Abstract
Objectives
To determine the feasibility of a rapid method of processing mandible bone margins for intraoperative histopathologic examination and to assess the relative value of fine, coarse, and core specimens in assessing bone margins.
Study design
Prospective histologic controlled study.
Setting
A tertiary level academic medical center histopathology laboratory.
Subjects and methods
Multiple bone samples were collected from fresh (< 12 hours post-mortem) human cadaveric mandible using a 1) standard 4 mm otolaryngologic cutting drill bit 2) diamond drill bit and 3) cutting core biopsy trocar. The specimens were placed in one of three decalcifying solutions (Decal A, Calex, EDTA Decal) from 15 to 75 minutes or control (fixation in 10% formalin). After each designated decalcification time period, specimens were cryosectioned or paraffin embedded and subsequently reviewed by a head and neck surgical pathologist. The specimens were assessed for overall quality, adequacy of decalcification, soft tissue quality, marrow quality, and presence of artifact.
Results
Bone margin specimens collected with a 4 mm burr and processed with EDTA Decal for 30 minutes yielded the highest quality histopathologic slides compared to the other methods in a similar time frame. The adequacy of decalcification directly impacted the quality of histopathologic assessment.
Conclusions
Mandible bone margins can be rapidly and safely prepared and adequately evaluated with only 30 minutes of decalcification. This method may provide acceptable intraoperative assessment of bone margins in patients with tumors which involve or approximate bone. We plan to examine this model in a prospective clinical study of patients with cancer invading mandibular bone.
1
Introduction
Of non-cutaneous head and neck cancers, thirty percent occur in the oral cavity, with approximately 22,000 patients who are newly diagnosed annually in the United States . Oral cavity cancers which invade the mandible have a higher T classification, portend a significantly worse prognosis and are likely to require more extensive and complex ablative and reconstructive surgery . Currently, there is no generally accepted method for intraoperative assessment of the surgical margin of bone resection. In cases of oral cavity cancers with gross mandibular bone involvement, surgeons usually submit a gingival mucosal margin and perhaps a tissue sample from the neurovascular structures in the infra-alveolar canal. However the cortical, trabecular and medullary bone margins are not histologically assessed intra-operatively because they cannot be adequately assessed without decalcification using the current frozen histopathology processing techniques. As a result, there are cases where a surgeon’s best intraoperative estimation and frozen pathology of gingival mucosa and the infra-alveolar canal at the surgical margins do not correctly determine the surgical margin of the mandible was free of tumor . In turn, these unfortunate patients usually require a return to the operating room for additional resection of the mandible using the same inexact process of assessing the intraoperative surgical margin and the significant challenge of reconstructing an additional segment of loss mandibular bone in the previously reconstructed site. This inability to fully assess the bony margin intra-operatively and the associated consequences also apply in tumors that arise from the mandible itself, such as ameloblastoma and osteogenic sarcomas.
In addition to the current incomplete method of tissue sampling for assessing freshly cut surgical bone margins, there is also no gold standard of imaging to assess for minor bone erosion by tumors with gingival invasion. In general, the use of computed tomography (CT) is believed to underestimate mandible erosion, while magnetic resonance imaging (MRI) and bone scintigraphy with single photo emission computed tomography (SPECT) may overestimate involvement . Moreover, CT images can be degraded by associated dental artifact while MRI reliability can be diminished by any significant movement by the patient.
Similar to the challenges observed in identifying early mandible erosion, there is no definitive way to predict the extent of mandible involvement by tumors transgressing into the medullary space. Pre-operatively, a combination of CT and MR imaging is typically recommended as part of the assessment. Intra-operatively, mandibular margins of at least one centimeter beyond any clinical or radiographic evidence of disease are thought to be appropriate. However, unlike soft tissue extension, a standard, safe, and simple method to rapidly assess early mandibular erosion or involvement of bone margins by carcinoma is currently not available. This inability to histologically confirm the presence or absence of pathology in the bony margin at the time of surgery may result in additional surgeries at best, or recurrence in the adjacent bone, at worst.
The current inability to rapidly determine histopathologic involvement of cancer in a bone margin is due to the prolonged time required to adequately decalcify the bony matrix of the mandible. Current histopathology methods take 12–24 hours to adequately decalcify a mandible specimen, which negates the option of a complete intraoperative assessment of the surgical margin. To develop a rapid, safe, and simple method to overcome these pitfalls in the histopathologic assessment of surgical margins in tumors involving the mandible, we tested several decalcifying solutions and tissue procurement techniques to determine which yielded the best quality.
2
Methods
After obtaining approval from the Indiana University Institutional Review Board, the mandible was harvested from a fresh cadaver (< 12 hours post-mortem). Using a standard 4 mm cutting burr or a 4 mm diamond burr, otologic drill, and suction irrigator with a collection bag and suction trap (Anspach; Palm Beach Gardens, FL), the mandibular bone was burred with contiguous saline irrigation. The burred bone slurry (“dust”) was collected and placed in labeled containers with 10% buffered formalin (Fisher Scientific; Pittsburgh, PA). Following 30 minutes of transport time to the laboratory, these specimens were placed in mesh cassettes ( Fig. 1 A ), saline rinsed and placed in one of three decalcifying solutions [Decal A (decal-bone; Tallman, NY), CalEX (Fisher Scientific; Pittsburgh, PA) or EDTA Decal (Richard-Allan Scientific; Kalamazoo, MI)]. The specimens were then soaked in their assigned decalcification solutions from 15, 30, 45, 60 or 75 minutes. They were subsequently embedded in OCT embedding medium (Thermo Scientific, Milwaukee, WI), cryosectioned, mounted on glass slides and stained with hematoxylin and eosin ( Fig. 2 ). The specimens processed for 75 minutes were subsequently embedded in paraffin. In addition, five core biopsy specimens of the mandible were taken with a 12 gauge core bone biopsy trocar ( Mekon core biopsy needle, Shanghai, China). Following 30 minutes of transport time in 10% formalin to the laboratory, these core specimens were rinsed with saline and placed in Decal A for 15, 30, 45, 60 or 75 minutes ( Fig. 1 B). All of these core bone specimens, except the 75 minutes, were subsequently embedded in OCT embedding medium (Thermo Scientific, Milwaukee, WI), cryosectioned, mounted on glass slides and stained with hematoxylin and eosin ( Fig. 2 ). The cored specimen processed in Decal A for 75 minutes was subsequently embedded in paraffin. There were three controls (fine burred, coarsely burred, and a cored bone sample) which were fixed in 10% formalin for 30 minutes, but were not processed in any decalcifying solution and were embedded in paraffin. The specimens were examined by a Head and Neck pathologist (DJS) who was blinded to all collection and processing information, including the type and time of exposure to the decalcifying agents. The pathologist evaluated each specimen’s overall quality for histopathologic assessment, adequacy of decalcification, quality of soft tissue if present, marrow quality if present and extent of artifact if present ( Table 1 ).
| Overall quality | Decalcification quality | Soft tissue quality | Marrow quality | Artifact | Comments | |
|---|---|---|---|---|---|---|
| No Decal (fine bone dust) | Unacceptable | None | NA | NA | None | No visible viable tissue |
| No Decal (core biopsy) | Acceptable, minor artifact | Partial | Acceptable, minor artifact | Acceptable, minor artifact | None | |
| No Decal (coarse bone dust) | Unacceptable | None | Acceptable, minor artifact | NA | None | Lots of fragments not decalcified |
| Decal A (fine bone dust) | ||||||
| 15 minutes | Unacceptable | Partial | Unacceptable | NA | Significant | Burn artifact |
| 30 minutes | Acceptable, minor artifact | Partial | NA | Acceptable, minor artifact | Minor | Burn artifact |
| 45 minutes | Unacceptable | Partial | NA | NA | Minor | Insufficient bone and soft tissue for diagnostic purposes |
| 60 minutes | Acceptable, minor artifact | Complete | Acceptable, minor artifact | NA | None | Slight burn artifact |
| 75 minutes | Unacceptable | Partial | NA | NA | None | Mostly grainy debris that is non-diagnostic |
| Cal-Ex (fine bone dust) | ||||||
| 15 minutes | Unacceptable | Partial | NA | Acceptable, minor artifact | None | Paucity of cells and tissue make it non-diagnostic |
| 30 minutes | No specimen | |||||
| 45 minutes | Unacceptable | Partial | NA | NA | None | No soft tissue and minute fragmented debris make it non-diagnostic |
| 60 minutes | Acceptable, minor artifact | Complete | Acceptable, minor artifact | NA | Minor | |
| 75 minutes | Acceptable, no artifact | Complete | Acceptable, no artifact | 3 | Minor | |
| EDTA (fine bone dust) | ||||||
| 15 minutes | Acceptable, minor artifact | Partial | Acceptable, no artifact | 3 | Minor | |
| 30 minutes | Acceptable, minor artifact | Complete | Acceptable, minor artifact | NA | None | |
| 45 minutes | Acceptable, minor artifact | Complete | Unacceptable | Unacceptable | Minor | |
| 60 minutes | Scant tissue, poorly preserved | |||||
| 75 minutes | Acceptable, minor artifact | Complete | Acceptable, minor artifact | Unacceptable | Minor | |
| Decal A (Core Biopsy) | ||||||
| 15 minutes | Acceptable, minor artifact | Partial | Acceptable, no artifact | NA | Minor | |
| 30 minutes | Acceptable, minor artifact | Partial | NA | NA | None | |
| 45 minutes | Acceptable, minor artifact | Partial | NA | NA | None | |
| 60 minutes | Acceptable, minor artifact | Partial | NA | Unacceptable | Minor | |
| 75 minutes | Acceptable, minor artifact | Complete | Acceptable, no artifact | Acceptable, no artifact | None | very nice sections |
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