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

August 1, 1996

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

ABSTRACT: We investigated the incidence of micrometastases from squamouscell carcinomas of the head and neck in neck dissection specimensoriginally staged as pN0. A total of 76 dissection specimens from60 patients were evaluated using serial microscopic sectioningin 10-mm intervals, H & E staining, and immunostaining withan antibody to pan-cytokeratin. Examination of 1,020 lymph nodesfrom 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 lymphnodes 3 to 6 mm in diameter. The surgeon should be aware of therelatively high incidence of micrometastases from oral and pharyngealcarcinomas, which are undetectable preoperatively or by routinehistopathologic examination. Primary tumor site (oral cavity andpharynx) and certain features of the primary can delineate a groupof patients with a higher risk of harboring occult metastaseswho may benefit from elective treatment of the neck. [ONCOLOGY10(8):1221-1226, 1996]

Introduction

The status of the cervical lymph nodes is the most important prognosticfactor in squamous cell carcinoma of the upper aerodigestive tract.The number of positive nodes and the presence of extranodal (orextracapsular) spread are the two most commonly used prognosticfactors. Other characteristics of prognostic importance are thesize of the node, level of positive nodes, and histologic responseto the tumor in the node. The extent of nodal disease in the neckhas prognostic significance not only for failure in the neck butalso 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 cervicalnodes is a major prognostic factor [4-6], the clinical implicationsof micrometastases remain unclear.

The demonstration of micrometastases in a neck dissection specimendeemed to be pathologically negative after routine examination,resulting in upstaging, seems to be more important than the findingof additional micrometastases in a resection specimen with overtnodal disease. We conducted a retrospective study to determinewhether serial sectioning and immunohistochemistry using an antibodyagainst cytokeratin, a marker of epithelial cells, could detectoccult micrometastases in initially pN0 neck dissection specimens[7].

Patients and Methods

Tissue Selection

Selected for reassessment of neck dissection specimens were theparaffin-embedded lymph nodes of 60 previously untreated patientswho presented from June 1986 to December 1990. These patientshad primary squamous cell carcinoma of the oral cavity, oropharynx,hypopharynx, or larynx; underwent laser microsurgery on the primarytumor and a unilateral or bilateral neck dissection; and wereclassified as pN0 after routine histopathologic examination. Ipsilateralneck dissection was carried out in 44 patients and bilateral dissectionin 16 patients, so that a total of 76 neck dissection specimenswere 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 andultrasonography. Computed tomography (CT), magnetic resonanceimaging (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 whichmetastases from the specific primary tumor are most likely tooccur [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, andIV were removed (lateral neck dissection) (Figure 1). The nonlymphaticstructures (sternocleidomastoid muscle, internal jugular vein,and spinal accessory nerve) were preserved.

Histopathologic Techniques

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

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

Since there is no agreed upon definition of micrometastases, theInternational Union Against Cancer (UICC) definition for axillarylymph node micrometastases from breast carcinoma was used. Accordingto this definition, micrometastases include metastatic depositsof up to 2 mm in diameter.

Results

Overall, 1,020 lymph nodes from 76 initially pN0 neck dissectionspecimens were examined. On average, 13.5 lymph nodes were foundin each of the specimens. The largest axial diameter of each nodewas recorded. The nodes were 1 to 22 mm in diameter. A total of5,999 sections were stained with H & E, and 1,261 sectionswere immunostained.

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

Reevaluation of the original slides revealed that three of theseeight metastases had been present but were overlooked. The overlookedmetastases were composed of 200 to 500 tumor cells each; the originalmicroscopic slides had shown 4 to 6 tumor cells (Figures 2 and3).

The eight micrometastases were found in six patients (Table 1),two of whom had two metastases. The primary tumor was locatedin the oral cavity in three patients, the oropharynx in two patients,and the piriform sinus in one patient. Neither serial sectioningnor immunostaining revealed micrometastases in patients with glotticor supraglottic primaries.

Two of the six patients with micrometastases had been subjectedto postoperative adjuvant radiotherapy because of positive microscopicresection margins, despite re-resection of the primary. Another4 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 patientsdeveloped recurrent metastasis in the neck. Local recurrencesdeveloped in seven patients, and a second primary tumor occurredin five patients. No distant metastases were observed.

Discussion

Detection and Incidence of Micrometastases

This study shows that the frequency of micrometastases in cervicallymph nodes is high. In 6 (7.9%) of 76 initially node-negativeneck dissection specimens, micrometastases were detected by serialmicroscopic sectioning, resulting in upstaging. We could not detectadditional metastases using immunohistochemistry for cytokeratin.In our opinion, the expensive and time-consuming immunohistochemicalmethod does not offer any benefit over conventional H & Estaining but does facilitate detection due to the intense redstaining of keratin-positive cells.

At routine "original" histopathologic examination, largermetastases and especially metastases with extracapsular spreadhad not been overlooked. Previous studies have reported that ex-tracapsularspread is present in about 20% of small (less than 10 mm) lymphnodes [12] and even occurs in lymph nodes less than 5 mm in diameter[13]. The observation that small metastases may show extracapsularspread may have important implications for the accuracyof clinicalstaging and prognosis.

Several studies have highlighted the limitations of routine histologicexamination in the detection of nodal disease. Investigationson the incidence of axillary lymph node micrometastases from breastcarcinoma have established that micrometastases are difficultto evaluate and that their incidence varies according to the methodsused for their detection. In breast carcinoma, the reported incidenceof micrometastases varies from 9% [14] to 37% [15].

To our knowledge, only one other study has assessed the incidenceof micrometastases in head and neck cancer. Using specimen radiography,van den Brekel et al [16] found an average of 15.9 lymph nodesin specimens obtained from mostly supraomohyoid neck dissections.These authors additionally sectioned lymph nodes at one deeperlevel of the paraffin block than they routinely did and employedH & E staining; they defined micrometastases as metastasesequal to or less than 3 mm in diameter. Based on that definition,they found 2 micrometastases (3%) among 62 initially negativeneck dissection specimens. In the present study, we examined acomparable number of nodes (13.5) from each selective neck dissectionspecimen and employed no special technique for improving the lymphnode harvest. We found a somewhat higher proportion of patientswith occult lymph node micrometastases (7.9%) than did van denBrekel et al. This may be due to the larger number of sectionsexamined.

Prognostic Significance of Micrometastases

Since serial sectioning of multiple cervical lymph nodes entailsadditional work, its therapeutic importance should be confirmedbefore it is recommended for routine use. The prognostic significanceof micrometastases in patients with head and neck cancer has neverbeen assessed. Our study assessed the incidence of micrometastaseswith no intention of examining their prognostic significance.However, in our cases, the detection of micrometastases has notinfluenced treatment decisions, as we see no indication for adjuvantradiotherapy if only micrometastases are present. The value ofsystemic therapy has not been established in head and neck carcinoma,as it has been in breast or colorectal cancer.

Although diseases are not comparable, reports on the prognosticsignificance of micrometastases in breast carcinoma are contradictory.Several studies have found that breast cancer patients with micrometastaseshave a prognosis similar to that in patients with node-negativedisease [17,18]. Other more recent studies show that patientswith axillary lymph nodes containing micrometastases have a higherdisease recurrence and lower overall survival than do patientswith tumor-free axillary nodes. This difference in survival isestimated to be approximately 10% to 20% [14,19-24]. Adjuvantchemotherapy is generally considered useful for patients withbreast carcinoma and axillary metastases (including micrometastases),and it seems likely that the therapeutic interventions employedasa result of the early detection of axillary micrometastases mayimprove outcome.

The incidence of micrometastases found by immunohistochemistryin lymph nodes in colorectal carcinoma is similar to the incidenceof micrometastases in breast cancer [25-27]. However, evidenceregarding the clinical significance of lymph node micrometastasesin colorectal cancer is conflicting. In colorectal carcinoma,the detection of occult micrometastases failed to correlate withsurvival in two studies [25, 26] but was prognostically significantin a third investigation [27].

Implications for Clinical Staging and Treatment

Micrometastases themselves cannot be detected preoperatively byany currently available staging technique. With palpation alone,the overall error in assessing the presence or absence of cervicallymph node metastases is reported to range from 20% to 30% [28].With the use of modern imaging techniques, such as ultrasonography,CT, and MRI, the error rate drops to below 10% to 15%. In general,lymph nodes more than 10 mm in diameter are regarded to be suspiciousfor malignancy. In our study, six metastases were located in nodes3 to 6 mm in diameter; nodes of this size are not reliably detectableby ultrasound, CT, or MRI. Thus, even the routine use of modernimaging techniques will not replace elective treatment of theneck.

As none of our patients with micrometastases experienced recurrentmetastasis in the neck, our study showed, on the one hand, thatselective neck dissection is therapeutically effective. On theother hand, the great majority of patients were subjected to anunnecessary therapy.

The rationale for elective treatment of the neck is that subclinicaldisease will develop into clinically manifest metastases. It iswell known that, despite close follow-up, some patients will developinoperable metastases when managed with a "wait-and-see"policy. Elective treatment of the neck nodes is considered justifiedin most institutions if the rate of occult lymph node metastasisexceeds 15% to 20% [29]. Furthermore, elective treatment is consideredin patients with necks that are difficult to examine clinically,when regular follow-up is impossible, or if the neck must be enteredfor exposure of the primary tumor.

With regard to the treatment modality used for elective treatmentof the neck, surgery is used for the neck if the primary tumoris treated surgically, and radiotherapy is employed if the primarytumor is irradiated.

Prediction of Cervical Metastasis

Several studies have attempted to correlate a number of histopathologicfeatures of the primary tumor with the risk of nodal metastasis.These studies modified conventional histologic grading to improvethe prognostic efficiency of the histologic assessment of theprimary tumor [30-33]. In an effort to identify patients who wouldactually benefit from neck surgery, we examined the relationshipof the pT category, cytologic grade, and maximum depth of invasionof the primary tumor to regional metastases [7]. In agreementwith others, we found that the maximum depth of tumor invasion,measured from a reconstructed mucosal line to the point of maximuminvasion, as described by Moore et al [34], was more importantthan the surface diameter [33,34-37]. We noted a strong correlationbetween the depth of invasion and nodal disease, whereas no influenceof the pT category as a parameter of surface extent and cytologicgrade was found.

Statistical calculation showed a depth of invasion of 4 mm tobe a valuable cut-off point for the occurrence of lymph node metastases.As the depth of tumor invasion is a pathologic finding, it isnot valuable for clinical staging or preoperative treatment planning.This may be the reason that this criterion has not gained widespreadacceptance.

Conclusions

In our opinion, the surgeon should be aware of the relativelyhigh incidence of micrometastases (7.9%) in patients with headand neck cancer. The majority of lymph nodes with micrometastaticdeposits cannot be detected preoperatively with modern imagingtechniques. Serial microscopic sectioning of the nodes revealsmicrometastases but is impractical for routine use. Selectiveneck dissection adds very little morbidity and is an effectivetreatment modality for early metastatic disease. Primary tumorsite (oral cavity and pharynx) and certain features of the primarytumor (eg, maximum depth of invasion) can delineate a group ofpatients with a higher risk of har-boring occult metastases whomay benefit from elective treatment of the neck.

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