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Intraoperative Microwave Processing of Bone Margins During Resection of Head and Neck Cancer
Edward C. Weisberger, MD;
Matthew Hilburn, MD;
Bradley Johnson, HT (ASCP);
Charly Nguyen, MD
Arch Otolaryngol Head Neck Surg. 2001;127:790-793.
ABSTRACT
Objective To develop an accurate method for performing histopathologic analysis
for a full cross-section of cortical bone within 2 to 3 hours.
Methods Microwave technology was used to augment and to more rapidly perform
fixation and decalcification of cortical bone.
Results Using the methods described, slides suitable for hispathologic analyses
regarding the presence or absence of malignant tumor were prepared in 2 to
3 hours and, in 10 patients studied, had a 100% correlation with slides prepared
in 7 days using the standard decalcification technique.
Conclusion Microwave technology allows accurate assessment of a full cross-section
of cortical bone regarding the presence or absence of malignant tumor within
the time limits required for resection of a malignant head and neck tumor
and reconstruction of the surgical defect.
INTRODUCTION
THE ABILITY to assess bone involvement by type of tumor has become increasingly
important in today's field of head and neck reconstructive surgery. When ablation
of a tumor requires composite resection of a bone such as the mandible that
has a relatively thick cortex, standard methods of cross-sectional histopathologic
analysis include a lengthy period for decalcification that can require 7 to
10 days. Forrest et al1 have reported intraoperative
analysis of cancellous bone curetted from the mandibular marrow space. However,
errors can occur from omitting a full cross-sectional analysis of the bone
margin.
PATIENTS AND METHOD
A technique of processing a full cross-section of mandibular or maxillary
bone was developed that allows an analysis of the histopathologic condition
within 2 to 3 hours. Nine patients with biopsy-proven squamous cell carcinoma
of the oral cavity and oropharynx and 1 patient with adenoid cystic carcinoma
of the maxillary sinus underwent composite resection of the primary tumor
and adjacent mandible or maxilla. An additional patient underwent 2 separate
composite resections. Patient demographics along with the histologic features
and primary sites of the neoplasm are given in Table 1. In all cases full cross-sectional intraoperative bone margins
were assessed using the process herein described that is facilitated via microwave
technology.
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Patients With Oral Cavity Cancer Who Had Microwave Processed Bone Margins
Performed Intraoperatively
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The entire surgical specimen containing the bone to be analyzed was
placed in 10% formalin and treated in a conventional microwave oven (model
8500 X5; Whirlpool "Time Master," Benton Harbor, Mich) at a medium setting
(corresponding to approximately 40°C) for 10 minutes. This initial fixation
allowed thin (1- to 2-mm) full cross-sections of the bone margins to be cut
with a 0.5-mm-thick, diamond-bladed band-saw (model 1180; Mar-med Inc, Cleveland,
Ohio) without fragmentation. These sections were further microwaved in formalin
at a medium setting for an additional 10 minutes. They were then microwaved
in rapid bone decalcification solution containing a proprietary mixture of
hydrochloric acid and formalin (Apex Engineering Products Corp, Plainfield,
Ill) at a medium-low setting (approximately 37°C) for 20 minutes. The
specimen was then evaluated for pliability (ie, the ability to distort or
bend it by a laboratory technician's digital pressure). The achievement of
pliability was the end point. This step was repeated, using 10-minute periods
of microwave irradiation until the end point was achieved. This usually required
4 to 7 repetitions regarding mandible margin sections. The sections were dried
by microwaving them at a medium setting in absolute alcohol for 10 minutes
and then cleared by microwaving them at a medium setting in isopropyl alcohol
for 15 minutes. The sections were microwaved at a medium setting in paraffin
for 3 cycles of 15 minutes each and then embedded, cut, stained with hematoxylin-eosin,
and placed on a cover glass. The entire process, from receipt in the laboratory
to interpretation by the pathologist required from 2 to 3 hours.2
Standard processing of a mandible for histopathologic analysis of cross-sectional
margins requires decalcification in Curtis Matheson Scientific protocol solution
B (0.135 g of sodium tartrate as a buffer in 1 L of 1% hydrochloric acid;
Biochemical Sciences Inc, Swedesboro, NJ) for approximately 7 days.
The accuracy of this analysis, in terms of correctly identifying the
presence or absence of tumor in the bone margins, was evaluated by comparison
with the analysis of the histopathology obtained from the examination of the
bone specimens prepared using standard techniques that required 7 days for
decalcification.
RESULTS
The microwave-facilitated "quick" bone margins were deemed positive
for tumor in one instance and suggestive of tumor in another. They were negative
for tumor in 9 additional resections. In the case in which a margin was suggestive
of tumor, an additional segment of bone was resected and the second margin
was negative for tumor. In other words, the margin suggestive of tumor was
treated as a positive result. There was a 100% correlation of the quick bone
margins prepared by microwave processing with those prepared using traditional
decalcification techniques and permanent sections. In all instances, the pathologist
judged that sufficient cytologic detail had been preserved to allow for adequate
analysis (Figure 1). This included
the ability to appreciate malignant neoplasm invading large neural structures.
Extension along the course of the inferior alveolar nerve can be an important
avenue for the spread of malignant neoplasms in the oral cavity.
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A, Bone marrow space and bone without tumor (hematoxylin-eosin, original
magnification x150). B, Bone with squamous cell carcinoma in bone marrow
space (hematoxylin-eosin, original magnification x150).
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COMMENT
One of the cardinal principles regarding surgical resection of malignant
neoplasms is the achievement of negative margins as an indication of adequate
removal of tumor. Frozen-section analysis using cryofixation has been a reliable
means of analyzing the margins of soft tissues. However, tissues that contain
a significant amount of hard calcium matrix, such as cortical bone, cannot
be sectioned by the cryotome and, therefore, cannot be evaluated using standard
frozen-section techniques.
Malignant neoplasms in the oral cavity can invade contiguous bony structures.
When this occurs, the prognosis for cure is diminished and resistance to radiotherapy
is increased.3-4 For this reason,
achieving clear margins of resection for bone might be even more important
than for soft tissues.
Moreover, reconstruction of bony defects of the oral cavity is frequently
accomplished using a microvascular free flap. To discover 1 week after surgery
that a composite free flap has been inserted into a mandibular defect with
tumor at the margin is discouraging and presents a dilemma regarding further
management. Radiotherapy offers a nonsurgical solution but is likely a compromise
in the effort to eradicate the malignancy because tumor invading bone is present
in the postoperative wound. Reoperation to achieve a negative bone margin
is an option, but the length of bone in the free flap has already been tailored
to fit a defect of specific dimensions. Furthermore, the end of the bone of
the free flap is against a segment of bone known to contain residual tumor
and is, therefore, potentially contaminated. Removal of the free flap at reoperation
usually requires replacement with another flap, imposing additional donor
site morbidity on the patient. This therapeutic dilemma can be avoided if
an accurate method of evaluating bone margins can be developed that can be
performed within the time limits of surgical resection with reconstruction.
Use of a microvascular free flap usually adds several hours to the length
of the surgical procedure, even when harvesting of the free flap commences
while ablation of the tumor is being completed. There is, therefore, adequate
time for analysis of the bone margin using the microwave technique described
earlier.
Several authors3-5
have identified 2 distinct patterns of mandibular bone involvement by oral
cavity squamous cell carcinoma. There is the erosive pattern with a sharp
interface between tumor and bone and the infiltrative pattern where fingerlike
projections of tumor invade, on an irregular front, with unpredictable lengths
of mandibular involvement, especially regarding the marrow space. The infiltrative
pattern is associated with a significantly lower prognosis for cure of cancer
and with a significantly higher rate of positive margins, identified on permanent
sections of the decalcified specimen. In patients with the infiltrative pattern,
Wong et al6 noted positive margins for tumor
in 47.6% of their patients. No intraoperative histologic monitoring of bone
margins was performed.
Microwaves have a frequency between 300 MHz and 300 GHz. The conventional
domestic microwave oven has a frequency of 2.45 GHz, corresponding to a wave
length of 12.2 cm.
Microwaves are generated by a magnetron and propagate at the speed of
light in a vacuum. They are reflected by most metals but pass through glass
and deeply into tissue samples. They cause rapid rotation (approximately 2.45
billion cycles per second) of dipolar molecules and of any molecules having
an asymmetric charge such as the polar side chains of many proteins. This
increased intramolecular and intermolecular motion produces heat, enhancing
chemical interaction, and facilitates diffusion of fluids in and out of tissues.
Because the microwave energy is not strong enough to alter covalent bonds
or to break hydrogen bonds, cellular architecture remains intact. Unlike more
conventional heating, which begins at the surface and depends on conduction
to reach deeper levels, microwave heating occurs anywhere the microwaves have
passed into the tissue and is more uniform throughout a greater volume of
tissue. Microwaves effect deeper levels more quickly.7-8
Microwaves facilitate important processes needed to prepare tissues for analysis
by the pathologist including fixation, dehydration, staining, and decalcification.
Microwave technology has been previously used to speed up and to improve immunohistochemical
reactions, special staining techniques, and decalcification of cadaveric temporal
bones.9-13
Decalcification of bone is necessary to allow cutting thin sections
from a tissue specimen using a microtome. This is accomplished by displacing
calcium using an acid solution. The rate at which this occurs depends on several
factors including the thickness of the bone involved. The thick cortical bone
of the mandible requires about 7 days for decalcification using standard processing
techniques. As we have shown, this can be reduced to about 2 to 3
hours when facilitated with microwave technology.
We are evolving our protocol so as to provide an answer regarding the
bone margin earlier in the procedure. For example, when the neck is N0 or
nodal disease is not bulky, the primary tumor can be resected en bloc with
the mandible and submandibular glands (ie, level I nodes). Preparation of
the bone margins can begin at this point while the extirpative surgical team
completes dissection of level II to V nodes, as indicated. Also, after initial
fixation, we have begun carefully cutting the bone marrow as a separate bloc
from the cortex and analyzing the marrow bloc first. Because less decalcification
is required for the thin bone spicules of the marrow bloc, analysis can be
completed in about 1 hour. The bone marrow is also the compartment most likely
to be positive for tumor involvement. If the bone marrow is shown to be positive
for tumor, decalcification of the cortical portion becomes irrelevant and
additional margins can be resected from the patient immediately.
Other methods of evaluating for the presence of bone involvement and
for the extent of bone that should be resected regarding tumors of the oral
cavity have been used. Radiographic techniques that have been used include
the following: standard pantographs of the mandible, computed tomography (CT),
and magnetic resonance imaging.14 Of these,
CT and magnetic resonance imaging scanning have proved to be the most accurate.14-15 Computed tomographic scans can reliably
detect erosion of cortical bone but can fail to show the extent of involvement
of the bone marrow. Also, CT scans are compromised when dental fillings are
present, resulting in significant metallic artifact. Magnetic resonance imaging
is better for showing abnormal marrow changes.
Radionuclide bone scans are of some value in predicting bone involvement
but are probably not as accurate as CT and magnetic resonance imaging scans.14 Clinical assessment by an experienced extirpative
head and neck surgeon can be of great value and at some institutions has been
remarkably accurate. Brown et al14 used direct
inspection of the resected specimen after periosteal stripping to judge the
adequacy of resection. Schusterman et al16
noted a 2% incidence of positive bone margins when clinical assessment alone
was used. However, as noted earlier, others have experienced a significantly
higher rate of abnormal bone margins with Wong et al6
noting abnormal bone margins in 47.6% of their patients with an infiltrative
pattern.
Forrest et al1 showed that curettage
of the bone marrow space had an overall accuracy of 97% in 33 patients. However,
of the 3 patients with margins shown to be positive for tumor on final decalcified
permanent section, only 2 (67%) of 3 were correctly identified on the basis
of intraoperative curettage (ie, there was a false-negative rate of 33% for
this subgroup).
We have used microwave technology to allow a quick analysis of a full
cross-section of the bone margin. This technique can also provide information
regarding involvement of neural structures and periosteum.
The microwave processing of bone margins requires some experience on
the part of the laboratory technician preparing the specimen. Using temperatures
that are too high during processing can result in the loss of cytologic detail.
The technician must be able to judge when the section is of adequate pliability.
While we were able to achieve satisfactory results with a conventional domestic
microwave oven, there are laboratory-grade microwave ovens available that
offer potential advantages such as more accurate assessment of the temperature
using temperature probes, and more accurate power cycles. Also, these ovens
often provide a venting system that reduces the risk of exposure of laboratory
personnel to potentially toxic fumes. The special bone saw used was equipped
with a diamond blade that provided thin sections, facilitating processing.
CONCLUSIONS
Microwave technology can speed up and enhance processing and decalcification
of full cross-sectional margins of bone. This allows accurate analysis within
the time limits of a surgical extirpation of an oral cavity cancer along with
microvascular reconstruction. This might improve the surgical eradication
of tumor and preserve a microvascular reconstruction that could be jeopardized
if a bone margin is found to be positive for tumor 1 week after the extirpative
surgery.
AUTHOR INFORMATION
Accepted for publication April 6, 2001.
Presented in part at the annual meeting of the American Head and Neck
Society, Fifth International Conference on Head and Neck Cancer, San Francisco,
Calif, July 30, 2000.
Corresponding author: Edward C. Weisberger, MD, Otolaryngology–Head
and Neck Surgery, Indiana University Medical Center, 702 Barnhill Dr, Suite
0860, Indianapolis, IN 46202.
From the Departments of Otolaryngology–Head and Neck Surgery
(Dr Weisberger), Surgery (Dr Hilburn), and Pathology (Mr Johnson), Indiana
University Medical Center, Indianapolis. Dr Nguyen is in private practice
in Mattoon, Ill.
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