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 .
Selected for reassessment of neck dissection specimens were the paraffin(Drug information on 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 ; 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.
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 . 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 3).
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.