes, liver lobes, and spleens. When neoplasms are present in these other “special” organs/tissues, we routinely assess proximal margins without an additional margin fee. A decalcification fee is applied for bony tissues. For example, for an amputated digit, the proximal margins are inked and we examine one soft tissue section taken through the mass (Fig. 5: A1) as well as sections representing the proximal skin margins (Fig. 5: A2 and A3). Then the toe is decalcified and a section is taken from the proximal bony margin (Fig. 5: A4). A longitudinal section through the length of the toe is also examined to assess for bone involvement (Fig. 5: A5).
When an entire lung or liver lobe is submitted with a neoplasm, the proximal margin is inked and at least one representative section that includes both the tumor and healthy tissue (radial section) is examined (Fig. 6: A3). One section is taken perpendicular to the grossly narrowest margin that includes the mass (radial section) (Fig. 6: A4). Then tangential sections from the surgical margin are taken by cutting off the inked edge and lying the resulting sections flat in a cassette inked side down (tangential sections) (Fig. 6: A5-A8).
Evaluation of Surgical Margins
As discussed above, surgical margins of tumor biopsies are evaluated on every routine biopsy in our laboratory. However, on routine samples the evaluation is limited to the extent of the neoplasm on 2 cross sections. A more complete margin evaluation is a complex process and has to be specifically requested for each biopsy submission. To avoid unnecessary costs for our clients and to provide detailed information on the extent of a neoplasm in a submitted section, we have revised a specific protocol explained within the following paragraphs. The following paragraphs and figures also illustrate how referring veterinarians can ink samples before submission, especially if there is a particular margin that is of strong concern.
Figure 7: To correctly identify surgical margins during the trimming process in the histology laboratory, it is necessary to paint (ink) the surgical margins. This can be done by the submitting veterinarian on unfixed samples or in our laboratory after the samples have been fixed. The procedure is simple and does not interfere with the microscopic evaluation. Besides ink, cotton swabs and wooden applicator sticks are all that is needed to perform this procedure. Courtesy of Veterinary Pathology.
Figure 8: Surgical margins of a biopsy are painted with a dye that adheres to the tissue and is visible under the microscope. There are many commercial dyes available, such as the one depicted from Cancer Diagnostics, Inc. Such kits contain multiple different colors (black, blue, green, red, yellow, etc.) for different aspects of mass orientation.
Figure 9: To save money, simple waterproof drawing ink can be purchased (Wal-Mart etc.) and such bottles will last several years.
Figure 10: The drawing ink can even be diluted with isopropyl alcohol (1:1). Isopropyl alcohol is also useful when submitting fixed tissue through the mail in winter months. The 10% neutral buffered formalin used for fixation of specimens is subject to freezing in very low temperatures. The addition of a small amount of isopropyl alcohol to the formalin specimen container (1 part alcohol to 10 parts formalin) will help to prevent damage to the tissue specimen related to freezing and thawing.
Figures 11 and 12: Biopsy margins can be painted on unfixed or fixed tissues. It is often an advantage for referring veterinarians to ink the margins on an unfixed tissue because they have performed the surgery and can best identify margins of concern. The mass should be placed on some absorptive material and needs to be blotted dry prior to painting the margins.
Figure 13: Different colors may be used to mark the cutaneous surface of a mass, which will help the histotechnician to correctly identify the orientation of the neoplasm. Such inking marks are superior to using sutures of different colors in identifying proximal and lateral margins of a mass. Courtesy of Veterinary Pathology.
Figure 14: Using a cotton swab facilitates even color distribution over the deep tissue margins. The referring veterinarians may decide to ink only margins of concern where they suspect incomplete removal. We will only evaluate the inked margins. Courtesy of Veterinary Pathology.
Figure 15: The biopsy margins of the mass may be inked with multiple colors too. Do not pour dye on the surface, but use the cotton swab or wooden applicator stick. A wooden applicator stick is especially helpful to ink the lateral margins by rolling it along these tissue margins. After inking the margins the dye should dry for 5-10 minutes prior to immersing the sample in formalin. Some dye will dissolve within the fixative, but this will not affect the evaluation. For large samples (thicker than 1-2 cm) incisions should be made into the mass to improve penetration of fixative. Courtesy of Veterinary Pathology.
Figure 16: After receiving the tissue, our technicians will palpate the section to determine where the mass/lesion comes closest to the surgical margins.
Figures 17 and 18: We will bisect the specimen vertically through the mass so the section extends through the margin closest to the identified mass and the center of the mass. A 2-6 mm full thickness plane/slab/piece will be cut from the cross section surface of the mass.
As previously illustrated in Figure 1, our laboratory uses the “Cross Method” (i.e. ½’s and ¼’s) as the standard trimming method for ellipse sections. The red line demonstrates the cross section of the mass and the associated closest specimen margin. The blue line demonstrates the quarter sections (1/4’s).
Figures 19 and 20: The inked margins are easy to recognize on the half section of the mass. Depending on the size of the mass, the half section may have to be split to fit into cassettes for further processing. Following embedding in paraffin and sectioning, the half sections of the mass will be placed on slides for microscopic evaluation (Fig.1). Courtesy of Veterinary Pathology.
Figures 21 and 22: Halves of the mass that have resulted from the cross section are vertically cut from the mass/lesion through the longest axis of the tissue and thin sections are placed into cassettes. These pieces demonstrate the mass in a different plane, and the association of the mass with surrounding normal long axis tissue margins
Figure 23: The number of slides necessary to identify whether the deep margins are clean is determined by the size of mass as illustrated. Evaluation of deep margins will be performed along the previously described half and quarter sections.
Figure 24: The half and quarter sections have been placed in cassettes for further processing.
Figures 25, 26 and 27: In addition to the deep margins, lateral margins will be evaluated. These margins are cut vertically to the cutaneous surface of the submitted section and trimmed into cassettes. Detailed evaluation of margins will only be performed following a written or oral request. The number of slides evaluating margins and the price for margin evaluation is based on the size of the excised tissue. As a general rule, sections smaller than 2 cm long will require 2 additional slides. A section of 2-4 cm length will require 6 additional slides, sections of 4-6 cm long need 9 additional slides and sections of 6-8 cm long will require 11 additional slides.
Figures 28 and 29: Inked tissue section margins (lateral margins: figure 24; deep margins: figure 25) are easily recognized on microscopic examination and will help the pathologist to determine complete surgical removal. Courtesy of Veterinary Pathology.
Matti Kiupel, Dr. vet. med. Dr. habil., PhD, Dipl ACVP
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