EPITHELIAL MEMBRANE ANTIGEN AND S-100 PROTEIN4ABELED CELLS IN PRIMARY AND METASTATIC LARYNGEAL CARCINOMAS Tadashi Nakashima, MD, Gen Yano, MD, ltsuroh Hayashi, MD, and Yasaburoh Katsuta, MD

lmmunohistochemical studies were done on the expression of S-100 protein-labeledcells in human laryngeal carcinoma of epithelial membrane antigen (EMA) and on the population density. EMA was detected in 65 of 76 (86%) squamous cell carcinomas and was usually more extensively positive in well and moderately differentiated carcinomas. Thus, EMA can serve as a marker of malignancy in laryngeal carcinomas. The population density of S-100 protein-labeledcells was high in well-differentiated tissues. In the metastatic tumors, the number of S-100 protein-labeled cells decreased as compared to findings ill the primary tumors. As a statistically significant difference in survival curve was noted between groups with low and intermediate density of S-100 protein-labeled cells, the infiltration of S-100 protein-labeledcells seems to be associated with a better prognosis. HEAD & NECK 1992;14:445-451 0 1992 John Wiley & Sons, Inc.

From Departments of Otolaryngology (Drs. Nakashima and Yano) and Pathology (Drs. Hayashi and Katsuta), National Kyushu Cancer Center, Fukuoka, Japan. Acknowledgments: Supported in part by a grant-in-aid for a comprehensive 10-year strategy for cancer control from the Ministry of Health and Welfare of Japan and a grant-in-aid for scientific research from the Fukuoka Cancer Society, Japan. The authors thank M. Ohara for helpful comments. Address reprint requests to Dr. Nakashima at the Department of Otolaryngology, National Kyushu Cancer Center, Notame 3-1-1, Minami-ku, Fukuoka 815, Japan. Accepted for publication May 18. 1992. CCC 0148-6403/92/060445-07 0 1992 John Wiley & Sons, Inc

EMA and 5-100 Protein-LabeledCells in Laryngeal Carcinoma

Although the curative rate of patients with laryngeal cancer is relatively favorable as compared to other head and neck cancers, the prognosis is poor in some cases. Size of the tumor, histologic type, and metastasis to regional lymph nodes or distant organs are all pertinent factors. To evaluate the nature of laryngeal carcinoma cells and their influence on the clinical course and prognosis, we examined the distribution of epithelial membrane antigen (EMA) and S-100 protein-labeled cells in human laryngeal carcinomas. EMA is a glycoprotein isolated from human milk fat globule membrane and is widely distributed in glandular Squamous epithelial cells in a normal state do not usually express this antigen but do show positive staining on the plasma membrane of adjacent epithelial cells, in various pathologies, including inflammation and ne~plasia.~,~ S-100 protein-labeled cells are recognized as Langerhans ~ e l l s ,and ~ . ~infiltration of these cells correlates with survival of patients with malignancies of the lung,6 na~opharynx,~ and esophagus.’ S-100 protein-labeled cells are thought to have a key role in cellular immunity by T lymphocytes and are defined as “T-zone histiocytes.’16 We examined the prognostic implication of clinical staging, histologic difference, and immunohistochemical staining pattern of EMA and

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diaminobenzidine tetrahydrochloride containing 0.01% (wt/vol) hydrogen peroxide in 0.05 M TrisHCl buffer (pH 7.6). Counter staining was done with methylgreen. For the controls, incubation with nonimmunized mouse (rabbit) serum was carried out. The staining intensity for EMA was visually MATERIALS AND METHODS graded from negative to intense. When the staining intensity of EMA was equal to that seen in Materials. The study group consisted of 79 Japthe glandular epithelium or acini, in the same anese patients (71 men, eight women), aged from tissue section, a weakly positive (+) value was 40 to 84 years (mean, 63.6 years), who were diaggiven. The number of S-100 protein-labeled cells nosed with laryngeal carcinoma and who underwas counted at the three most densely infiltrated went total laryngectomy at the National Kyushu areas and averaged as a population density gradCancer Center between April 1981 and March ing according t o density per high-power field 1991. No patient had been given preoperative raunit, as follows: low, 0 to 6 cells; intermediate, 7 diotherapy or chemotherapy. Macroscopic examito 15 cells; high, more than 16 cells.' The countnation of the removed larynges revealed that 48 ing was done under the microscopic field of X400 were supraglottic, 29 glottic, and two subglottic. According to the 1987 UICC TNM classifi~ation,~ magnification, using a binocular Nikon X2 microscope. tumor stage was as follows: stage I, 10 cases; stage 11, 24; stage 111, 27; stage IV, 18. MeStatistics. The survival curve was calculated tastases were present in 27 patients (26 regional by the Kaplan-Meier life table method.12 Surlymph nodes metastases and three distant mevival time was compared using the generalized tastases). Pathologic examination revealed 76 Wilcoxon test, and differences were considered (26 well, 40 moderately, 10 poorly differentiated) to be statistically significant when the p value squamous cell carcinomas, one verrucous carciwas less than 0.05. noma, one adenocarcinoma, and one spindle cell carcinoma. The clinical follow-up ranged from 12 RESULTS to 115 months (mean, 46 months per patient). At this writing, 52 of 79 patients (65.8%) are Immediately upon excision, the tumors were alive and disease free, 2 1 have died of the displaced in 10% formalin, embedded in paraffin, ease, and six others have died of nonrelated and processed conventionally. Consecutively cut causes, without a local recurrence or a distant sections, 5-pm thick, were stained with hematoxmetastasis. The corrected survival curves of paylin & eosin, or immunohistochemically. tients with the disease of each stage, according to the 1987 TNM classification, are shown in lmmunohistochemical Staining of EMA and S-I00 Figure 1. There was a significant difference beProtein. Serial sections were stained using the tween stages I and 111, stages I and IV, stages I1 avidin- biotin-peroxidase complex method deand 111, stages I1 and IV, but not between stages scribed elsewhere. Briefly, the deparaffinized I and I1 or stages I11 and IV ( p < 0.05). The sursections were treated with 70% methanol (containing 0.3% hydrogen peroxide) for 15 minutes at room temperature to eliminate endogenous peroxidase activity, and then washed in phosphateStage I (n=10 1 buffered saline (PBS, pH 7.4). The primary mouse monoclonal antibody to epithelial membrane antigen (EMA; BioGenex Laboratories, California, USA) or rabbit polyclonal antibody to S-100 protein (Nichirei, Tokyo) was applied overnight at 4"C, in humidified chambers. The sections were then exposed to biotinylated secondary antibody cn and streptavidin- peroxidase complex (BioGenex) for 30 minutes, respectively, at room tempera1 2 3 4 5 6 7 8 9 Years after operation ture. The peroxidase reaction was performed by a 5- to 10-minute incubation in 0.05% (wt/vol) 3.3'FIGURE 1. Survival curves of patients with laryngeal cancer.

S-100 protein-labeled cells, as related to survival time for patients with laryngeal cancer. To exclude the influence of therapeutic agents, studies were done only on the tumors of larynges excised from patients given no preoperative radiotherapy or chemotherapy.

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viva1 pattern was more favorable for those in the early stage of the disease. In 76 laryngeal lesions histologically diagnosed as squamous cell carcinoma, staining intensity of EMA and distribution of s-100 proteinlabeled cells were determined. EMA was always present on the luminal surfaces of normal secretory glandular cells scattered throughout the section (Figures 2a and 2b). The staining was intense in the serous-type glandular acini. In the normal stratified squamous epithelium, EMA was detected only on the linear surface of the mucosa. In the laryngeal carcinomas, weak to intense reaction to EMA was evident in 65 of 76 tumors (85%),and the remaining 11 squamous cell carcinomas were negative for EMA (Table 1, Figure 24. EMA-negative tumors were predominant in advanced stage tumors (stages I11 and

IV, Table la). Well and moderately differentiated squamous cell carcinomas were usually more extensively positive than were the poorly differentiated carcinomas (Table lb). In EMApositive tumors, staining was intense in the outer layer of the invading carcinomas. In the keratinizing samples, nests of parakeratotic cells were almost invariably intense (Figure 2d). In metastatic skin lesions, positivity for EMA aided in detecting microscopic metastasis. The population density of 5-100 protein-labeled cells was separated into three degrees (low, intermediate, high) and analyzed in correlation with tumor stage and histologic differentiation (Table 2). The density of S-100 protein-labeled cell infiltration was low in 31 tumors, intermediate in 30, and high in 15. The labeled cells had an irregular shape and

FIGURE 2. lmmunostaining for EMA in laryngeal carcinomas. (a) Invasion of moderately differentiated squamous cell carcinomas (arrow) beneath the intact laryngeal epithelium. Hematoxylin & eosin staining; original magnifications (a,b), x 10. (b) Adjacent section to (a) stained for EMA. Staining intensity of the carcinomas for EMA (arrow) was determined “weak” because it was almost equal to that seen in the serous-type glandular acini (G). (c) Moderately differentiated carcinoma of the larynx. EMA is present on the surface of the tumor (arrow), but the cancer cells (*) are “negative” for EMA. Original magnification, x 50. (d) Invasion of well-differentiated squamous cell carcinoma. The keratinizing cancer cells are “intensely” stained for EMA (arrows). Original magnification, x25.

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Table 1. Intensity of staining for EMA and clinicopathologic features. (a) Stage Staining intensity for EMA

I (10)

II (23)

111 (26)

IV (17)

Total (76)

2 7 1

1 17 5

4 17 5

4 10 3

11 51 14

Negative Weak Intense

(b) Histologic differentiation

Negative Weak Intense

Well (26)

Moderate (40)

Poor (10)

Total (76)

3 17 6

4 29 7

4 5 1

11 51 14

~

Numbers in parentheses indicate lhe number of lissues examined

size and dendritic features, which included extended cytoplasmic processes, in variable numbers and lengths (Figure 3a). These labeled cells were scattered among the cancer cells and formed clusters in some areas of the stroma. The area with a greater cell density showed no particular features in routine histologic stainings (Figure 3b), nevertheless, there were a larger number of S-100 protein reactive cells in close proximity to the neoplastic tissue (Figure 3c). No correlation was found between the stage of tumor and the population density of S-100 protein-labeled cells. In contrast, density of the labeled cells was high, mostly in well or moder-

ately differentiated carcinomas, and six of 10 poorly differentiated squamous cell carcinomas contained only a few cells reactive to S-100 protein (Figure 3d). Effect of the staining pattern of EMA and the distribution of S-100-labeled cells on survival time were also noted. We found no significant difference between each group showing EMA staining intensity of negative, weak, and intense (Figure 4). In contrast, there was a statistically significant difference in the survival curve between groups with low and intermediate density of S-100 protein-labeled cells but not between low and high density (Figure 5 ) . For 27 patients with metastases in regional lymph nodes or distant organs, we examine the staining pattern for EMA or S-100 protein (Table 3, Figure 6). In the 27 primary tumors, EMA was negative in six and weakly or intensely positive in 21. In 26 nodal metastases, EMA was negative in five and positive in 21. All three distant metastases were intensely positive for EMA. The population density of S-100 protein-labeled cells was low in 12 and high in six of 27 primary tumors, whereas 20 of 26 metastatic lymph nodes had a small population density, none of the metastatic lesion had a high population density. Three distant metastases also showed only a lowgrade infiltration of S-100 protein-labeled cells. With only two exceptions, the population density of S-100 protein-labeled cells in the metastatic carcinoma was smaller than in the primary lesion, in the same patient (Figure 6). DISCUSSION

We directed our attention to EMA and S-100 protein because EMA is a marker antigen of neoTable 2. Population density of S-100 protein-labeled cells and clinicopathologic features. (a) Stage

Density of $100 protein labeled cells

I (10)

II (23)

111 (26)

IV (17)

Total (76)

Low Intermediate High

3 7 0

11 6 6

10 11 5

7 6 4

31 30 15

(b) Histologic differentiation

Low Intermediate High ~

~~~~

Well (26)

Moderate (40)

Poor (10)

Total (76)

11 10 5

14 17 9

6 3 1

31 30 15

~~

Numbers in parentheses mdicate number of tissues examined

448

Table 3. Intensity of staining for EMA and population density of S-100 protein-labeled cells in patients with metastasis to reaional lymph nodes or distant organs. Primary (27)

Regional (26)

Distant (3)

6 18 3

5 15 6

0 0 3

12 9 6

20 6 0

3 0 0

Staining intensity for EMA Negative Weak Intense Density of S-100 protein-labeled cells Low Intermediate High ~~~~~

Numbers in parentheses indicate number of tissues examined

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FIGURE 3. lmmunostaining for S-100 protein in laryngeal carcinomas. (a) S-100 protein-labeled cells have markedly infiltrated, and the population density is graded as “high.” The S-100 protein-labeled (Langerhans) cells are irregularly shaped with dendritic cytoplasmic processes (arrows). Original magnifications (a,b), x 100. (b) Adjacent section to (a) stained with hematoxylin 8, eosin. Infiltration of the Langerhans cells is not so evident in this section. (c) S-100 protein-labeled cells concentrated in the nest of tumor cells (arrows). Original magnification, x50. (d) In this poorly differentiated tissue, only a few 5-100 protein-labeled cells are detected throughout the section and were graded, “low.” Original magnification, x 100.

plastic change of squamous epithelium3 and S-100 protein-labeled cells (Langerhans cells) are considered to play a key role in host immunity.6 EMA is present on the luminal membrane of glandular epithelia, but is not demonstrable in normal states of the squamous epithelium. This antigen is expressed strongly in infiltrative squamous carcinomas, as well as in a variety of non-neoplastic tissues.13 The absence of stainable EMA in malignant melanoma and in basal cell carcinoma supports the notion that EMA may be useful for a differential diagnosis of malignant tumor of epidermal origin. Sloane and co-workers reported that of eight laryngeal carcinomas, three showed a positive reaction to EMA but five were n e g a t i ~ e However, .~ we found no

EMA and S-100 Protein-Labeled Cells in Laryngeal Carcinoma

precise documentation of the expression of this marker antigen in human laryngeal carcinomas. We were surprised to see that 65 of the 76 (85%) squamous cell carcinomas of the human larynx we examined were positive for EMA. EMA tended to be expressed in well-differentiated tissues, and well and moderately differentiated laryngeal carcinomas were more uniformly and consistently stained than poorly differentiated ones, findings which are consistent with observations of a d e n o c a r ~ i n o m a .However, ~.~~ there was no correlation between the expression (and staining intensity) of EMA and stage of the tumors, nor was there a significant correlation between the expression of EMA and the prognosis. The population density of S-100 protein-labeled cells did not correlate with tumor stage or

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I

100)q,

!I -

I

0

20t \

( + + ) n.l.4 (-) I

n=ll

( + ) n=51

L

50

.-

o ' i ; ; i ; ; ; i ; Years after operation

FIGURE 4. Survival curves of groups of patients with laryngeal carcinomas divided into negative (-), weak (+), and intense (++), according to intensity of staining for EMA. There is no statistical difference between each two groups of three.

histologic differentiation. However, the density of S-100 protein-labeled cells decreased considerably in metastatic lesions, lymph nodes, or in distant organs. Thus, tumor cells that metastasize are less immunostimulatory to, for example, T lymphocytes.6 The significant difference seen in the survival curve between groups of low density and intermediate density of S-100 protein-labeled cells indicates that the appearance of S-100 protein-labeled cells is an important prognostic factor and represents the immunity of the host. These findings support the results of Gallo et al. who noted a statistically longer survival of laryngeal cancer patients with concentrations of high or intermediate density of Lan erhans cells than in those with a low density.& They also observed that the number of Langerhans cells tended to increase in patients with lymphocyte infiltration and suggested that S-100 protein-labeled cells can probably present anti-

& 10 n

E

3

Z

0

Primary

Metastat ic

FIGURE 6. Comparisonof S-100 protein-labeledcells in primary and metastatic tissues from the same patient with laryngeal carcinoma. With only two exceptions (*), the population density of S-100 protein-labeled cells in the metastatic lymph node (0)or distant organ (a)was smaller than that in the primary lesion

(0).

gen to T lymphocytes. We observed that the density of S-100 protein-labeled cells almost consistently decreased in the metastatic lesion, as compared to findings in the primary lesion. In conclusion, while EMA is a marker antigen of malignancy and an indicator of differentiation in laryngeal carcinomas, it does not possess a prognostic factor for survival. 5-100 protein-labeled cells are predominant in welldifferentiated tissues and are associated with a better prognosis. Decrease in the number of S-100 protein-labeled cells in metastatic lesions may indicate a change in host defense mechanisms against the cancer.

( + ) 1-1-30

h

REFERENCES

I Hyderman E, Steele K, Ormerod MG. A new antigen on (2) n=31

O

'

L

;

i

;

;

Years after operation

;

G

FIGURE 5. Survival curves of groups of patients with laryngeal carcinomas divided into low (r), intermediate (+), and high (++) according to the populationdensity of S-100 protein-labeledcells. The difference between low and intermediate was statistically sig nificant (p < 0.05, generalizedWilcoxon test).

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the epithelial membrane: its immunoperoxidase localization in normal and neoplastic tissues. J Clin Pathol 1979;32:35-39. Sloane JP, Hughes F, Ormerod MG. An assessment of the value of epithelial membrane antigen and other epithelial markers in solving diagnostic problems in tumour histopathology. Histochem J 1983;15:645-654. Sloane JP, Ormerod MG, Carter RL, et al. An immunocytochemical study of the distribution of epithelial membrane antigen in normal and disordered squamous epithelium. Diugn Histopathol 1982;5:11- 17. Cocchia D, Michetti F, Donato R. Immunochemical and

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5. 6. 7.

8.

9.

immunocytochemical localizatian of S-100 antigen in normal human skin. Nature 1981;294:85-87. Lauriola L, Michetti F, Sentinelli S, Cocchia D. Detection of of S-100 protein labelled cells in nasopharyngeal carcinoma. J Clin Pathol 1984;37:1235- 1238. Furukawa T, Watanabe S, Kodama T, et al. T-zone histiocytes in adenocarcinoma of the lung in relation to postoperative prognosis. Cancer 1985;56:2651-2656. Nomori H, Watanabe S, Nakajima T, et al. Histiocytes in nasopharyngeal carcinoma in relation to prognosis. Cancer 1986;57:100-105. Matsuda H, Mori M, Tsujitani S, et al. Immunohistochemical evaluation of squamous cell carcinoma antigen and 5-100 protein-positive cells in human malignant esophageal tissues. Cancer 1990;65:2261-2265. Hermanek P, Sobin LH. International union against cancer. TNM classification of malignant tumors, 4th ed. Berlin, Heidelberg: Springer Verlag, 1987.

EMA and 5-100 Protein-Labeled Cells in Laryngeal Carcinoma

10. Hsu SM, Raine L, Fanger H. Use of a avidin-biotinperoxidase complexes (ABC) in immunoperoxidase technique. A comparison between ABC and unlabelled antibody (PAP)procedures. J Histochem Cytochem 1981;29: 577-580. 11. Nakashima T, Inamitsu M, Uemura T, Sugimoto T. Immunopathology of polymorphic reticulosis of the larynx. J Laryngol Otol 1989;103:955-960. 12. Kaplan EL, Meier P.Non-parametric estimation from incomplete observation. J A m Stat Assoc 1958;53:457-481. 13. Sloane JP, Ormerod MG. Distribution of epithelial membrane antigen in normal and neoplastic tissues and its value in diagnostic tumor pathology. Cancer 1981;47: 1786- 1795. 14. Gallo 0, Libonati GA, Gallina E, et al. Langerhans cells related to prognosis in patients with laryngeal carcinoma. Arch Otolaryngol Head Neck Surg 1991;117:10071010.

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