85 @ 1990 The Japanese Society of Pathology

Immunohistochemical Demonstration of Nonspecific Cross-reacting Antigen in Normal and Neoplastic Human Tissues Using a Monoclonal Antibody Cornpa rison with Ca rcinoembryo nic Antigen Loca liza t io n

Yutaka Tsutsumi', Noboru Onodal, Mitsuko Misawa', Masahide Kuroki2, and Yuji Matsuoka2 Nonspecific cross-reacting antigen (NCA) immunoreactivity was localized in normal and neoplastic human tissues using a monoclonal antibody to 55, 90 and 95 kDa molecules of NCA. This was compared to the localization of immunoreactive carcinoembryonic antigen (CEA) as demonstrated by polyclonal and monoclonal antibodies. In frozen sections, CEA was localized in normal surface epithelium of the stomach and colon where NCA was only weakly detected. Type 1 and type 2-like pneumocytes were positive for NCA, while CEA was localized only in type 2-like pneumocytes. CEA and NCA were both demonstrated in ductal cells of frozen pancreatobiliary and mammary tissues. The antigenicity of CEA and NCA in normal tissues was significantly lost after paraffin embedding as compared to frozen sections. NCA was consistently demonstrated in eccrine sweat glands embedded in paraffin. In various tumor tissues, CEA and NCA were colocalized and expression increased sufficiently to be detected in paraffin sections. Adenocarcinomas of the stomach and colon and cystadenocarcinoma of the pancreas, as well as neuroendocrine carcinomas of the lung and thyroid, showed a CEA predominance over NCA. In ductal adenocarcinomas of the pancreas and breast and in cholangiocarcinoma, NCA reactivity was greater than CEA. Keratiniring foci of most squamous cell carcinomas of mucosal origin and some adenocarcinomas equally expressed both. Hepatocellular carcinoma, lobular mammary carcinoma and papillary thyroid carcinoma were positive only with unabsorbed polyclonal antibody which widely recognizes CEA-related substances. Renal cell carcinoma, prostatic adenocarcinoma, transitional cell car-

Received July 24, 1989. Accepted for publication September 22, 1989. 'Department of Pathology Tokai University School of Medicine, Isehara. 2Departmentof Biochemistry, Fukuoka University School of Medicine, Fukuoka. Mailing address: Yutaka Tsutsumi, M.D. (@ Department of Pathology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1 1, Japan.

z),

cinoma, anaplastic carcinomas, choriocarcinoma and basal cell carcinomas showed little or no immunoreactivity. Hence the relative ratio of CEA/NCA expression in tumors was dependent on the tissue of origin and histologic type. The cytoplasmic granular staining of NCA in cancer cells was a noteworthy difference from the plasma membraneassociated localization of CEA. Acta Pathol Jpn 40: 8597, 1990. Key words : Nonspecific cross-reacting antigen, Carcinoembryonic antigen, lmmunohistochemistry, Monoclonal antibody, Human carcinoma

INTRODUCTION The presence of a group of substances immunologically cross-reactive with carcinoembryonic antigen (CEA) has complicated its immuno histoc hemica I demonst ration. A dozen molecules and genes categorized in the CEA family have been identified by both immunologic (17) and molecular (8-1 5) techniques. A well-known member of the family of CEA-related substances is nonspecific cross-reacting antigen (NCA) of varied molecular weights (2, 3, 5, 7, 12, 14). Several studies have demonstrated NCA immunoreactivity in normal and neoplastic tissue sections (6, 16-22). NCA reactivity is often shown indirectly by subtracting CEA-specific staining from staining obtained with antibodies which recognize antigenic sites common to CEA and NCA. In only a few studies polyclonal antisera have been used to identify NCA, and this has been in a limited number of organs (16, 17, 20). In the present study, we have localized NCA immunoreactivity in a variety of normal and neoplastic human tissues using a monoclonal antibody (mAb) for NCA of specific molecular sizes, and compared this to the localization of immunoreactive CEA in the same tissue using both

86

CEA and NCA in Normal and Neoplastic Tissues (Tsutsumi et a/.)

polyclonal antibodies (pAbs) and mAbs. In normal tissues, a significant loss of antigenicity of CEA and NCA was observed after formalin fixation and paraffin embedding as compared to frozen sections. The relative ratio of CEA/NCA expression in the tumors studied was dependent on both the tissue of origin and histologic t Y Pe.

ride (Wako Pure Chemical Co., Osaka, Japan), 6 5 m g sodium azide and 0.003% hydrogen peroxide per dl of 0.05 M Tris-HCI buffer, pH 7.6. As a negative control, the primary antibody was replaced by a non-immune rabbit or mouse serum at a 1 : 5 0 0 dilution. All sections were counterstained with 5% methyl green (pH 4.0).

RESULTS MATERIALS AND METHODS Normal or non-neoplastic regions of human antral and fundic mucosa of the stomach, colonic mucosa, lung, pancreas, breast, kidney, liver and thyroid were examined. Adenomas and adenocarcinomas of the stomach and colon were also sampled. Fresh frozen sections (6 p m thick) were post-fixed in cold acetone for 1 0 min. Additionally, gastric and colonic tissues were pre-fixed in periodate-lysine-4% paraformaldehyde (PLP) at 4°C for 6 h, washed overnight in 0.01 M phosphate-buffered saline containing 10-20% sucrose (pH 7.2), and 6 pm-thick frozen sections cut. Formalinfixed, paraffin-embedded tissues from 100 tumors, with adjacent non-tumorous tissues, were serially cut a t 4 p m thickness. The type of non-neoplastic and neoplastic tissues studied is shown in Tables 1 and 2, respectively. CEA-related antigens were localized by an indirect immunoperoxidase method using two pAbs and four mAbs. Rabbit pAbs (Dako Co., Copenhagen, Denmark) were used at a 1 : 1,000 dilution with or without extensive absorption by a perchloric acid extract of human spleen (20) (a rich source of NCA (23)). The specificity of the mouse IgG mAbs CM005 and CMOlO (Mochida Pharmaceutical Co., Tokyo, Japan) has been described previously (20) : CM005 detects a peptide epitope common to both CEA and NCA, while CMOlO is specific for an epitope present on the peptide sequence of CEA. The mouse IgG mAb F33-104 recognizes a similar epitope on the peptide sequence of CEA (24, 25). Neither CMOlO nor F33-104 react with NCA (20,24,25). The mouse IgG mAb F106-88 reacts with a peptide epitope on NCA55, NCA-90 and NCA-95 (the 55, 90 and 9 5 k D a subtypes of NCA, respectively) but not with NCA-160 and CEA (Kuroki and Matsuoka, in preparation). All mAbs were used a t a 1 : 500 dilution, and horseradish peroxidase-la beled ant i-ra bbit and anti- mouse immunoglobulins (Dako) a t a 1 : 5 0 dilution. Prior to immunostaining, all sections were dipped in 0.5% periodic acid solution for 10 min followed by 3 mM sodium borohydride for 10 min, to break down sugar moieties and inactivate endogenous peroxidases. Antibody incubation and rinsing were each carried out for 3 0 min. The working solution for the peroxidase reaction contained 30 m g 3, 3’-diaminobenzidine tetrahydrochlo-

Frozen sections of normal gastric and colonic mucosa, either postfixed in acetone or prefixed in PLP, gave comparable results. The surface epithelial cells were clearly positive with anti-CEA pAbs and mAbs. In addition to these cells, unabsorbed pAb and mAb CM005 strongly stained granulocytes in the stroma. The staining of surface epithelial cells was most intense, while cells at the generative zone (the glandular neck region in the stomach and the bottom one third of the colonic crypt) were negative in the stomach and weakly positive in the colon. Gastric glands reacted only with unab sorbed pAb, and reactivity was weak. No neuroendocrine-type cells were stained. By contrast, anti-NCA mAb F106-88 only faintly stained gastric and colonic surface epithelium, and cells at the generative zone were negative. Stromal granulocytes were positive. The neoplastic cells were strongly positive for both CEA and NCA immunoreactivities, as were non-neoplastic epithelial cells adjacent to the cancer. A representative feature of colonic adenoma and adjacent non-tumorous mucosa is shown in Fig. 1. In frozen sections of the lung, immunoreactive NCA as defined by mAb F106-88 was identified in the cytoplasm and on the plasma membrane of alveolar pneumocytes, including both type 1 and type 2-like cells, and focally in ciliated bronc hia I epithelium, while CEA immunoreact iv it y as defined by mABs CMOlO and F33-104 was noted on the plasma membrane of type 2-like pneumocytes only (Fig. 2). Type 1 pneumocytes were also strongly positive with unabsorbed pAb but were negative with mAb CM005. The cytoplasm of granulocytes and alveolar macrophages reacted consistently with unabsorbed pAb and mAbs CM005 and F106-88 and occasionally with absorbed pAb. In frozen sections of the pancreas and breast, only the apical surface of ductal epitheliaf cells reacted with mAbs to CEA or NCA. Unabsorbed pAb recognized a wider range of epithelial cells: The luminal surface of centroacinar cells of the pancreas were positive (Fig. 3a), and the cytoplasm of Langerhans islets were faintly stained. Almost all epithelial cells in the breast, including myoepithelial cells, were decorated by unabsorbed pAb (Fig. 3b), while absorbed pAb reacted with duct-lining cells but scarcely with myoepithelial cells.

Acta Pathologica Japonica 40 (2) : 1990

87

Figure 1. Localization of immunoreactive CEA (a) and NCA (b) in PLP-prefixed frozen sections of adenomatous (left half of a, b) and adjacent non-tumorous (right half) colonic mucosa, demonstrated by the indirect immunoperoxidase method using anti-CEA mAb F33-104 and anti-NCA mAb F106-88. CEA immunoreactivity is localized in the entire thickness of both adenomatous and non-tumorous mucosa, with stronger staining seen in the epithelium lying in the upper part of the mucosa and in adenoma. lmmunoreactive NCA is only weakly localized in non-tumorous surface-covering epithelium, but is strongly positive in adenomatous epithelium. Stromal granulocytes are positive for NCA.

In the kidney, unabsorbed pAb decorated most epithelial cells including those of the glomerulus. Bile canaliculi formed by hepatocytes reacted with unab sorbed pAb. In the kidney and liver, however, no cells were stained with mAbs. In the thyroid, follicular epithelial cells reacted only focally with pAbs and were negative with mAbs. Parafollicular-like cells were immunoreactive for CEA (recognized by pAb and mAb) but not for NCA. Although all of the antibodies demonstrated reactive sites in formalin-fixed, paraffin-embedded tissues, a significant loss of antigenicity was observed in processed

tissues as compared to frozen sections, especially so for normal tissues. Table 1 summarizes immunohistochemical localization of CEA and NCA in paraffin-embedded non-neoplastic tissues, in combination with results o b served in frozen sections of the above-mentioned organs. Staining with mAbs CMOlO and F33-104 gave similar results. In certain paraffin-embedded tissues (see Table l), immunoreactive substances were infrequently detected in areas where the epithelial cells were inflamed, regenerative or hyperplastic, or where they lay adjacent to cancer. In paraffin sections, normal colonic crypt cells and

88

CEA and NCA in Normal and Neoplastic Tissues (Tsutsumi et a/.)

Figure 2. Localization of immunoreactive CEA (a) and NCA (b) in acetone-postfixed frozen sections of normal adult lung, visualized by the indirect immunoperoxidase method using mAbs F33-104 and F106-88. CEA immunoreactivity is localized on the plasma membrane of type 2-like pneumocytes, while immunoreactive NCA is distributed in the cytoplasmic granular structures and on the plasma membrane of both type 1 and type 2like cells. The cytoplasm of anthracotic alveolar macrophages (arrows) is positive for NCA but negative for CEA. Part of ciliated mucosa of the terminal bronchiole is also stained for NCA (arrowhead).

Figure 3. lmmunoperoxidase staining with unabsorbed pAb in acetone-postfixed frozen sections of normal pancreas (a) and breast (b) (Inset: pancreatic lobule). Unabsorbed pAb recognizes the widest range of epithelial cells. The luminal surfaces of centroacinar cells (short arrows), intralobular ductule-lining cells (long arrows) and interlobular ductlining cells in the pancreas are positive, as are mammary ductal components including myoepithelial cells (arrowheads).

89

Acta Pathologica Japonica 40 (2): 1990 Table 1.

lmmunostaining for CEA and NCA in Non-neoplastic Tissues Using Various Paraffin Sections and Some Frozen Sections Polyclonal

Monoclonal

[CEAI

[CEAI

[Immunogen]

Cell

Tissue

unabs.

a bs.

CM005 -

Granulocyte Macrophage Epidermis skin Non-keratinizing squamous epithelium gum, esophagus anus, exocervix Ciliated epithelium lung nasal mucosa Transitional epithelium urinary bladder renal pelvis urethra Columnar surface epithelium stomach small bowel colon gallbladder endocervix Gland (ductal component) eccrine gland apocrine gland salivary gland intrahepatic bile duct pancreas breast renal convoluted tubule renal collecting tubule Gland (acinar component) stomach Brunner‘s gland salivary gland pancreas esophageal gland bile duct gland bronchial gland nasal gland endocervical gland endometrium prostate type 1 pneumocyte type 2-like pneumocyte hepatocy t e glomerulus thyroid follicle Neuroendocrine cell thyroid parafollicular cell Langerhans islet alimentary tract

it/+

+/+

(+I

+ it it/+ it

-/(+I -/(+I (+I

-/-/-

-

it/+ it

+/+

+/+

it/+

(+)

-

(+I

it

+

+ +

+

(+I

it

+

-

-

+

(+I

-

+/+

-/+ -/+

-/+ -/+ -/+ -/-1-

-/+

(+)I+

(+I/+ (+I

+ (+)/it + + + + + + (+I +/U

+I+ +I+ -/+ -/+

+/+ -/U

+

(+I

+I+ +/+

F106-88

+/+

+

it

-/+

“CAI CMOlO & F33-104

it it

-

+

+/+

Monoclonal

(+I/+ -/-/+

~

-

(+I (+I/+ (+I/+ -/+ -/-1-

-/-

-/+ -

(+I -

(+I -

-/+/+ -/-1-/+

-

-

-1-

+I+ +/+ -/-/-/-

(+)/+

-/-

-1-/-

+/+

(+I/+

-/+f

-/-

-/+ -/-

- l-

-1-

-/-

-/-/-/-

Bold data after slash : results using acetone-postfixed frozen sections, unabs. : unabsorbed antibody, abs. : antibody absorbed with a perchloric acid extract of spleen. it : Consistently positive, : Inconsistently positive, (+) : Infrequently positive (especially in inflamed, regenerative or hyperplastic epithelial cells and in cells adjacent to cancer), - : Negative, f : Faint staining.

+

90

CEA and NCA in Normal and Neoplastic Tissues (Tsutsumi et a/.) Table 2.

lmmunostaining for CEA and NCA in a Variety of Paraffin-embedded Tumor Tissues Polvclonal

Monoclonal

-

-

Monoclonal “CAI

Tissue

Histology

No. of cases

1. Adenoma and adenocarcinoma Colon M, W adenoma Stomach W, scirrhous ca. M adenoma Pancreas ductal ca. (M, W) ductal ca. (M) cystadenoca rcinoma W Gallbladder in situ Liver hepatoma cholangioca. Salivary gland mixed tumor mucoepidermoid ca. acinic cell ca. Breast ductal ca. ductal ca. lobular ca. Prostate M, W Kidney clear cell ca. Thyroid papillary ca. W Lung Nasal cavity P W, P Endometrium W Endocervix Ovary serous cystadenoca. mucinous cystadenoca. mucinous cystadenoma Skin mucinous adenoca. apocrine adenoca. 2. Squamous cell carcinoma and related tumor Esophagus M basaloid ca. Anus cloacogenic ca. Gum ameloblastoma Nasal cavity W Epipharynx lymphoepit helio ma Lung M, W Uterine cervix non-keratinizing non-keratinizing Skin M, W basa lioma 3. Transitional cell carcinoma Urinary bladder W, P Renal pelvis in situ 4. Anaplastic and embryonal-type carcinoma Thyroid sarcomatous ca. Lung large cell ca. Oviduct choriocarcinoma Alimentary tractaAFP-producing ca. 5. Neuroendocrine tumor Uterine cervix small cell ca. Lung oat cell ca. small cell intermediate ca. ca rcinoid carcinoid

F106-88

unabs.

a bs.

4 1 3 1 2 3 1 2 1 1 2 1 2 1 1 2 1 1 2 2 2 2 1 2 1 3 1 1 1 1

4-5 4 4-5 5 4 5 5 4-5 5 4 3-4 4 1-2 2 0 3 1 3 0-1 0 2 4 4 0-2 5 0-2 4 4 1 2

4-5 4 4-5 5 4 3-5 5 3-5 5 3 0 3 1 2 0 2 1 0 0 0 0 3 4 0-2 5 0-2 4 3 0 2

4-5 4 3-5 5 3-4 4-5 5 4-5 5 3 0 4 1 2 0 2 0 0 0 0 0-2 4 4 0-2 5 0-2 4 0 2

4-5 4 3 5 5 2 -4 3-4 3 3 5 5 3 0 2 0 1 0 1 0 0 0 0 0 2 4 0-1 2 0-1 3 0 0 0

1 1 2 1 1 2 3 2 1 2 2

2 0 2-3 2 3 0 2-3 2-3 4 2 0

2 0 2-3 2 3 0 2-3 2-3 4 0-1 0

2 0 2 1 3 0 2-3 2-3 4 0 0

1

1

0 0-2 0 3 0 2-3 1-3 4 0 0

0 0 0 2 0 2-3 2-3 2 0 0

2 1

0 3

0 2

0 2

0 0

0 0

1 3 1 3

0 0-1 0 2-3

0 0-1 0 2

0 0-1 0 1-2

0 0 0 1

0 0 0 0

1

4 5 2 0-1 3

4 5 2 0-1 3

4 5 2 0-1 3

4 5 2 0 3

0 1 1 0 0-1

1 1 5 2

0

2-3 2 2 3 5 0-2 4-5 3 1 5 1 0 4 1 2 0 2 0 0 0 0 0 1-2 0 0 5 0 3 0 0 0

91

Acta Pathologica Japonica 40 (2): 1990 Table 2. Continued

Polyclonal

Monoclonal

[CEAI

[CEAI

[ Immunogen] Tissue Thyroid Appendix Duodenum Rectum Gallbladder Pancreas

Histology

:&,":

unabs.

a bs.

CM005

medullary ca. adenocarcinoid malignant ca rci noid carcinoid ca rcinoid islet cell tumor

3 1 1 3 2 2

5 5 4 0-2 2 0

5 5 4 0 1-2 0

5 5 4 0 1-2 0

Monoclonal "CAI

~ ~ ~ - ' ~ 4 F106-88 & 5 5 4 0 1 0

1-4

3 4 0 0 0

unabs. : unabsorbed antibody, abs. : antibody absorbed with a perchloric acid extract of human spleen, W : well-differentiated carcinoma, M : moderately differentiated carcinoma, P : poorly differentiated carcinoma, ca. : carcinoma, AFP-producing ca. : alpha-fetoprotein-producing carcinoma =either embryonal carcinoma or a special histologic type composed of glycogen-rich tumor cells with a medullary growth pattern, a : two arising from the stomach and one from the colon. Scoring criteria are as follows: 5 : Most of the tumor cells are positive. 4 : More than half of tumor cells are positive. 3 : Less than half of tumor cells are positive. 2 : Less than 10% of tumor cells are positive. 1 : Less than 1% of tumor cells are positive. 0 : No tumor cells are positive.

normal gastric foveolar cells reacted relatively weakly with CEA antibodies, particularly with mAbs CMOlO and F33-104; NCA was negative. Staining of granulocytes by mAbs CM005 and F106-88 was also often rather faint and inconsistent in paraffin-embedded tissues. CEA reactivity demonstrated by mAbs CMOlO and F33-104 was further positive in the surface layer of paraffin-embedded non-keratinizing squamous mucosa.

lmmunoreactivity was only infrequently noted in hyperkeratotic epidermis, nasal ciliated epithelium, gallbladder mucosa or in eccrine sweat gland ducts. Immunoreactive NCA was also identified only infrequently in the apical surface of salivary gland ducts, intrahepatic bile ducts, pancreatic ducts, gallbladder mucosa and bronchial ciliated mucosa, or in the surface layer of transitional mucosa and non-keratinizing squamous mucosa in

Figure 4. Localization of immunoreactive NCA in paraffin-enbedded normal eccrine sweat glands (b), immunostained using mAb F106-88. Compare with negative CEA staining by mAb F33-104 (a). NCA immunoreactivity is localized on the apical surface of duct-lining cells.

92

CEA and NCA in Normal and Neoplastic Tissues (Tsutsumi et a/.)

paraffin sections. We observed an interesting exception where mAb F106-88 clearly and consistently stained the luminal surface of eccrine sweat gland ducts in paraffin sections, in which CEA immunoreactivity was expressed

only occasionally (Fig. 4). Apocrine sweat glands were negative for CEA-related substances. The acinar component of glandular tissues embedded in paraffin generally reacted only with unabsorbed pAb,

Figure 5. Localization of immunoreactive CEA (a) and NCA (b) in paraffin-embedded colonic adenocarcinoma. well-differentiated. The indirect immunoperoxidase method was done using mAbs F33-104 and F106-88. NCA immunoreactivity is focally expressed in the cancer tissue while CEA expression is widespread and stronger.

Figure 6. Localization of immunoreactive CEA (a) and NCA (b) in paraffin-embedded pancreatic adenocarcinoma, ductal type. The indirect immunoperoxidase method was performed using mAbs F33-104 and F106-88. NCA expression is more prominent than CEA. In addition t o the plasma membrane localization, NCA shows a granular cytoplasmic reactivity.

Acta Pathologica Japonica 40 ( 2 ) : 1990

which consistently stained bile canaliculi of hepatocytes and alveolar pneumocytes (type 1 and type 2-like cells). The alveolar pneumocytes were stained inconsistently with mAb F106-88, and only type2-like cells reacted

93

infrequently with mAbs CM005, CMOlO and F33-104. Endometrial glands and bile duct-associated glands were stained rarely with absorbed pAb and/or mAb CM005. Thyroid follicular cells in paraffin sections were unre-

Figure 7. Localization of immunoreactive CEA (a) and NCA (b) in paraffin-embedded gastric adenocarcinoma, moderately differentiated. The indirect immunoperoxidase method was done using mAbs CMOlO and F106-88. Most tumor cells are positive for both CEA and NCA immunoreactivities. Note cytoplasmic granular staining of NCA.

Figure 8. Localization of immunoreactive CEA (a) and NCA (b) in paraffin-embedded pulmonary squamous cell carcinoma, keratinizing type, by the indirect immunoperoxidase method using mAbs F33-104 and F106-88. Keratinizing cancer cells are strongly labeled for both CEA and NCA immunoreactivities. lmmunoreactive CEA is more plasma membrane-associated than NCA. Anuclear keratin pearls are decorated only for NCA.

94

CEA and NCA in Normal and Neoplastic Tissues (Tsutsumi et a/.)

active with any antibody studied, while calcitonin-positive parafollicular cells reacted with pAbs and mAb CM005. As seen in Tablel, mAb CM005 and absorbed pAb occasionally decorated more epithelial cells than did mAbs CMOlO, F33-104 and/or F106-88. Unabsorbed pAb reacted with the greatest variety of epithelial cell types. In contrast to non-neoplastic tissues, cancer tissues commonly exhibited accentuated staining for CEA or CEA-related substances even after paraffin embedding. Table 2 summarizes results of immunostaining in a variety of paraffin-embedded tumor tissues. Anti-CEA mAbs CMOlO and F33-104 showed identical staining patterns. Many tumors co-expressed both CEA and NCA immuno react ivit ies. Five patterns of expression were identified in the different tissues. The first was demonstrated by adenomas and adenocarcinomas of the colon and stomach, CEA reactivity with mAbs CMOlO and F33-104 being more prominent than NCA reactivity with mAb F106-88 (Fig. 5). Adenocarcinoma of the nasal cavity, cystadenocarcinoma of the pancreas, adenocarcinoma in situ of the gallbladder, one of three non-keratinizing squamous cell carcinomas of the uterine cervix, neuroendocrine carcinomas of the lung, thyroid and uterine cervix and some carcinoid tumors were also included in this group. The second was demonstrated by ductal adenocarcinoma of the pancreas (Fig. 6), cholangiocellular carcinoma of the liver and endocervical adenocarcinoma, NCA reactivity being more prominent than CEA. Focal expression of both substances with a relative predominance of NCA was seen in two of three ductal carcinomas of the breast and in a mucoepidermoid carcinoma of the salivary gland. The third staining pattern was characterized by an equal degree of expression of both CEA and NCA reactivities. This group included one of four adenocarcinomas of the stomach (Fig. 7), one of four ductal adenocarcinomas of the pancreas, an adenocarcinoma of the gallbladder, a mucinous cystadenocarcinoma of the ovary, keratinizing foci in most of the squamous cell carcinomas arising from mucous membranes (Fig. 8 ) and a metastasizing duodenal carcinoid. Hepatocellular carcinoma, lobular carcinoma of the breast, papillary thyroid carcinoma and squamous cell carcinoma of the skin were negative with CEA-specific mAbs CMOlO and F33-104 and NCA-specific mAb F106-88 but positive with unabsorbed pAb. These tumors were grouped in the fourth category (Fig. 9). The fifth group, in which little or no immunoreactivity was observed with any antibody, encompassed renai cell

Figure 9. Localization of CEA-related (non-CEA and non-NCA) su bsta nce(s) in paraff in-embedded hepatocellula r carcinoma by the indirect immunoperoxidase method using unabsorbed pAb. Bile canaliculi formed by neoplastic hepatocytes are stained with unabsorbed pAb. Absorbed pAb and all mAbs failed to react with norma I and neoplastic hepatocytes.

carcinoma, prostatic adenocarcinoma, acinic cell carcinoma of the salivary gland, lymphoepithelioma of the nasopharynx, basal cell carcinomas of the skin and esophagus, transitional cell carcinoma of the urinary bladder, anaplastic carcinomas of the lung and thyroid, choriocarcinoma, islet cell tumor and some carcinoid tumors. The intracellular localization of CEA and NCA reactivities varied. While immunoreactive NCA was usually localized on the apical plasma membrane of nonneoplastic ductal and surface lining columnar cells, in tumor tissues and in normal pneumocytes, neutrophils and macrophages it was less plasma membrane-associated. The granular and cytoplasmic staining pattern of immunoreactive NCA is shown in Fig. 2 and Figs. 6-8. In all tissues CEA immunoreactivity was predominantly associated with the plasma membrane. In those cases of carcinoma overexpressing CEA, however, cytoplasmic staining was often diffuse. Control sections incubated with the non-immune serum were uniformly negative, and the complete block of endogenous peroxidases was confirmed.

DlSCUSStON Our immunohistochemical study has confirmed that a variety of epithelial tumors co-express both CEA and

Acta Pathologica Japonica 40 ( 2 ) : 1990

NCA reactivites, and that the relative predominance of immunoreactive NCA is dependent on the tissue of origin and histologic type. For example, in adenocarcinomas of the stomach and colon, as in neuroendocrine carcinomas of the lung and thyroid, fewer cells expressed immunoreactive NCA than CEA, while in ductal adenocarcinomas of the pancreas and breast and in adenocarcinomas of the bile duct and uterine cervix NCA expression predominated. In cystadenocarcinoma of the pancreas, in particular, the predominance of CEA expression may be indicative of a metaplastc nature of this histologic subtype towards gastrointestinal epithelium. CEA and NCA immunoreactivities were equally expressed in some adenocarcinomas, and in keratinizing foci of most of the squamous cell carcinomas of mucosal origin. The expression pattern in these tumors and in non-neoplastic tissues was similar, although in the latter immunoreactive CEA with or without NCA was often inconsistent in paraffin sections, and reactivity was weak as compared to frozen sections. Such pitfalls in apply ing CEA immunohistochemistry have previously been shown in the pancreas (20). In normal tissues, the loss of antigenicity of CEA and NCA after formalin fixation and paraffin embedding may be due to relatively smaller amounts of antigens than in the tumor tissues. Since NCA immunoreactivity was consistently demonstrated in paraffin-embedded normal eccrine sweat glands, skin adnexal tumors would be a good target of study on CEA and NCA expression. The demonstration of immunoreactive NCA in paraffin sections appeared relatively specific for tumor tissues. Undetectable levels of NCA in paraffin-embedded normal gastrointestinal epithelial cells and positive expression in tumors indicate a relative specificity. Even in frozen sections, the expression level of NCA was very low in normal gastric and colonic epithelial cells. By contrast, CEA immunoreactivity was detectable in paraffin-embedded normal tissues(20). Active production of CEA by normal colonic mucosa has been shown in an organ culture experiment (26). Similarly, Sat0 et a/. have recently reported the specificity of NCA for colon tumor messenger ribonucleic acid (mRNA) by the Northern hybridization technique (27) : CEA mRNA was present in both normal and tumor tissues, whereas NCA mRNA was found only in the tumor extract. The mAb F106-88 is specific for 55, 90 and 9 5 kDa molecules of NCA (NCA-55, NCA-90 and NCA-95) but not for a 160kDa molecule (NCA-160) (Kuroki and Matsuoka, in preparation). In frozen sections, which are most suitable for detection of CEA-related substances, mAb CM005 did not react with NCA-like substances in type 1 pneumocytes; these were stained with unab sorbed pAb and mAb F106-88. Since subtypes of NCA

95

are thought to be encoded by genes that are separate from those that encode CEA (12,28) and their expression purportedly varies from tissue to tissue (6), further studies using mAbs specific for subtypes of NCA (6, 7) would clarify expression of different NCA subtypes in normal and neoplastic tissues. The antiserum obtained by immunization of animals with purified CEA molecules reacts widely with sub stances in the CEA family, including CEA, NCA and biliary glycoprotein-l (BGP-I) (20, 29). BGP-I is normally localized in bile canaliculi formed by hepatocytes (4, 30). The absorption with a perchloric acid extract of human spleen almost eliminated the reactivity of pAb with NCA, BGP-I, and other CEA-related substances which may be present, for example, in lobular carcinoma of the breast. However, differences in absorption procedures among laboratories are likely to influence the effectiveness of NCA elimination. The incomplete elimination was shown in our study by granulocytes which in frozen sections were not perfectly negative with absorbed pAb although granulocytes in paraffin sections were. The present results suggest that even extensively absorbed pAb is not strictly specific for CEA, and that mAb CM005 may react with a sequence common t o CEA, subtypes of NCA and other CEA-related sub stances. Evaluation of NCA immunoreactivity by a subtraction method (as, for example, the difference between staining with unabsorbed pAb or mAb CM005 and that with absorbed pAb or mAb CMO10) may, therefore, be difficult. Except for few reports that have described NCA production by cancer cells (27, 31), identification has been made mostly by this indirect method (6, 18-22). Alternatively, NCA pAbs have been used but these may not be monovalent (16,17,20). Failure to identify either the tissue-type or histologictype dependency of CEA/NCA expression ratio in tumors may be explained by each of these technical limitations. The results of the present study indicate that the mAb F106-88 provides a specific method for evaluating NCA expression and exression of its subtypes in various tissues. The cytoplasmic granular staining of NCA immunoreactivity in most cancer tissues, and in some normal cells, was in sharp contrast to the plasma membraneassociated localization of immunoreactive CEA in both normal and tumor tissues, and of immunoreactive NCA in normal ductal and surface-lining columnar cells. Recently, we have found NCA localization in autophagic vacuoles in cultured lung cancer cells, particularly in quiescent cells (32). In proliferative cells, NCA expression was predominantly plasma membrane-associated (32). The cytoplasmic localization may thus indicate change in NCA-immunoreactive membrane metabolism

96

CEA and NCA in Normal and Neoplastic Tissues (Tsutsumi et a/.)

in cancer cells. The plasma membrane anchoring mechanism of NCA may be different f r o m the glycophospholipid-mediated membrane anchoring t o the carboxylterminal domain of CEA (26, 33). A n independent intracellular transport, functions and a fate of CEA and NCA are also hypothesized. To help settle these questions, further immunoelectron microscopic studies are now in progress. Acknowledgement : The manuscript was reviewed by Associate Prof. Rosemary C. Jones, Ph. D., Department of Pathology, Harvard University School of Medicine, Boston, MA, USA, and Stephen P. Naber, M.D., Ph. D., Department of Pathology, Tufts University School of Medicine, Boston, MA, USA.

11.

12.

13.

14.

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Immunohistochemical demonstration of nonspecific cross-reacting antigen in normal and neoplastic human tissues using a monoclonal antibody. Comparison with carcinoembryonic antigen localization.

Nonspecific cross-reacting antigen (NCA) immunoreactivity was localized in normal and neoplastic human tissues using a monoclonal antibody to 55, 90 a...
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