Intraoperative Consultation in Head and Neck Pathology
Case 1 -
Invasive Squamous Cell Carcinoma
Bruce M. Wenig, M.D.
Mary S. Richardson, D.D.S., M.D.
Intraoperative consultation (frozen section) plays an important part in the treatment of the head and
neck cancer patient. The appropriate use of intraoperative consultations (frozen sections) usually
results in a definitive diagnosis with immediate therapeutic impact while the patient is in the operating
room. The accuracy of frozen sections has been analyzed for general surgical cases, as well
as specifically for head and neck surgery. The studies on general surgical cases and
otolaryngologic cases show similar findings with a 95 to 98 percent diagnostic accuracy rate, a 2 to 4
percent error rate (including less than 2 percent false negative and less than 1 percent false positive)
and less than 4 percent of cases being deferred for permanent section evaluation. Diagnostic errors may
occur due to improper sampling, technical flaws, interpretive inaccuracies and in faulty communication.
Among the determinations made by frozen section analysis include the evaluation of adequacy of
surgical margins of resection; the differentiation between nonneoplastic, benign and malignant
proliferations; the evaluation of lymph nodes for the presence of metastatic disease; the determination
of specimen identification and specimen adequacy, including the verification of such organs as the
parathyroid glands; and the determination of whether a given case requires special diagnostic testing
best performed on frozen material, such as for lymphomas.
The 7 cases presented in this course represent selective head and neck lesions that
the surgical pathologists can expect to confront in daily practice whether in a community hospital
setting or in an academic center. Irrespective of the diagnostic issue at hand, a key component to
performing intraoperative consultations is the communication between the pathologist and the surgeon.
A 45 year old female with a known history of a T1 oral tongue
squamous cell carcinoma treated by irradiation presented 2 years later with a 2.8 cm lateral tongue mass
in the location of the previous tumor. The mass was resected.
The specimen received included a single roughly
elliptical-shaped tissue fragment measuring 2.1 x 1.5 x 0.6 cm. The surface of the tissue fragment
appeared tan-pink, smooth and shiny with an ill-defined and irregular appearing white area with focal
ulceration extending to but did not grossly appear to involve the designated (by the surgeon) "medial
margin" but not to the designated "lateral margin" of resection. The medial, lateral and deep margins of
resection were inked and a representative portion of the specimen was submitted for frozen section
squamous cell carcinoma comprised of cohesive nests and cords of invasive carcinoma with an endophytic
growth originating from the surface epithelium with infiltration into the submucosa and into subjacent
skeletal muscle. The neoplastic infiltrate varied in appearance from round to oval to angulated tumor
nests with associated stromal desmoplasia and a mild nonspecific chronic inflammatory cell reaction. In
the deeper aspects of the tissue (including at the deep margin) the carcinoma had a diffuse growth
pattern with small groups and cellular dissociation; further, there was less evidence of squamous cell
differentiation although individual cell keratinization was evident. The neoplastic were composed of
pleomorphic and hyperchromatic nuclei with prominent nucleoli, increased nuclear-to-cytoplasmic ratio and
increased mitotic activity, including atypical mitoses; intercellular bridges could be seen.
- - carcinoma was present at the deep margin of resection;
- - carcinoma came within 3mm of the medial margin of resection;
- - mild dysplasia was evident at the posterior margin of resection;
- - all other margins of resection were negative for carcinoma/dysplasia.
Intraoperative Diagnosis :
Invasive keratinizing squamous cell carcinoma involving
the deep margin of resection and coming within 3mm of the medial margin of resection. The posterior
margin showed mild dysplasia. All other margins, including lateral and anterior were negative for
The resection was extended medially and within the depth of the
lesion; subsequent medial and deep margins of resection showed clearance of greater than 1cm.,
respectively. The surgeon chose not to take additional tissue from the posterior margin. Biopsies of
all other surgical margins were negative. The procedure was a subtotal glossectomy with selective
(supraomohyoid) neck dissection; the latter were not sent for frozen section analysis.
Diagnosis on Permanent Section:
Invasive squamous cell carcinoma; all
margins were negative for carcinoma. Twelve regional lymph nodes were negative for carcinoma. AJCC
staging was T2N0M0 (Stage II).
Indications for Intraoperative Consultation of Mucosal Lesions
The indications for intraoperative consultation in mucosal (squamous cell) lesions of the upper
aerodigestive tract include:
1) render a histologic diagnosis (e.g., carcinoma/dysplasia) when
definitive therapeutic intervention is planned immediately;
2) assessment of the adequacy of resection (i.e., surgical resection
3) preliminary assessment of the nature of a planned procedure based
on the extent and distribution of the neoplasm (e.g., subtotal versus total laryngectomy);
4) adequacy for diagnostic purposes;
5) determination for special handling (e.g., immunohisotochemistry,
flow cytometry, microbiologic cultures, other);
6) determination of neurotropism, lymph-vascular space invasion (LVI)
or bone involvement that may necessitate resection of the involved bone (e.g., mandibulectomy);
7) if lymph nodes are excised then a frozen section may be requested
to exclude the presence of metastatic disease and the need for a neck dissection.
Surgeon's Expectations of the Intraoperative Assessment of Mucosal Lesions
1) establish the diagnosis of carcinoma/dysplasia and differentiate it
from look-alike lesions;
2) confirm presence of absence of lesional tissue at the margins of
3) when applicable, identify the presence of osseous involvement;
4) when applicable, identify the presence of nodal metastasis.
Specimen Handling and Orientation
The evaluation of surgical margins of resection for the presence or absence of lesional tissue falls
under the purview of the surgical pathologists. However, how the specimen is removed and the orientation
of the specimen is the responsibility of the surgeon. This is particularly true for those cases in which
the tumor is initially excised and the designated margins are separately removed, the tumor is removed in
multiple parts or the specimen is a complex en bloc excision requiring proper orientation by the surgeon
for those margins that are of critical concern. Once removed and properly oriented, the specimen becomes
the responsibility of the surgical pathologist.
There is no standard method by which surgeons remove tissue and thereby request intraoperative
consultation of the surgical resection margins. Some approaches include:
1) excision of the entire lesion, designation of specific margins and
tissue selection by the pathologist for frozen section. There are some centers in which all of the
circumferential resection margins are submitted for frozen section irrespective of the number of frozen
sections that may be required to completely evaluate the circumferential margins. In other centers, a
limited number of frozen sections (e.g., up to four – anterior, posterior, medial and lateral) are
submitted as determined by the pathologist;
Note: The anatomy of the head and neck is complex and in any given
resection the risk posed by removing the entire lesion and surrounding structures for intraoperative
2) submission of biopsies from areas of clinical concern following the
main resection and these biopsies are entirely submitted for frozen section.
- loss of the 3-dimensional orientation of the
- erroneous sampling of the areas of concern.
Right Angle (Perpendicular) versus Parallel (En-Face) Sections
Right angle section advantages include:
Right angle section disadvantages include:
- technically easier to obtain full thickness
- distance of the lesion to the resection
margin can be viewed and measured.
Parallel section advantages include:
- allows for evaluation of a relatively small
area of the lesion/margin.
Parallel section disadvantages include:
- allows for evaluation of a larger area of the
lesion/margin to include an entire margin if necessary.
Note: If parallel sections are made, the recommendation is to embed the
tissue with the true surgical margin deepest in the block.
- if the surgical margin is uninvolved the
distance of the lesion to the resection margin cannot be viewed and/or measured.
- due to retraction from the underlying
connective tissues the superficial layers (i.e., mucosa and submucosa) may not be optimally seen.
Surgical Resection Margins
Arguably, the most common request of the pathologist by the head and neck surgeon at the time of
intraoperative consultation is the assessment of the surgical margins. Successful local control of a
malignant tumor depends on complete surgical excision of all the disease. Undoubtedly the presence of
gross residual cancer results in local persistence of disease and increased morbidity and mortality.
There are many factors that impact on the assessment of the surgical margins, including the type of
surgical specimen, proper orientation of the specimen, proper sectioning of the specimen and obviously
the correct interpretation of the histopathologic changes. Unfortunately, even in the best situations an
intraoperative report of negative margins may be followed a few days later by a permanent section
diagnosis of positive margins.
Byers et alreported on the results of frozen section in 216 patients with neoplasms of the
oral cavity, oropharynx and hypopharynx. Three groups were identified, including 68 percent of the
patients in whom the tumors were initially adequately resected on the basis of negative surgical margins,
23 percent of the patients who had positive surgical margins that necessitated additional surgical
resection to assure the presence of negative surgical margins, and the remaining 9 percent of patients in
whom negative free margins by frozen section could not be obtained. The follow-up of these three groups
included local recurrence rates of 14.4 percent, 20 percent and 80 percent, respectively, with the third
group having the worst survival rates.Byers and colleagues concluded that the probability of
local recurrence in head and neck cancer is reduced when the resection margins are determined by
intraoperative frozen section consultation. The use of intraoperative frozen sections will also allow
the surgeon to extend the surgical resection without loss of orientation of the operative field, a
potential problem when additional surgery is required in a second operation.Further,
carcinoma remnants that have not been completely removed in the initial operation often are difficult to
identify macroscopically, making their removal in a second operation more difficult. Spiro and
colleagues reported an intraoperative frozen section diagnostic accuracy of 89% for oral tongue cancer.
These authors found that the diagnostic accuracy was the same whether the sample was taken from the
patient or from the surgical specimen.
Errors in interpretation and in sampling accounted for discrepancies. Cooley et al found five
discrepancies in 249 frozen sections of laryngeal squamous cell carcinoma in which the frozen diagnosis
was negative but the permanent sections revealed dysplasia or carcinoma in situ. These authors found
that insufficient leveling of the frozen block resulted in these discrepancies. Their recommendation was
that in examining margins for laryngeal squamous cell carcinoma the frozen section tissue should be
completely sampled by examining several levels at the time of frozen section. Spiro et al reported that
of the 131 samples taken intraoperatively whose margins were reported as negative, 13 were later reported
to be positive following permanent sections. While the frozen section diagnosis of surgical margins are
extremely accurate they are not entirely reliable in eliminating positive margins in the final diagnostic
Definition of a "Positive" Margin
Specimens in which no tumor or dysplasia is present at the surgical margins of resection, are
considered as completely excised. "At the margin of resection" means that the neoplastic cells are seen
in contact with or lie within millimeters of the pigment that was painted along the margin prior to
sectioning (i.e., tumor across or up to the resection margin). In this situation, the specimen is
considered incompletely excised, requiring a wider excision in order to be assured that all viable tumor
cells have been adequately removed. Batsakis (1999) in a comprehensive review from the pathologist's
perspective in the assessement of surgical excision margins in HNSCC made a number of cogent
1) the sole reliance of margins as assessed on the resected specimen
should be discouraged and, when feasible, the intraoperative evaluation of tissue surrounding the
specimen should be made and have that regarded as the "true margin";
2) the histologic definition of what constitutes a "positive" should
be uniformly accepted and applied. There is discrepancy in the literature as to what constitutes a
positive or negative resection margin. While some authors only include invasive carcinoma at the margin
as positive excluding carcinoma in situ, dysplasia and gross residual disease, most pathologists would
agree with the classification of positive margins, as defined by Loree and Strong:
Suffice to say that the evaluation of dysplasia under optimal circumstances can be subjective let
alone at the time of frozen section where tissue distortion and artifact create additional diagnostic
difficulties potentially resulting in misdiagnoses, including overdiagnosis and underdiagnosis. The
diagnosis of severe dysplasia has equal biologic significance with carcinoma in situ and microinvasive
carcinoma. A diagnosis of mild dysplasia also can be very problematic but is of questionable
significance. In fact, the progression of low-grade dysplasia to higher grade epithelial lesions is so
low as to make this diagnosis of little clinical significance, and often of minimal concern to the head
and neck surgeon. However, the progression of moderate dysplasia to a higher grade lesion is nearly
similar to that of severe dysplasia, (Barnes) so that a frozen section diagnosis of moderate dysplasia
results in a therapeutic approach similar to that of severe dysplasia.
- lesional tissue within 0.5 mm of the surgical
margin (so-called close margins) with the exception of laryngeal lesions (see below);
- dysplastic epithelium at the margin;
- carcinoma in situ at the margin;
- invasive carcinoma at the margin.
Adequacy of Resection Margins
The presence of lesional tissue within 5mm of the inked surgical margin irrespective of whether it is
invasive carcinoma or carcinoma in situ/severe dysplasia places a patient at a nearly equal risk for
local recurrence (Batsakis, 1999). These margins are associated with approximately 80% incidence of
recurrent disease, so that failure of local control is not inevitable with such positive margins (Byers
et al). The absence of positive margins does not guarantee local control of disease nor is it a reliable
guide to the biologic behavior of a tumor. Spiro et al report that the presence of positive margins
increased the likelihood of local recurrence but did not impact on survival since subsequent surgery
and/or irradiation controlled tumor recurrence in some of their patients. The incidence of positive
surgical margins is quite variable ranging from as low as 3 percent (Looser et al) in some studies to as
high as 60 percent in other studies (Mantravadi et al).
The question of how wide a tumor should be excised is the responsibility of the surgeon. However, for
some specimens such as the laryngeal squamous cell carcinoma, free margins up to 5 mm may be sufficient
while a similar tumor at another extralaryngeal site, such as the oral cavity and pharynx (oro-,
hypopharynx) wider margins (1.0 cm) are optimal.
Factors Impacting of Margin Status and Prognosis
Certainly, this variability in "positive surgical margins" is dependent on multiple factors, not the
least important of which may be the definition of what constitutes a positive margin. The factors that
may impact on whether a margin is positive or negative, and in prognosis (control of disease) include:
More recently, Brandwein-Gentsler et al suggests that resection margin status alone is not an
independent predictor of local recurrence nor should resection margin status alone be used as the sole
variable in deciding whether adjunctive radiation therapy is required; rather the need to give adjunctive
radiation is suggested to be based on the following parameters:
- clinical stage;
- tumor size;
- pattern of invasion;
- adjuvant therapy;
- histopathologic evaluation;
- site dependency;
- soft tissue margins.
According to Brandwein-Gensler et al, histologic assessement results in stratification of patients
into low-risk, intermediate-risk and high-risk categories that define recommendations for adjuvant
radiotherapy (Table 1).
- positive margins;
- perineural invasion;
- osseous invasion;
- histologic risk assessment that includes
score based on worst pattern of invasion:
- Patterns of invasion include:
- 1 = invasion in broad pushing front;
- 2 = invasion in "finger-like" broad pushing pattern
or separate large tumor islands;
- 3 = invasive tumor islands of tumor at periphery
greater than 15 cells/island;
- 4 = invasive tumor islands of tumor at periphery
smaller than 15 cells/island or strands of tumor cells in single cell filing pattern regardless of island
- 5 = satellites of dispered tumor infiltrates of any
size with 1mm or greater distance of intervening normal (non-fibrotic) tissue at the tumor-host
- perineural invasion;
- lymphocytic response;
Table 1. Risk Assessment for Oral Squamous Cell Carcinoma*
*: Adapted from Brandwein-Gensler et al. **: Patterns of invasion: 1 = invasion in broad pushing
front; 2 = invasion in "finger-like" broad pushing pattern or separate large tumor islands; 3 = invasive
tumor islands of tumor at periphery greater than 15 cells/island; 4 = invasive tumor islands of tumor at
periphery smaller than 15 cells/island or strands of tumor cells in single cell filing pattern regardless
of island size; 5 = satellites of dispered tumor infiltrates of any size with 1mm or greater distance of
intervening normal (non-fibrotic) tissue at the tumor-host interface. *** = sum of all points; LR =
Local recurrence; RT = Radiotherapy.
|Histologic Variable ||0 ||1 ||3|
|Perineural invasion ||None ||Small nerves ||Large nerves|
|Lymphocytic response ||Continuous band ||Large patches ||Little to none|
|Worst pattern of invasion at interface** ||1, 2 or 3 ||4 ||5|
| || || |
|Risk Score*** ||Risk for LR ||Overall Survival Probability ||Adjuvant Treatment with RT|
|0 ||Low ||Good ||No benefit|
|1 or 2 ||Intermediate ||Intermediate ||No benefit|
|3 - 9 ||High ||Poor ||Beneficial regardless of 5 mm margins|
Jacobs et al found that of the entry population of 696 patients in their study, 112 patients (16
percent) with stage III and IV operable tumors had positive surgical margins. These authors indicate
that the 16 percent represented a national average rather than an institutional incidence.In
comparison to patients with negative margins, patients with positive margins had a significantly higher
rate of local failure (21% versus 9%, p=0.0003), and distant failure (20% versus 12%, p=0.42) but a
similar incidence of regional (nodal) failure. Patients with positive surgical margins were most often
seen in nonglottic primary cancers and with increasing incidence as the N stage increased. The
positive-margin patients had a higher rate of distant metastasis and died more rapidly than
positive-margin patients with lower nodal status. The survival of the positive-margin patients was
approximately one half of the margin-negative patients. The addition of chemotherapy did not
significantly alter the survival of positive-margin patients. These authors reported a median survival
of 19 months in patients with positive margins essentially equating to the expected outcome for patients
with inoperable cancer.
Tumor Size ("T" Staging)
Scholl et al reported that in 54 of 268 patients (20.1%) with lingual squamous cell carcinoma, the
carcinoma was not initially completely removed (i.e., positive margins at frozen section). When
additional surgery resulted in negative margins, the local recurrence rate was worse than if the initial
margin was negative. Scholl et al also found that positive mucosal margins were more often present in T1
and T2 tumors and soft tissue margins were more common in T3 and T4 lesions. Looser et al reported that
71% of patients with positive margins had recurrent disease at the primary site in comparison with 32% of
patients with the tumor-negative margins. Loree and Strong found the incidence of positive margins was
directly proportional to the increasing tumor size. Batsakis (1988) notes that from 5% to 10% of
carcinomas resist the surgical goal of clear margins regardless of T stage. He further notes that the
survival of patients with free margins is related to the T stage of their carcinoma. All in all, the
lower the clinical stage and/or pathologic class the better the ability to achieve local control and the
overall better survival rates.
Pattern of Invasion
The pattern of invasive squamous cell carcinoma and the involvement of resection margins in oral
squamous cell carcinoma were evaluated in a study by Spiro et al. The patterns of invasion were graded
1-4 as follows: Grade 1 – invasion in a "pushing" manner with well-delineated border; Grade 2 – invasion
along the advancing edge in solid cords, bands or strands; Grade 3 – invasion in small groups or cords of
infiltrating cells; Grade 4 – marked cellular dissociation in small groups and/or single cells. These
authors found that those carcinomas invading with Grade 3 or 4 patterns were associated with an increase
in nodal and distant metastasis, and a significant decrease in survival. The Grade 3 and 4 invasive
patterns tended to be larger tumors (higher T stage) and occurred in younger patients (median of 56 years
as compared to Grade 1 and 2 invasive patterns with a median age of 62 years).
Other studies have shown that implications of a positive surgical margin for squamous cell carcinoma
of the head and neck are associated with increased local failure and decreased survival rates. Loree and
Strong reported that the overall local recurrence rate in the group of tumor-positive margins was 36% as
compared with 18% for the tumor-negative margin group. Further, these authors showed a statistically
significant difference in the five-year survival rate between the tumor-positive margin patients (52%
5-year survival) as compared to the tumor-negative margin patients (**52% 5-year survival). Although
postoperative radiotherapy was found to reduce the local recurrence rate in the positive-margin patients,
the overall 5-year survival rates between margin-positive and margin-negative patients was not affected
by the postoperative radiotherapy.
Scholl and colleagues in their evaluation of oral tongue squamous cell carcinoma indicate in the
presence of complete surgical removal of all disease to include macroscopic disease and histologically
negative margins without neural invasion that adjunctive therapy does not improve local control as
compared to surgery alone.
Beitler and colleagues showed that the addition of adjuvant external radiation therapy and
brachytherapy in patients with unexpectantly unsatisfactory surgical margins (i.e., microscopically
positive or close margins) after surgical resection improves local control.
van Es and colleagues reported that for T1 and T2 carcinomas of the mobile tongue and floor of mouth
the single most important parameter in determining local recurrence is the histopathologic evaluation of
margin status and that other histopathologic parameters were essentially irrelevant in predicting
recurrence. The presence of positive margins does not always translate into failure of local control;
however, the overwhelming majority of patients with recurrent disease at the primary site have positive
margins. The ability to completely resect a squamous carcinoma of the head and neck weighs heavily in
the surgeon's decision to utilize surgery in the attempt to eradicate the cancer, and the presence of
disease (gross or microscopic) at the surgical margins represents a key prognostic feature for patient
The identification of negative margins is not a guarantee for the absence of local recurrence. The
larynx perhaps is an outlier in regard to positive-margins and local recurrence. As compared to
extralaryngeal mucosal sites, patients with primary laryngeal squamous cell carcinoma with positive
surgical margins have a significantly lower incidence of local recurrence. Bauer et al reported that of
a total population of 111 patients with laryngeal squamous cell carcinoma 39 (35%) had positive surgical
margins. Of these 39 patients, seven (18%) developed local recurrence while four of the remaining 72
patients (6%) with negative margins developed local recurrence. These findings suggest that margin
status and local recurrence are site dependent and assists in explaining why surgeons are more apt to
accept nearer margins for laryngeal carcinoma (free margins up to 2 mm) but require wider margins (5-10
mm) for carcinomas of extralaryngeal mucosal sites. The factors that may contribute to the lower
incidence of local recurrences in laryngeal squamous cell carcinoma with positive margins supporting
organ sparing (conservative) laryngectomy may include:
Among the extralaryngeal sites with significant recurrence rates following negative surgical margin
determination include the oral cavity and pharynx. A factor that contributes to the recurrence rates of
pharyngeal and oral cavity carcinomas in the face of negative surgical margins is the tendency for
submucosal spread by these carcinomas. Intraoral sites with negative margins but significant recurrence
rates include the palate, tonsil, buccal mucosa, tongue, gingiva, floor of mouth and lip.
- these patients have early stage carcinoma
associated with a more favorable prognosis;
- the submucosa of the glottic region has
(quantitatively) less lymph-vascular spaces thereby decreasing the incidence of spread and lowering the
incidence of locoregional failure.
Soft Tissue Margins
Mucosal margins are not the only tissue margins. Surgical margins of resection may include all soft
tissue components including adipose tissue, skeletal muscle, bone and neural structures. The latter are
of particular concern in those tumors that have a propensity for neurotropism, such as adenoid cystic
carcinoma of salivary glands. For oral cavity carcinomas, the presence of dentition significantly
influences nerve-related spread. In evaluating spread of carcinoma within the mandible, McGregor and
MacDonald (1988, 1989) found a fourfold increase in cancer spread related to the inferior alveolar nerve
in edentulous, nonirradiated mandibles as opposed to partially dentate, nonirradiated mandibles. Nerve
involvement was associated with extensive spread of carcinoma in the medullary parts of the bone.
Bone, in particular the osseous margins of resection of the mandibular region pose an especially
significant issue in regard to carcinomas of the alveolar ridge, floor of mouth, lower buccal sulcus and
lower retromolar region. Carcinomas of these sites are among those with the highest rate of recurrence.
As such, the evaluation of the mandibular bone for the presence and extent of involvement is a key
determinant in patient management (Cleary and Batsakis).The methods of evaluating for osseous
involvement include: preoperative radiologic assessment; gross inspection; frozen section or imprint
evaluation of bone from the resected osseous stump; frozen section of adherent soft tissues. The
clinical determination of mandibular involvement is not reliable since one third of histologically proven
carcinomatous invasion of the mandible showed no clinical indication of (preoperative) bone involvement
(Weisman and Kimmelman).Spread of oral carcinoma to the mandible typically occurs by direct
invasion rather than by metastasis, lymph-vascular space spread or via nerves. Spread of carcinoma is
nearly always through cancellous bone and its marrow spaces. The frozen section assessment is difficult
given the inherent difficulties in performing frozen section on bone. Touch preparations or imprints of
the cancellous bone of the stump or frozen sections of curetted material can be performed, and have shown
with varying degree of success. Forest et al performing frozen sections on curetted material were able
to accurately predict the adequacy of mandibular resection in 97% of the margins (31 of 32 margins).
Alternatively, a portion of the soft tissue immediately adjacent to the periosteum may be submitted for
frozen section; if negative this might indicate that there is sparing of the bone and if positive would
indicate that there is bone involvement. However, the e valuation of the nonosseous soft tissue is not a
reliable indicator vis-à-vis osseous involvement. Further, in irradiated mandibles it may not be
possible to separate the periosteum. The size of the carcinoma does not appear to influence the
incidence of bone involvement but proximity to bone does. In an evaluation of local control of oral and
oropharyngeal carcinomas with clinically determined mandibular bone involvement, Dubner and Heller found
a 19% recurrence rate following marginal mandibulectomy and 6% following segmental mandibulectomy. In
this study local recurrence was not dependent on tumor size, N stage, invasion of the mandible or
radiotherapy. In those patients who recurred following segmental mandibulectomy, the recurrent disease
was present within extraosseous soft tissues.
Tissue Shrinkage and Surgical Margins
The reliability of measuring the resection margins is impacted by postremoval changes especially
related to tissue shrinkage. It is obvious that obtaining adequate tumor free surgical margins is
critical for the successful management of the cancer patient. Any discussion on surgical resection
margins would not be complete without a discussion on tissue shrinkage in the pathologic processing of
resected tissues. Disparate surgical margin lengths of resected specimens between the in vivo
measurements by the surgeon and the in vitro measurements by the pathologist have been reported for head
and neck resection specimens (Johnson et al; Beaumont and Hains) and non-head and neck resection
specimens (Gardner; Goldstein; Silverman; Sondenaa and Kjellvold).Johnson et al found a mean
tissue shrinkage of 31% (p<0.0001) from the initial in-situ measurement by the surgeon to the final
microscopic assessment of oral cavity and lingual surface mucosal margins by the pathologist. These
authors reported that to obtain 5 mm of histopathologically clear margin an in-situ margin of resection
of at least 8 to 10 mm needs to be taken. In their evaluation of oral squamous cell carcinomas Beaumont
and Hainsfound a reduction of 46% from the planned surgical margin before resection to the
microscopically measured margin following pathologic preparation (minimum of 10 mm measured in situ
surgical margin and average of 5.8 mm margin following fixation). These authors reported significant
differences in the longitudinal diameter of the whole specimen from in situ to fresh states (p <
0.0004) and in the diameter of the tumor from the fresh state to fixed states (p < 0.0000) with the
most significant shrinkage reported from the fresh state to the fixed state with a mean shrinkage of 4.82
mm. Goldstein et alin their evaluation of colorectal resection specimens found that bowel
segments shrank to a median length of 3 cm or 40% of their original in vivo length after being removed
from the patient and left in an unfixed state for 10 to 20 minutes. After formalin fixation the
free-floating ends shrank an additional 0.85 cm. Overall, after fixation colon segments shrank 57% of
their original in vivo length. However, the majority of the tissue shrinkage occurred within the first
few minutes after removal of the specimen and only 30% of the tissue shrinkage was attributed to formalin
fixation. Similarly, Johnson et alfound that the greatest proportion of shrinkage occurred
immediately upon resection. Although there are very few studies assessing the shrinkage of tissue
margins it is apparent that a significant amount of tissue shrinkage occurs from the moment the tissue is
excised to the time the pathologist reviews the histologic preparation of the excised tissues. Such
tissue shrinkage should be taken into account and accommodated for by the surgeon at the time of the
Another issue relative to the intraoperative histological evaluation of tumor resection margins is the
time factor. The identification of negative surgical margins by frozen section may require a large
number of samples. The result of numerous frozen sections in conjunction with the possibility of neck
dissection may be the prolongation of surgery. In a prospective study of 24 patients with oral squamous
cell carcinoma, Bähr and Stoll advocate the removal of the regional lymphatic drainage while the frozen
section examination of the surgical margins is being performed in order to reduce the time of surgery.
Microinvasive, Superficial or "Early" Invasive Squamous Cell Carcinoma (SCC)
Microinvasive SCC is a cancer that infiltrates into the superficial compartment of the lamina propria.
For laryngeal lesions, some authors consider microinvasive cancer to include the presence of scattered
malignant cells within the submucosa just below the basement membrane or within 1-2 mm of the basement
membrane while other authors feel that microinvasive carcinoma is present when tongues or discrete foci
of malignant epithelium invades through the basement membrane. Irrespective of its specific definition,
a diagnosis of microinvasive carcinoma excludes those lesions that are restricted to the surface
epithelium or carcinoma in situ (Tis) and those carcinomas that are deeply invasive into muscle and
cartilage, and extralaryngeal structures (T2 or greater tumors). Of note, extension of the dysplastic
process to involve the seromucinous glands is still considered as CIS and not invasive carcinoma.
Clinical manifestations and appearance are similar to those of carcinoma in-situ. In the larynx, full
cord mobility is present. Any dysfunction in vocal cord mobility (fixation) by definition means muscle
invasion, which excludes a diagnosis of microinvasive cancer.
Histologically, microinvasive carcinoma can occur in two unrelated phases. The first is the
development from and as a continuum of carcinoma in situ. The second is invasion from an epithelium
demonstrating no evidence of CIS. In the upper aerodigestive tract, particularly in the larynx, severe
dysplasia (i.e., carcinoma in situ) is not a prerequisite for the development of an invasive squamous
cell carcinoma. Such invasive carcinomas "drop off" or "drop down" from the basal cell layer with the overlying mucosa showing no evidence
of dysplasia. In all examples of invasive carcinoma the invasive nests must be cytologically malignant
including dysplastic changes, dyskeratosis and mitotic figures, including atypical forms. The tumor
nests have an irregular outline with infiltrative borders. The presence of invasive cancer generally
results in a desmoplastic host response that includes edematous change immediately around the tumor nests
with granulation tissue and fibrosis.
Microinvasive cancer is a biologically malignant lesion potentially capable of gaining access to
lymphatic or vascular channels in the lamina propria that may result in metastatic disease. For
microinvasive carcinoma of the laryngeal glottis, several studies have shown that the clinical
significance is similar to CIS/severe dysplasia that and with appropriate therapy (excision and/or
radiotherapy) progression of disease from a microinvasive to a more invasive carcinoma does not occur.
This may be due to the earlier clinical manifestations produced by glottic cancers leading to an earlier
diagnosis of cancer before it has invaded into deeper aspects of the larynx. Glottic microinvasive
cancers are generally not associated with metastatic disease due to the fact that the glottic portion of
the larynx has quantitatively less lymph-vascular spaces as compared to the supra- and subglottis. In
contrast to the laryngeal glottis, supraglottic microinvasive carcinomas are associated with metastatic
disease in approximately 20 percent of patients.
Histologic Prognostic Indicators
Among the histologic findings that may impact on the prognosis in head and neck squamous cell
carcinoma include: 1) status of the surgical resection margins, 2) tumor size, thickness and location of
the lesion; 3) pattern of invasion; 4) involvement of lymph-vascular spaces, 5) invasion of soft tissue
structures including nerves, bone and cartilage, 6) nodal metatsasis with or without extranodal extension
of tumor; 7) distant metastasis; 8) host response; 9) neovascularization, and 10) presence of multiple
Pitfalls in the Intraoperative Assessment of Squamous Cell Carcinoma/Dysplasia (Differential Diagnosis)
Frozen section consultations on mucosal surface lesions can be useful especially in differentiating
inflammatory and neoplastic lesions. Histologic grading of a mucosal malignancy (i.e., squamous cell
carcinoma) may be problematic and is not advocated by frozen section. Artifactual distortion and
sampling limitations may lead to erroneous conclusions relative to the histologic differentiation of the
1) Reactive Epithelial Changes
Reactive, infectious and neoplastic lesions (e.g., granular cell tumor) may be associated with an
exuberant epithelial response (pseudoepitheliomatous hyperplasia [PEH]) that may be mistaken for squamous
cell carcinoma. Despite this marked epithelial proliferation, there typically is an absence of
significant cytomorphologic atypia and evidence of invasion with associated tissue response (i.e.,
desmoplasia) allowing for differentiating PEH from squamous carcinoma.
2) Postirradiation Changes
Postirradiation alterations may lead to false positive diagnosis due to the presence of bizarre
cytologic alterations in the epithelium, minor salivary glands, fibroblasts, skeletal muscle and
Distinguishing invasive squamous cell carcinoma from radiation change is challenging. Obviously,
knowledge of prior radiation treatment would be imperative in the evaluation of a given specimen. If
this information is known prior to surgery and intraoperative consultation then comparison of any
previous material (e.g., biopsy) with the frozen section under review would be beneficial. Ultimately,
the differentiation of radiation effect versus squamous cell carcinoma is based on histologic review.
During the acute postirradiation phase (days to weeks) biopsies are seldom obtained. Biopsies taken in
the later postirradiation period (6- 7 weeks following therapy to years later) show variable histologic
changes, including a thinner than normal surface epithelium, surface ulceration, squamous epithelial
atypia, atrophy of minor salivary gland acini, pseudoepitheliomatous proliferation of minor salivary
glands, (atypical) squamous metaplasia of minor salivary glands, submucosal fibrosis, vascular
alterations characterized by telangiectatic capillaries often with prominent (plump) endothelial cells,
myointimal proliferation, foamy histiocytes within the intima and thrombosis, as well as atypical
(bizarre) fibroblasts, and bizarre striated muscle degeneration. The retention of the lobulated outline
of the minor salivary glands, presence of "smudged" appearing nuclei of the atypical fibroblasts and
absence of cohesive cellular grouping of the atypical fibroblasts scattered in scar formation or
inflammatory tissue assists in distinguishing radiation change from carcinoma. Delayed radiation injury
generally is not characterized by cellular inflammatory infiltrate. Familiarity with these findings
should allow for their recognition and prevent misinterpretation. As previously indicated, the
information that a patient received prior radiation to a given region being sent for intraoperative
consultation should be a requirement.
3) Juxtaoral Organ of Chievitz
Normal microscopic structure of unknown function found in the located bilaterally at the angle of the
mandible (in the retromolar trigone of the oral cavity) near the buccotemporalis fascia and intimately
associated with branches of the buccal nerve. Histologically, this structure is composed of nests or
clusters of squamous cells with intercellular bridges surrounded by basaloid cells; the latter may show
nuclear palisading. Keratinization is not present although duct-like lumina may be seen. The cells are
uniform with low nuclear-to-cytoplasmic ratio, minimal pleomorphism and absent mitotic figures;
desmoplasia is absent. These structures may easily be misinterpreted as invasive carcinoma with
perineural invasion. Familiarity with these structures, their characteristic location and overall
relatively bland histomorphology should allow for distinguishing these normal benign structures from
invasive carcinoma. Of course, detailed conversation with the surgeon would be imperative in identify
these structures and not misdiagnosing them as invasive carcinoma.
As with any surgical procedure, there are contraindications for the utilization of frozen sections.
Frozen section consultation should not be used:
- when the frozen section diagnosis will not
have any impact on surgery or no immediate decision, such as satisfying the curiosity of the surgical
- if the tissue specimen is small and additional
sampling is not planned (in which situation, frozen sections may be equivocal and/or the material is
artifactually distorted by the frozen section technique hampering histologic evaluation following
- for heavily calcified or ossified tissue;
- for certain lesions such as small cutaneous
melanocytic lesions and lymphoproliferative lesions requiring special handling or extensive histologic
evaluation for diagnosis.
Intraoperative "Rapid" Immunohistochemical Assessment
Intraoperative immunohistochemical assessment consisting of touch smear cytologic preparations and
cytokeratin staining in 20 minutes from the time of tissue sampling. Limited studies to date (Matsusaka)
on gastric mucosal margins in patients undergoing gastrectomy for gastric (non-signet ring cell)
carcinomas show accurate and rapid assessment of tumor margins. To date, such analyses have not been
published relative to mucosal margins of the upper aerodigestive tract.
Molecular Biology in the Assessment of Surgical Margins ("Molecular Margins")
Brennan et al utilized molecular biologic markers in assessing histopathologic negative surgical
margins and negative lymph nodes for patients with squamous cell carcinoma of the head and neck. In 52
percent of the patients studied, p53 mutations were found in the tumor margins that were identifed as
free of tumor by conventional histologic examination. p53 mutations were found in 21 percent of the lymph
nodes that were negative for tumor by conventional histologic examination. In 38 percent of the patients
with p53 positive margins, the tumor recurred locally. The authors concluded that the presence of p53
mutations in surgical margins and in lymph nodes that were negative for tumor by light microscopic
examination portended a substantially higher risk of local recurrent disease than those patients without
p53 mutations in their surgical specimens. In their opinion, molecular biologic studies augmented
conventional light microscopy in identifying cancer at surgical margins and in lymph nodes, and also may
improve the prediction of local tumor recurrence.
Subsequent to the study by Brennan and colleagues, Ball et al evaluated p53 immunostaining of surgical
margins in predicting local recurrence of oral and oropharyngeal SCC. These authors found that there was
sample odds ratio test predicting a 5.333 times higher chance of local recurrence in patients with at
least one p53 positive surgical margin.
eIF4E is a translation initiation factor and powerful oncogene when overexpressed in model cell lines
(Franklin et al) eIF4E has been reported to be elevated in HNSCC but not in benign lesions
and overexpression of eIF4E may represent an early step in malignant transformation.
Immunohistochemical analysis for eIF4E expression has shown it to be consistently elevated in patients
with SCC. In a study by Franklin et al of laryngeal and hypopharyngeal carcinoma 92% (12 of 13 cases) of
patients with negative eIF4E in histologically negative surgical margins were free of locoregional
recurrences while 67% (12 of 18 patients) with overexpressed eIF4E in the surgical margins developed
recurrences disease-free interval of 31.95 months). These authors reported significantly differences in
the Kaplan-Meier survival curves for eIF4E-positive and eIF4E-negative margins (p=0.0002). Molecular
margins as presently constituted have not yet supplanted conventional methods of evaluating surgical
In general, the accuracy of frozen section diagnosis in head and neck surgery is high, and when
deferred diagnoses are excluded, the reported accuracy ranges from 97 to 99% (Gandour-Edwards et al
Ord and Aisner; Remsen et al; Spiro et al).
Several errors and other factors may account for any potential discrepancies in the frozen
section-to-permanent section diagnosis. Errors can be divided into four categories including:
In addition, there are other factors that may influence the result of a frozen section, including (but
not limited to):
- sampling errors;
- interpretative errors;
- technical errors;
- communication errors.
- cooperation between the surgeon and
- quality assurance program allowing for
continuous analysis and critical overview of the frozen section standards;
- availability of other pathologists for
consultation in challenging cases;
- availability of technically competent staff;
The frozen section diagnosis of lymph nodes is considered to be extremely accurate. Gnepp in a review
of the literature reported that an accuracy rate of 98.9 percent, excluding deferred diagnoses, with a
0.1 percent false-positive rate and a 1 percent false negative rate. It should be noted that the lymph
nodes represents the most frequently deferred specimen in frozen section diagnosis, especially in the
diagnosis of a lymphoma. In general, the diagnosis of a carcinoma in a lymph node is not problematic at
frozen section. Lymph node frozen section has also been utilized for accurate staging of the head and
neck patient. Rassekh et al compared intraoperative node examination (palpation and inspection) to
frozen section diagnosis in the identification of metastatic disease to cervical lymph nodes, thereby
evaluating the surgeon's ability to predict nodal stage. These authors prospectively studied 108 necks
in 79 patients and reported that the overall reliability for intraoperative staging (palpation and
inspection) was 59.3 percent (64 of 108) as compared to 92.3 percent (24 of 26) for frozen section
biopsy, representing a highly significant difference (p < .005).Although frozen section
biopsy was not performed on all cases, Rassekh et alconcluded that upstaging the neck without
frozen section biopsy is much less reliable, and that frozen section biopsy is needed prior to converting
a selective node dissection to a radical or modified radical neck dissection. Manni and van den Hoogan
reached a similar conclusion.
Sentinel Lymph Nodes in Head and Neck Squamous Cell Carcinoma
There is increasing literature documenting the efficacy of sentinel lymph node evaluation in patients
clinically staged as N0 necks (cN0) with diagnostic accuracy of over 90% and with few reported false
negatives. Elective treatment of cN0 necks by surgery and/or irradiation is controversial with some
authorities advocating a conservative (watch and wait) approach and other authorities advocating
treatment. The overall risk of occult metastases or neck recurrence in cN0 neck ranges from 10 to 30%
prompting arguments for and against elective treatment. The majority of patients with cN0 necks likely
do not harbor occult metastases, but patients with undetected and untreated metastases will experience
high failure rates with increased morbidity and mortality; for this reason, sentinel lymph node procedure
is gaining more support in the treatment of cN0 neck. To date, there have been limited studies of
sentinel lymph node procedure in head and neck squamous cell carcinoma showing:
Conceptually, lymphatic drainage from a specific anatomic site is assumed to be predictable in the
lymphatic drainage; therefore, metastatic tumor cells from a specific location will become deposited into
the first (sentinel) node in a lymph node chain. Pathologic work-up, including handling of the gross
resection specimen, number of histologic sections, use of special stains (i.e., immunohistochemistry) for
the sentinel lymph node procedure for head and neck squamous cell carcinoma has not to date been
standardized. Larger clinical trials are needed to determine the efficacy of the sentinel lymph node
procedure for head and neck squamous cell carcinoma for staging, treatment and survival of the patient
with cN0 neck. The application of sentinel lymph node procedure to head and neck squamous cell carcinoma
has not as yet reached the level of standard of patient care.
- a sensitivity of greater than 90% in
identifying at least one sentinel lymph node;
- false negative rate of less than 10% (mean,
- the clinical significance of identifying
micrometastatic disease (i.e., isolated tumor cells and/or clusters of tumor cells measuring less than
.2mm as defined in breast cancer) relative to head and neck squamous cell carcinoma remains to be
clarified but likely is significant in its prognostic import.
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