Detection of Nodal Micrometastases in Head and Neck Cancer by Serial Sectioning and Immunostaining

Detection of Nodal Micrometastases in Head and Neck Cancer by Serial Sectioning and Immunostaining

ABSTRACT: We investigated the incidence of micrometastases from squamous cell carcinomas of the head and neck in neck dissection specimens originally staged as pN0. A total of 76 dissection specimens from 60 patients were evaluated using serial microscopic sectioning in 10-mm intervals, H & E staining, and immunostaining with an antibody to pan-cytokeratin. Examination of 1,020 lymph nodes from 76 neck dissection specimens revealed 8 micrometastases (7.9%) in 6 specimens from 6 patients with oral and pharyngeal primaries, resulting in upstaging. Six micrometastases were located in lymph nodes 3 to 6 mm in diameter. The surgeon should be aware of the relatively high incidence of micrometastases from oral and pharyngeal carcinomas, which are undetectable preoperatively or by routine histopathologic examination. Primary tumor site (oral cavity and pharynx) and certain features of the primary can delineate a group of patients with a higher risk of harboring occult metastases who may benefit from elective treatment of the neck. [ONCOLOGY 10(8):1221-1226, 1996]


The status of the cervical lymph nodes is the most important prognostic
factor in squamous cell carcinoma of the upper aerodigestive tract.
The number of positive nodes and the presence of extranodal (or
extracapsular) spread are the two most commonly used prognostic
factors. Other characteristics of prognostic importance are the
size of the node, level of positive nodes, and histologic response
to the tumor in the node. The extent of nodal disease in the neck
has prognostic significance not only for failure in the neck but
also for the development of distant metastases and, possibly,
for recurrence at the primary site [1-3].

Histopathologic examination of neck dissection specimens, therefore,
provides basic information for diagnosis, staging, and prognosis.
Clinicians seldom question the reliability of this examination.
Although extranodal spread of metastatic carcinoma from cervical
nodes is a major prognostic factor [4-6], the clinical implications
of micrometastases remain unclear.

The demonstration of micrometastases in a neck dissection specimen
deemed to be pathologically negative after routine examination,
resulting in upstaging, seems to be more important than the finding
of additional micrometastases in a resection specimen with overt
nodal disease. We conducted a retrospective study to determine
whether serial sectioning and immunohistochemistry using an antibody
against cytokeratin, a marker of epithelial cells, could detect
occult micrometastases in initially pN0 neck dissection specimens

Patients and Methods

Tissue Selection

Selected for reassessment of neck dissection specimens were the
paraffin-embedded lymph nodes of 60 previously untreated patients
who presented from June 1986 to December 1990. These patients
had primary squamous cell carcinoma of the oral cavity, oropharynx,
hypopharynx, or larynx; underwent laser microsurgery on the primary
tumor and a unilateral or bilateral neck dissection; and were
classified as pN0 after routine histopathologic examination. Ipsilateral
neck dissection was carried out in 44 patients and bilateral dissection
in 16 patients, so that a total of 76 neck dissection specimens
were evaluated. The primary sites were as follows: oral cavity
(19 patients), oropharynx (6), hypopharynx (9), glottis (13),
and supraglottis (17).

Clinical Staging and Neck Dissection

Preoperative staging of the neck nodes was done by palpation and
ultrasonography. Computed tomography (CT), magnetic resonance
imaging (MRI), and aspiration cytology were not employed routinely.
The neck dissections, either elective (prophylactic) or therapeutic
(when positive nodes were clinically suspected), were selective
[8]; ie, they were confined to the lymph node groups in which
metastases from the specific primary tumor are most likely to
occur [9,10]. In patients with laryngeal and oropharyngeal primaries,
levels II and III were removed, whereas in those with oral carcinoma,
levels I, II, and III were dissected (supraomohyoid neck dissection),
and in those with hypopharyngeal primaries, levels II, III, and
IV were removed (lateral neck dissection) (Figure 1). The nonlymphatic
structures (sternocleidomastoid muscle, internal jugular vein,
and spinal accessory nerve) were preserved.

Histopathologic Techniques

For routine examination, the neck dissections specimens were fixed
in formalin. All visible or palpable lymph nodes were dissected
free of surrounding fat. All nodes ³ 5 mm were cut in half
and were then embedded in toto for histologic examination. The
"original" surgical pathologic diagnosis was based on
microscopic examination of two to three 1-mm sections from each
node stained with hematoxylin and eosin (H & E).

Subsequent to this "original" examination, the paraffin-embedded
lymph nodes were totally sectioned into 1-mm sections. Every 10th
section was stained with H & E, and every 50th section was
obtained for immunostaining. A mouse monoclonal antibody to pan-cytokeratin
was used for immunostaining. This antibody reacts with human cyto-keratin
of 40, 46, 52, 56, 58, and 65 to 67 kD. To make the antigen (cytokeratin)/antibody
reaction visible, the classic alkaline phosphatase antialkaline
phosphatase (APAAP) technique was used [11]. In this technique,
the APAAP complex is stained with neufuchsin, which causes the
keratin-positive cells to turn red. The remaining tissue is made
visible by counterstaining with hematoxylin.

Since there is no agreed upon definition of micrometastases, the
International Union Against Cancer (UICC) definition for axillary
lymph node micrometastases from breast carcinoma was used. According
to this definition, micrometastases include metastatic deposits
of up to 2 mm in diameter.


Overall, 1,020 lymph nodes from 76 initially pN0 neck dissection
specimens were examined. On average, 13.5 lymph nodes were found
in each of the specimens. The largest axial diameter of each node
was recorded. The nodes were 1 to 22 mm in diameter. A total of
5,999 sections were stained with H & E, and 1,261 sections
were immunostained.

Eight lymph nodes (.8%) harbored metastases of a squamous cell
carcinoma, shown in the H & E-stained sections. All metastases
were micrometastases, located in the subcapsular sinus of the
lymph node, and all were found in nodes from level II (upper jugular
group). Six micrometastases were found in lymph nodes < 10
mm in diameter. Two metastases were detected in nodes 20 mm in
diameter. The examination revealed no larger metastases and no
extranodal spread. Im-munostaining found the same eight metastases
but no additional metastases.

Reevaluation of the original slides revealed that three of these
eight metastases had been present but were overlooked. The overlooked
metastases were composed of 200 to 500 tumor cells each; the original
microscopic slides had shown 4 to 6 tumor cells (Figures 2 and

The eight micrometastases were found in six patients (Table 1),
two of whom had two metastases. The primary tumor was located
in the oral cavity in three patients, the oropharynx in two patients,
and the piriform sinus in one patient. Neither serial sectioning
nor immunostaining revealed micrometastases in patients with glottic
or supraglottic primaries.

Two of the six patients with micrometastases had been subjected
to postoperative adjuvant radiotherapy because of positive microscopic
resection margins, despite re-resection of the primary. Another
4 of the total 60 patients had been irradiated for the same reason.
With a median follow-up period of 37 months, none of the 60 patients
developed recurrent metastasis in the neck. Local recurrences
developed in seven patients, and a second primary tumor occurred
in five patients. No distant metastases were observed.


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