Expression of thomsen-friedenreich-related antigens in primary and metastatic colorectal carcinomas....

1700

Expression of Thomsen-Friedenreich- Related Antigens in Primary and Metastatic Colorectal Carcinomas A Reevaluation

Yi Cao, M.S.,* U w e R. Karsten, Ph.D.,* Winfrid Liebrich, Ph.D.,* Wolfgang Haensch, M.D.,*,t Georg F. Springer, M.D., D.Sc.h.c.,S and Peter M. Schlag, M.D.*,t

Background. Expression of the pancarcinoma Thom- sen-Friedenreich (TF) carbohydrate antigen or, more correctly, hapten, in colorectal carcinomas is not gener- ally agreed on. Furthermore, its suggested role in liver metastasis so far has not been substantiated by direct immunohistochemical evidence.

Methods. Cryostat sections from 52 primary tumors (20 with adjacent transitional mucosa), 22 liver metastases of colorectal carcinomas, and 17 samples of normal mucosae were examined immunohistologically with a panel of at least two monoclonal antibodies (mAbs) each to TF, to its precursor, Tn, and to sialosyl-Tn, among them two newly developed anti-TF mAbs.

Results. Of the primary colorectal carcinomas, 60% expressed TF. Staining was more intense with TF-alpha/ beta-reactive than with exclusively TF-alpha- or TF- beta-reactive mAbs. Normal and transitional mucosae were negative.

Liver metastases were positive for TF in a significantly higher percentage of cases (91%) than primary carcinomas. Patients with TF-positive primary tumors had

Presented in part as a poster at the 13th Meeting of the European Association for Cancer Research, Berlin, September 25-28,1994.

From the *Max Delbriick Centre for Molecular Medicine, Berlin- Buch, Germany, tRudolf-Virchow-Klinikum, Humboldt University, Berlin, Germany, and the $H.M.Bligh Cancer Research Laboratories, UHS/Chicago Medical School, Chicago, Illinois.

Supported by the Deutsche Forschungsgemeinschaft (Grant Ka 921/1) and Grant CA 22540 from the U.S. National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

The authors thank Prof. Dr. P. Stosiek (Cottbus) for providing normal colonic tissues, Prof. Drs. H. Clausen (Copenhagen) and P. Driber (Prague) for providing the monoclonal antibodies, and Mrs. S. Grigull for her assistance.

Address for reprints: Uwe Karsten, Ph.D., Max Delbriick Centre for Molecular Medicine, Robert-Rossle-Str. 10, D-13125 Berlin-Buch, Federal Republic of Germany.

Received March 21, 1995; revisions received June 2, 1995, and July 17, 1995; accepted July 17, 1995.

a significantly higher risk to develop liver metastases compared with patients with TF-negative tumors (57% vs. 14%, respectively).

Tn and sialosyl-Tn were expressed concomitantly in most primary (85%) and metastatic (95%) colorectal carcinomas. These antigens also were detected in transitional mucosae (Tn in 25%, sialosyl-Tn in 55% of cases). Normal mucosae were negative.

Conclusions. These results prove unequivocally the presence of exposed TF epitopes in a majority of colorectal carcinomas in which both anomers of TF are expressed. These data further suggest that TF favors liver metastasis and that its expression in primary colorectal carcinomas is a significant risk factor for the development of liver metastasis. Cancer 1995: 76:1700-8.

Key words: colorectal carcinomas, immunohistochemistry, Thomsen-Friedenreich antigen, Tn, sialosyl-Tn, liver metastasis, prognosis.

Thomsen-Friedenreich (TF)-related histo-blood group antigens (TF, Tn, sialosyl-Tn) are a group of tumor-associated carbohydrate epitopes on glycoconjugates whose expression (or absence) on cancer cells has been found to correlate with prognosis-relevant processes, such as aggressiveness and meta~tasis.'-~ The presence of these antigens in colorectal carcinomas has been described,4-" but there remain conflicting reports on this i ~ s u e ' ~ ~ ' ~ that require further investigation. The aims of our contribution were fourfold. First, five monoclonal anti-TF antibodies (mAbs), two of which were newly developed in our laboratory, were used to examine whether the reported lack of expo>ed TF antigen oc- curred for technical reasons or because of the use of an inappropriate mAb. In addition, with the fine specificities provided by this panel of anti-TF mAbs, we were able to study the relative proportions of the alpha and

TF Antigen in Colorectal Carcinomas/Cao et al. 1701

beta anomers of TF. Second, TF and Tn antigens may play a role as ligands of the asialoglycoprotein receptors found in the l i ~ e r ' ~ , ' ~ in establishing metastatic deposits in this organ. Although a number of in vivo and in vitro experiments support this direct immunohistochemical evidence from human liver metastases is unavailable. Therefore, a major point of this study was to compare the expression of TF-related antigens on primary tumors and liver metastases. Third, we were interested in the relative proportions of expression of the biosynthetically linked TF, Tn, and sialosyl-Tn antigens to gain some insight into the alterations of glycosylation in colorectal carcinomas. Finally, we compared the expression of the TF-related epitopes on neoplastic colorectal tissues with that of apparently healthy por- tions of colonic tissue adjacent to the tumor as well as with normal colonic mucosa.

Materials and Methods

Tissues

Primary colorectal carcinoma tissues were obtained from 52 patients undergoing surgical resection. Among these, 35 patients had a clinical follow-up for at least 1 year and were divided into two groups of 21 and 14 patients according to the occurrence or absence of liver metastases during this time, respectively. In 20 cases, the uninvolved mucosa immediately adjoining the tumor was also collected. This mucosa is referred to as transitional mucosa. Transitional mucosa is grossly normal but usually exhibits microscopic abnormalities, such as elongated and branched crypts, but no cellular atypia. Liver metastases of colorectal carcinomas were obtained from 22 patients. The 17 normal colonic mucosae included 13 specimens from patients with cancer taken at surgery or colonoscopy from apparently normal parts of the colonic wall distant from the tumor and 4 specimens taken at autopsy from persons without colonic disease. These 17 mucosae showed normal histo- morphologic and cytomorphologic findings.

All samples were immediately frozen and stored at -8OOC. Cryosections were cut at 4-pm thickness.

Classification of primary carcinomas was done with hematoxylin-eosin-stained sections according to International Union Against Cancer and World Health Organization recommendations, respec- t i~e ly . '~ , '~ Lymph node metastases were recorded on the basis of the pathologic examinations. ABO blood groups were also documented.

Antibodies

A panel of mAbs against TF, Tn, and sialosyl-Tn was used, consisting of at least two well established mAbs

each. Specifications and references are listed in Table 1. A78-G/A7 and A68-B/A11 (both IgM, K ) are newly developed anti-TF mAbs from our laboratory. HH8 and TKH2 mAbs were kindly provided by Dr. H. Clausen (Copenhagen, Denmark) and mAb TEC-02 by Dr. P. Driber (Prague, Czech Republic). MAb HB-TI was pur- chased from Dako (Hamburg, Germany), BM22 from Accurate Chemicals (Westbury, NY), and B72.3 from Biogenesis (Bournemouth, UK). For comparison, peanut agglutinin (PNA, biotinylated, Sigma Chemical, Dei- senhofen, Germany) also was applied. Optimal dilutions were determined in preliminary experiments. Su- pernatants were used as follows: A78-G/A7 150, HH8 1:5, TKH2 1:4, and TEC-02 1 : l O . Other preparations were diluted accordingly: HB-TI 1:50; BM22 1:5; BaGS- 6, B72.3, and PNA 1 : l O O .

Irnmunohistologic Procedures

Staining of tissue sections was performed by the avidin- biotin-peroxidase complex method with a commercial kit (Vectastain ABC Elite Kit, Vector Laboratories, Burl- ingame, MA) as follows. Frozen sections were air dried at room temperature and fixed with 10% formalin in phosphate buffered saline (PBS) for 15 minutes at room temperature. Endogenous peroxidase activity was elim- inated by treatment with 3% hydrogen peroxide in PBS for 30 minutes at room temperature. Nonspecific binding sites were blocked with normal rabbit serum preabsorbed with neuraminidase-treated blood group 0 erythrocytes. After washing them with PBS, we incubated sections with mAbs or PNA in appropriate dilutions for 1 hour at room temperature. The thoroughly washed sections (except those stained with biotinylated PNA) were treated with biotinylated anti-mouse immu- noglobulin antiserum for 30 minutes at room temperature and thereafter with the avidin-biotin-peroxidase complex. Color development during incubation with the peroxidase substrate (diaminobenzidine) was con- trolled under the microscope with a positive control section. Counterstaining was done with hematoxylin. Pos- itive control sections originating from a single suitable tissue specimen were included in all batches. Negative controls were incubated with a comparable dilution of an IgM from an irrelevant mouse plasmocytoma (MOPC 104E, Sigma) instead of with the mAb in each case.

For the detection of sialosyl-TF, sections were incubated with neuraminidase from Vibrio cholerae (Serva, Heidelberg, Germany) at a concentration of 0.02 U/ml in PBS containing 0.01 M Caf+ for 1 hour at room temperature to remove sialic acid (NeuAc), washed, and reacted with anti-TF mAbs. Erythrocytes present in the cryosections served as an internal positive control.

1702 CANCER November 25,2995, Volume 76, No. 10

Table 1, Thomsen-Friedenreich-Related Carbohydrate Epitopes and Monoclonal Antibodies or Lectins Used in This Study

Monoclonal Antigen Epitope structure antibody or lectin Reference

Thomsen-Friedenreich (TF) GalPl-3GalNAcal-R* (TFa; type 3 core)

Galpl-3GalNAcpl -R (TFP; type 4 core)

Tn

Sialosyl-Tn

Sialosyl-TF

GalNAcal-R

NeuAca2-6GalNAcal -R

Gal@l-3(NeuAca2- 6)GalNAca 1 -R

A78-G/A7 HH8

BM22 Peanut agglutinin?

A78-G/A7

Peanut agglutinin BaGS-65

TKH2 872.3

HH87

HB-T1

A68-B/All

HB-TI

TEC-02

A78-G/A77

25 26

Dako 27 28 $

$

29 30 31

* R may stand for, e.g., O-Thr-linked epithelial mucins or O-Ser/Thr-linked glycoproteins (TFa, Tn, sialosyl-Tn), blood group A-related type 3 g l y ~ o l i p i d s ~ ~ (TFa, Tn), or ganglioside (type 4) residues (TFB). t Not strictly TF-specific. $ Unpublished results. 5 Murine ascites anti-Tn evoked with 0 Tn red blood cells; see reference 2. ll After treatment of the tissue sections with neuraminidase from V . cholerue.

All sections were treated with antibodies A78-G/ A7 and HH8 for TF, BaGS-6 and TEC-02 for Tn, and TKH2 and 872.3 for sialosyl-Tn. The lectin PNA that was not strictly TF specific was always included, because this reagent had been used in a number of earlier studies. Three additional anti-TF mAbs were applied in a number of cases to obtain additional information or confirmation. Tissue sections from liver metastases (after the incubation step with normal preabsorbed rabbit serum) were additionally treated with the Avidin/Bio- tin Blocking Kit (Vector Laboratories) according to the manufacturer’s instructions to block endogenous biotin present in hepa t~cytes .~~ Briefly, sections were incubated with the avidin blocking solution for 15 minutes at room temperature, rinsed with PBS, and incubated with the biotin blocking solution for another 15 minutes. Scoring was performed as follows: - = all cells negative; + = less than 30% positive cells; 2+ = 30- 60% positive cells; 3+ = more than 60% positive cells. For normal mucosa and transitional mucosa, the percentage of positive crypts was counted, whereas in carcinomas the percentage of positive cells in several opti- cal fields (12.5X) was estimated.

Statistical Analysis

Data were analyzed with either the chi-square test or Fisher’s exact probability test.33 Differences were con- sidered significant if the probability was less than 0.05.

Results

Comparison of Antibodies of Similar Specificity: Basic Patterns

Colorectal carcinomas were found to be reactive with anti-TF mAbs in a substantial number of cases. The mAbs A78-G/A7 (Fig. 1, top right) and HB-TI yielded similar results with respect to localization and staining intensity of positive cells. In glandular adenocarcinoma, TF was detected predominantly at the cell membranes and glandular lumina (Fig. 1, top right), whereas in nongland-forming areas of tumors, diffuse cytoplasmic or membrane staining was observed.

In the case of Tn, BaGS-6 (Fig. 1, center right) always stained more cells in the same area than mAb TEC-02. Results obtained with the two anti-sialosyl-Tn mAbs B72.3 (Fig. 1, bottom right) and TKH2 on tumor tissues were very similar. In transitional mucosa, TKH2 detected vessel endothelia of blood group A patients,26 whereas 872.3 did not. Because the reactivity in transitional mucosa (as well as in carcinoma tissues) was always identical with both mAbs to sialosyl-Tn, one can assume with certainty that the results reflect the presence of sialosyl-Tn.

The type of staining was similar for Tn and sialosyl- Tn. In glandular adenocarcinomas, membrane and cytoplasmic reactivity was observed (Fig. 1, center right,


Figure 1. Immunohistochemistry of cryosections from (all left) transitional colonic mucosa and (all right) colon carcinomas with monoclonal antibodies to TF-related antigens. (Top left and right) A78-G/A7 (anti-TF); (center left and right) BaGS-6 (anti-Tn); (bottom left and right) B72.3 (anti-sialosyl-Tn). Note that the transitional mucosa shown in top left was from a patient of blood group A1 (original magnification, XI00 [all left, top right] and X200 [center and bottom right]).

bottom right), whereas in solid carcinomas diffuse cytoplasmic staining prevailed.

Among the carcinomas negative for TF, 6 of 21 cases of primary tumors and 2 of 2 liver metastases became positive after neuraminidase treatment. These are referred to as expressing "sialosyl-TF" (see Table I). The pattern observed in these cases was characterized by a large number of positive cells with diffuse membrane and cytoplasmic reactivities.

An overview is given in Table 2, where, for reasons

of clarity, identical results obtained with mAbs of the same nominal specificity have been combined.

Comparison of Reactivities of Anti-TF-alpha/ beta, Anti-TF-alpha, and Anti-TF-beta Specific Monoclonal Antibodies

Antibody HH8 (anti-TF-alpha) reacted in most cases with the same tissue areas as HB-T1, A78-G/A7, and PNA, which recognize both anomers. We examined


Table 2. Expression of Thomsen-Friedenreich-Related Antigens in Normal Mucosa, Transitional Mucosa, Primary Colorectal Carcinomas, and Liver Metastases*

~~ ~ ~~

Tissues TF Sialosyl-TF Tn Sialosyl-Tn

Primary carcinomas 31/52 (60)t 6/52 (W$ 44/52 (85)$ 44/52 (85)$ Liver metastases 20/22 (91)t 2/22 (lo)$ 21/22 (95)$ 21/22 (95)$ Transitional mucosa 0/20§ 6/20 (30)n 5/20 (25) 11/20 (55) Normal mucosa 0 /17 6 /17 (3517 0/17 0/17 Values are no. of cases reactive/total no. of cases examined (with percent in parentheses). t P < 0.05. $ P > 0.05. 5 One case reacted with PNA. ll Very weak staining compared with carcinomas.

mAb BM22 (anti-TFa) in one TF-positive colorectal carcinoma in different dilutions and found it to be only marginally reactive. The mAb A68-B/Allt which (in immunohistochemistry) is specific for the beta-anomer of TF, stained 10 of 17 tumor tissues studied. All A68- B/Al1 positive cases were also reactive with A78-G/ A7. The order of staining frequency and intensity of anti-TF mAbs with primary colorectal carcinomas was TF-alpha/beta > TF-alpha > TF-beta (see Fig. 2). This probably reflects the distribution and relative amounts of the respective antigens.

Comparison of Expression of TF-Related Antigens in Primary Tumors and Liver Metastases

Among the liver metastases from colorectal carcinomas, the percentage of TF-positive cases was significantly higher than in the group of primary tumors (91% vs. 60%, respectively). Tn and sialosyl-Tn antigen expres-

3+

2+

1+ Hi 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Patient no.

Figure 2. Comparison of the reactivity of (circle) anti-TF-alpha/beta, (square) anti-TF-alpha, and (diamond) anti-TF-beta in individual patients with primary colorectal carcinomas. (For a definition of scoring, see the Materials and Methods section.)

sion were identified in 85% of primary colorectal carcinomas and in 95% of liver metastases, respectively; this difference was not statistically significant. Table 2 sum- marizes the results.

Patients with TF-positive tumors had liver metastasis at a significantly higher rate than those with TF-negative tumors (P = 0.016, Fig. 3). The respective results for Tn and sialosyl-Tn were as follows: 47% of 30 patients with tumors carrying these antigens, but none of 5 patients with tumors lacking these antigens, were prone to have liver metastasis (P = 0.134).

Simultaneous Expression of TF, Tn, and Sialosyl-Tn in Primary Colorectal Carcinomas and in Liver Metastases

The patterns of simultaneous expression of these three biochemically related antigenic structures are shown in Table 3. A majority of primary colorectal carcinomas expressed a11 three epitopes. Of 14 primary carcinomas and 2 metastatic tissues reactive with Tn and sialosyl- Tn mAbs but not with TF reagents, 6 and 2 cases, respectively, became positive for TF after neuraminidase treatment. Among the seven tumors lacking any TF-related antigens, five cases were poorly differentiated carcinomas. Tn and sialosyl-Tn were simultaneously exposed in every case, In general, there was a correlation between all three antigens with respect to the intensity of expression (tumors strongly positive for TF were also intensely stained for Tn and sialosyl-Tn and vice versa). Within a given tumor, regardless of the grade of differentiation, Tn and sialosyl-Tn antigens were detected in more cells than was TF. Moreover, Tn and sialosyl-Tn were found more often than TF in the cytoplasm of well differentiated carcinomas.

Comparison of Colorectal Carcinomas with Adjacent Transitional Mucosa

We did not detect TF, Tn, or sialosyl-Tn in the examined cases of normal colonic mucosa. However, we observed


60 tistically significant higher percentage than poorly differentiated carcinomas (P < 0.05; Table 5).

0 Patients with TF(-) carcimrntu Patients with TF(+) cmioorntu

Figure 3. Comparison of the frequencies of liver metastasis in 14 patients with TF-negative carcinomas and in 21 patients with TF- positive carcinomas (P < 0.05).

a partial occurrence in the transitional mucosa adjacent to colorectal carcinomas. As shown in Tables 2 and 4, regardless of whether the primary colorectal carcinomas were positive for TF, the corresponding transitional mucosa was always negative with anti-TF mAbs (Fig. 1, top left). Only one transitional mucosa was reactive with PNA in the supranuclear cytoplasm. After neuraminidase treatment, 6 of 20 cases of transitional mucosa became very weakly positive for TF (weak supranuclear cytoplasmic, apical and membrane staining). Tn and sialosyl-Tn were partially expressed in the transitional mucosa. The percentage of cases positive for Tn and sialosyl-Tn in transitional mucosa was significantly lower than in the corresponding tumor (P < 0.05). No correlation was found between the presence of Tn (or sialosyl-Tn) in the tumor and its adjacent transitional mucosa (P > 0.05). Tn and sialosyl-Tn-carrying antigens in the transitional mucosa (Fig. 1, center and bottom left) were mostly localized in the cytoplasm, pri- marily in the supranuclear region. In addition, goblet cell vacuoles were found to be positive. In two and three cases, respectively, the transitional mucosa was positive for Tn and sialosyl-Tn, whereas the primary tumor was negative; all these tumors were poorly differentiated.

Correlation of TF-Related Antigens wi th Staging and Grading Features

Discussion

The TF-related antigens (or, more precisely, haptens) have been structurally categorized as histo-blood group antigen types 3 and 4.34 Type 3 antigens so far have been found mostly in m u c i n ~ ~ ~ and type 4 antigens exclusively in glycolipids. Tn, TF-alpha, and sialosyl-TF (type 3 antigens) are linked as consecutive steps in their biosynthetic pathway.36

The TF antigen is well established as an oncodevel- opmentally regulated, cancer-associated antigen that is occluded from normal cells of adults and widely distrib- uted among human tumors.'-3 Colorectal carcinomas, which are one of the most common cancers in developed countries, have been described to express TF in several s t u d i e ~ , ~ - ~ ~ ~ * " but this finding has been ques- tioned in other s t ~ d i e s . ' ~ ' ~ ~ Longenecker et al. con- cluded that colon cancers express exclusively TF-beta."

Our results clearly show that colorectal carcinomas carry exposed TF hapten (60% of 52 primary tumors), thus confirming earlier data (mainly obtained with PNA or polyclonal antisera). In a recent study using mAb AH9-16, 71% of 29 colon carcinomas were reported to be TF positive.' In contrast to our data, a weak reaction was seen in this study in the supranuclear cytoplasm of cells in transitional mucosa. Biochemical data published very recently provide further evidence of the presence of exposed TF in colorectal carcinoma^.^^

In a single recent case, we studied the colon carcinoma and its liver metastasis with pools of affinity-pu- rified human monospecific polyclonal anti-TF and anti- Tn as well as with pools of rodent monoclonal anti-TF and anti-Tn ascites. The TF and Tn epitopes were observed in the primary tumor and to a greater extent in the metastasis (Springer GF, Wang B. Unpublished data, 1995).

Table 3. Extent of Simultaneous Expression of Thomsen-Friedenreich (TF)-Related Antigens in Primary Carcinomas, Liver Metastases, and Transitional Mucosa*

Primary Liver Transitional TF Tn S-Tn carcinomas metastases mucosa

No significant correlations were found between the expression of any of the three antigens and such parameters as wall penetration, lymph node status, and localization of the tumor within the gastrointestinal tract (Table 5). Well or moderately differentiated colorectal carcinomas expressed TF, Tn, and sialosyl-Tn at a sta-

+ + + 30/52(58) 19/22(86) 0/20 - + + 14/52 (27) 2/22 (10) 5/20 (25)

- - + 0/52 0/22 6/20 (30) - - - 7/52 (13) 0/22 9/20 (45)

* Values are no. of cases applicable/total no. of cases examined (with percent in parentheses).

+ - - 1/52 (2) 1/22 (4) 0/20


Table 4. Expression of Thomsen-Friedenreich (TF)- Related Antigens in Colorectal Carcinomas: Comparison of Primary Carcinomas and Adjacent Transitional Mucosa*

Transitional Primary mucosa carcinomas TF Tn S-Tn

+ + 0/20 3/20 (15) 8/20 (40) + - 0/20 2/20 (10) 3/20 (15) - + 11/20 (55) 13/20 (65) 8/20 (40) - - 9/20 (45) 2/20 (10) 1/20 (5)

*Values are no. of cases reactive/total no. of cases examined (with percent in parentheses).

All anti-TF-alpha/-beta mAbs used in the current study reacted qualitatively similarly; however, we noted that the TF-alpha specific mAbs (HH8 and BM22) and the TF-beta specific mAb A68-B/A11 stained colorectal tumors considerably weaker than HB-T1 and A78-G/A7, which react with both anomers of TF.25 On the basis of this direct immunohistochemical evidence, we believe it to be likely that colorectal carcinomas express both anomers of TF, TF-alpha and TF-beta. The PNA-binding glycoproteins have been identified in colorectal carcinoma^.^ Because naturally occurring TF- beta structures so far are known only from gly~olipids,~~ it is likely that glycolipids (type 4 histo-blood group antigens) are among the TF-carrying glycoconjugates present in colorectal carcinomas. Considering the abil- ity to recognize TF-alpha as well as TF-beta and the low background staining observed, we believe that the new anti-TF mAb A78-G/A7 is a powerful tool in immunohistochemistry. A68-B/All (Karsten U, Cao Y, Stolley P, Hanisch F-G, Unpublished data, 1995) is to our knowledge the first mAb specific for TF-beta in immunohistochemistry .

Liver metastasis is the most common blood spread in colon cancer. Tumor cells enter the liver through the portal vein. Because liver sinusoids are an open vascular bed, these cells can contact hepatocytes directly. Spe- cific cell adhesion to receptors (lectins) present on hepatocytes and Kupffer's cell^'^^'^ is thought to play an im- portant role in this p r o c e ~ s . ' ~ - ~ ~ However, most data available so far have been derived from animal models. In the current study, we found that liver metastases of colorectal carcinomas expressed TF at a significantly higher rate (91 Yo) than primary colorectal carcinomas (60%) and that patients with TF-positive tumors developed liver metastasis at a higher percentage than patients with TF-negative primary tumors. The differences between these groups indicate that TF-positive tumor cells are more likely to metastasize to the liver, thus supporting the hypothesis that TF plays an impor- tant role in the process of metastasis of human cancer

cells into the liver. It follows that the expression of TF on primary tumors appears to represent a significant risk factor for the development of liver metastases, es- pecially with gastrointestinal and probably also with pancreatic cancer. If our data, which must be categorized as a pilot study, are confirmed in further studies, such patients would deserve very close follow-up of their liver status.

Other tumor-associated carbohydrate epitopes, for example, sialosyl-Le" and sial~syl-Le",~~ apparently also contribute to the process of metastasis of colorectal tumors to the liver.

Our results with the closely related antigens Tn and sialosyl-Tn were similar in tendency to those obtained with TF but were not statistically significant. Data from the literature about the expression of these antigens in colorectal carcinomas and their metastases are inconsis- tent. Hasegawa et al.39 found no evidence for stronger expression of Tn and sialosyl-Tn in liver metastases and primary lesions with metastases than in primary tumors and primary lesions without metastases, respectively, whereas Itzkowitz et al.9 proposed sialosyl-Tn as a prognostic factor in patients with colorectal cancer. Considering the frequent coexpression of all three TF- related antigens found in our study, a correlation between the expression of sialosyl-Tn and the development of liver metastasis could be expected.

A correlation of PNA-reactive (TF-positive) tumors with unfavorable prognosis also has been observed in a number of studies on urinary bladder ~ancer.~' ,~ '

The mechanisms responsible for the aberrant glycosylation patterns observed in cancer4' are not fully

Table 5. Relationship of Thomsen-Friedenreich (TF)- Related Antigens to Several Pathologic Parameters of Primary Colorectal Carcinomas*

~

TF Tn Sialosyl-Tn

Differentiation Well or moderately

differentiated 29/44 (66)t 41/44 (93)t 41/44 (93)t Poorly differentiated 2/8 (25)t 3/8 (38)t 3/8 (38)t

T1, T2 11/27 (65) 14/17 (82) 14/17 (82) T3, T4 17/32 (53) 28/32 (88) 28/32 (88)

Absent (NO) 17/29 (59) 25/29 (86) 25/29 (86) Present (Nl-3) 11/20 (55) 17/20 (85) 17/20 (85)

Rectum 10/16 (63) 13/16 (81) 13/16 (81) Sigmoid 5/8 (63) 6/8 (75) 6/8 (75) Other 13/25 (52) 23/25 (92) 23/25 (92)

* Values are no. of cases reactive/total no. of cases examined (with percent in parentheses). t P < 0.05.

T stage (wall penetration)

Lymph node metastases

Localization

TF Antigen in Colorectal Carcinomas/Cao e t al. 1707

understood. Our data revealed multimodal alterations in glycosylation among colorectal carcinomas. Three patterns of expression of TF, Tn, and sialosyl-Tn in primary colorectal carcinomas emerged. First, the most common phenotype found in colorectal carcinomas consisted of the simultaneous expression of TF, Tn, and sialosyl-Tn. We were surprised by this finding, because it implicates the concomitant accumulation of interme- diates at different stages of biosynthesis. Second, a smaller group of colorectal carcinomas expressed Tn and sialosyl-Tn but not TF (TF alone was expressed in only one case). Third, around one-tenth of tumors did not express any TF-related antigens. Most tumors in this group were poorly differentiated. The existence of these three phenotypes points to different mechanisms of al- teration in, for example, the glycosyltransferases responsible and could be a starting point for further biochemical studies. In conclusion, we have presented evidence that TF antigen is not only present on colorectal carcinomas, but also may as an adhesion molecule be actively involved in the development of liver metastasis.

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