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Expression of CEACAM6 in Resectable Colorectal Cancer: A Factor of Independent Prognostic Significance

Peter Jantscheff, Luigi Terracciano, Adam Lowy, Katharina Glatz-Krieger, Fritz Grunert, Burkhard Micheel, Jens Brümmer, Urs Laffer, Urs Metzger, Richard Herrmann, Christoph Rochlitz

From the Kantonsspital Basel, Departments of Research, Molecular Cancer Research—Oncology, Pathology, and Medical Oncology for the Swiss Group for Clinical Cancer Research (SAKK), Basel; Swiss Group for Clinical Cancer Research (SAKK), Bern; Chirurgische Klinik, Spitalzentrum Biel, Biel; Department of Surgery, Triemlispital Zurich, Switzerland; University of Freiburg, Institute of Molecular Medicine, Freiburg; University of Potsdam, Institute of Biology and Biochemistry, Golm; University of Hamburg, University Hospital Eppendorf, Hamburg, Germany.

Address reprint requests to Peter Jantscheff, PhD, Albert-Ludwigs-University of Freiburg, Department of Experimental Urology, Stefan-Meier-Str 8, D-79104 Freiburg/Br, Germany; e-mail: jape{at}sun11.ukl.uni-freiburg.de.

	ABSTRACT

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Purpose: CEACAM6, CEACAM1, and human carcinoembryonic antigen (CEA) are coexpressed in normal colorectal epithelia, but show deregulated expression in colorectal cancers (CRC). Upregulation of CEACAM6 expression in hyperplastic polyps and early adenomas represents one of the earliest observable molecular events leading to colorectal tumors. The aim of our study was to evaluate the prognostic relevance of CEACAM6, CEACAM1, and CEA tissue expression in patients with CRC.

Patients and Methods: Immunohistochemical analysis was carried out on tissue microarrays from 243 paraffin-embedded biopsies from a randomized controlled clinical trial (Swiss Group for Clinical Cancer Research [SAKK] 40/81) of adjuvant fluorouracil-based chemotherapy with CEACAM-specific monoclonal antibodies. The median follow-up was 8 years. Overall survival (OS) and disease-free survival (DFS) were calculated using Kaplan-Meier estimates and hazard ratios (HRs) estimated using Cox proportional hazards models.

Results: Tissue expression of CEACAM6, CEACAM1, and CEA was enhanced in 55%, 58%, and 94% of patients, respectively. Multivariate Cox analysis including sex, age, tumor site, stage, differentiation grade, treatment, and nodal status as covariates showed that CEACAM6 overexpression independently predicted poor OS (HR, 1.86; P = .0100) and DFS (HR, 2.00; P = .0028), whereas CEACAM1 or CEA were not significantly related to these outcomes. The data did not provide evidence for or against the hypothesis that the CEACAM6 effect on survival differs according to treatment.

Conclusion: Expression of the cell adhesion molecule CEACAM6 in CRC is an independent prognostic factor allowing subdivision of patients into low- and high-risk groups. Whether CEACAM6 or CEA and CEACAM1 might be useful as predictive markers of chemotherapy benefit remains unclear.

	INTRODUCTION

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COLORECTAL CANCER (CRC), with its high incidence and significant morbidity and mortality, remains one of the leading causes of cancer-related death worldwide. Identification of high-risk markers in this disease would offer the possibility to select patients for intensive postoperative follow-up protocols, and to look for new adjuvant treatment strategies for such patients.

Several members of the human carcinoembryonic antigen (CEA) family are intercellular adhesion molecules^1–4 and belong to the immunoglobulin superfamily.⁵ They are coexpressed in normal colorectal epithelia,^6–8 but show deregulated cell surface expression in CRCs.^7–14 While CEACAM6 and CEA are overexpressed in many colorectal and other human cancers,^{7,9,12,14–18} CEACAM1 and CEACAM7 are usually downregulated in these tumors.^7,8,10,11

The significance of tissue overexpression of CEA as a prognostic tool in colorectal and other carcinomas remains controversial.^{16,17,19–21} Recently, CEACAM1 expression has been shown to predict significantly poorer overall survival (OS) and disease-free survival (DFS) in lung cancer patients and to be strongly associated with metastatic spread in cutaneous malignant melanoma.^22–24 An association between tissue expression of CEACAM6 or CEACAM7 and long-term survival has not been investigated until now. The frequently observed deregulated cell surface expression of CEACAM6, CEA, CEACAM1, or CEACAM7 in tumor cells, however, has indicated a functional role of these adhesion molecules in tumorigenesis.^8,10–12,15 Recently, various in vitro and in vivo studies confirmed the tumor growth inhibiting as well as stimulating properties of these molecules.^25–35 Therefore, we have performed a retrospective immunohistochemical study of the expression of CEACAM family members in tumor microarrays from paraffin-embedded CRC tissues. Expression levels of these molecules were correlated with DFS and OS in a group of 243 patients from a Swiss Group for Clinical Cancer Research (SAKK) randomized controlled clinical trial of a single course of adjuvant intraportal chemotherapy.³⁶

	PATIENTS AND METHODS

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Patients
Between 1981 and 1987, the SAKK enrolled 505 patients with adenocarcinoma of the colon or rectum who underwent surgery for CRC in a randomized study of a single course of perioperative adjuvant intraportal chemotherapy with fluorouracil (FU) and mitomycin C.³⁶ Formalin-fixed, paraffin-embedded biopsies were available from 264 of these patients. Characteristics of patients included in the present study are shown in Tables 1 and 2. The 243 of 264 patients in whom immunohistochemistry was technically assessable (see below) and whose data were included in the immunohistologic analyses reported here, were representative of the 505 patients in the clinical trial with respect to age and sex distribution, localization of disease, and nodal stage (Tables 1 and 2). Additionally, 105 formalin-fixed, paraffin-embedded tumor biopsies available from CRC patients from other studies were included to determine the incidence of loss of expression or overexpression of CEACAM molecules. However, these latter samples were not included in the survival analysis because follow-up data were not available.

Immunohistochemistry
Sections were deparaffinized in xylene and rehydrated in graded alcohol to Tris-buffered saline (50 mmol/L Tris, 150 mmol/L NaCl, pH 7.4). Monoclonal anti-CEACAM antibodies of different specificity were used. First, optimized immunohistochemical protocols were established by staining representative control histopathology sections of healthy and CRC tissue with various anti-CEACAM monoclonal antibodies (mAbs). Tissue expression of CEACAM6, CEACAM1, or CEA in normal colorectal tissue was homogenous and of low intensity, and was subjectively graded as positive (+; not shown). Next, we compared immunohistological staining of routine pathology sections and of TMAs from selected areas. Evaluating antibodies to individual molecules of the CEACAM family, we did not observe any differences in staining intensities between TMAs and routine sections from tumorous areas of various biopsy samples. This confirms our previous findings with many different antibodies and target proteins that microarray technology is a highly reproducible and representative way to define tissue expression of tumor-associated proteins.³⁸ Immunostaining was enhanced by treating sections in a microwave oven in 10 mmol/L citrate buffer (pH 6.0) at 90°C for 60 minutes. The ABC method (Vectastain, Burlingame, CA) was used as detection technique as described by the manufacturer. Appropriate positive and negative controls were used in every staining run. Finally, the slides were counterstained with hemalaun and mounted with glycerine/gelatin. For technical reasons we lost approximately 10% of colorectal biopsies for interpretation.³⁷ We could unambiguously interpret staining of 243 biopsies from SAKK 40/81, and of 92 unrelated colorectal tumor biopsies.

Antibodies
The specificity of anti-CEACAM mAbs has been characterized in detail previously and has been confirmed during the Leukocyte Typing Workshops V and VI.^18,9–44 Therefore, antibody specificities are designated by the CD nomenclature as CD66a, CD66b, CD66c, and CD66e, respectively.^43,44 The antigens, however, were designated by the redefined CEACAM nomenclature recently proposed from a collaborative working group (CEA/PSG Workshop held in Ratzeburg, Germany) to solve nomenclature problems (http://www.ukl.uni-freiburg.de/immz/cea/human/ceacam/cea_fam_hum_ceacam.htm).⁴⁵

Monoclonal antibodies used for staining the tumor array slides are summarized in Table 3. All antibodies were of the immunoglobulin G1 (IgG1) isotype. CEACAM staining of individual tumor tissue arrays by mAbs was compared and subjectively graded into four groups from negative (-) to strongly positive (+++) by two independent pathologists in a blinded fashion, where (-) indicated cytoplasm negative; (+) indicated cytoplasm of scattered cells was weakly positive; (++) indicated cytoplasm of most cells was weakly to moderately positive; and (+++), cytoplasm of all cells was strongly positive. Tissue expression of CEACAM6, CEACAM1, and CEA was determined using the antigen-specific mAbs 13H10 (CD66c+), 4D1/C2 (CD66a+), and D11-CC11 (CD66e+), respectively (Fig 1A and 1B). CEA/CEACAM1 cross-reacting antibodies (4/3/17 and 12–140–4) stained all tissues expressing one of the antigens, whereas a CEACAM8-specific mAb (BIRMA17C) served as an internal negative control (data not shown).

View larger version (79K): [in this window] [in a new window]   Fig 1. Tissue expression of CEACAM6, CEACAM1, and human carcinoembryonic antigen (CEA). (A) Tissue microarrays. (B) Higher magnification (x 400) of immunostained CEACAM6 or CEACAM1 negative (-) to strongly positive (+++) arrays. (C) Different antigen expression patterns.

	RESULTS

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Biopsies of two groups of patients (SAKK 40/81)—untreated (141 patients) or treated with a single course of perioperative intraportal FU/mitomycin C chemotherapy (102 patients)—were analyzed (Tables 1 and 2). OS and DFS of these patients significantly differed as a function of adjuvant chemotherapy (Fig 2).³⁶

View larger version (16K): [in this window] [in a new window]   Fig 2. (A) Overall and (B) disease-free survival in colorectal cancer patients — fluorouracil- (FU-) treated and untreated (as-treated analysis). HR, hazard ratio.

When applying the staining protocol described above to 243 patients from the SAKK 40/81 trial and to 92 additional colorectal tumor samples, we found enhanced levels (+ + +/+ +) of CEACAM6 and CEACAM1 tissue expression in 55% and 58% of CRC patients (SAKK 40/81), respectively (Fig 1A, 1B, and 1C). In contrast, CEA showed enhanced tissue expression in the large majority (94%) of CRC patients (Fig 1C). While CEACAM6, CEA, and CEACAM1 showed similar expression levels in normal colorectal tissue (not shown),^6–8 we observed strongly varying expression of individual antigens and all possible combinations of expression of the three CEA family members in tumor biopsies from our patients (Fig 1C). In 40.7% of patients, all three molecules showed enhanced expression of CEACAM1, CEACAM6, and CEA (+ + +), whereas in 14.9% and 15.6% of patients, respectively, CEACAM6 and CEA (+ - +) or CEACAM1 and CEA (- + +) were found to be enhanced. Tumor tissue arrays from 23.1% and 1.6% of patients, respectively, showed enhanced CEA (- - +) or CEACAM1 (- + -) expression only, whereas in 4.1% of patients, normal or downregulated expression levels of all three molecules (- - -) were found (Fig 1C). Similar results have been observed in the 92 additionally investigated unrelated colorectal tumor control biopsies (data not shown).

To perform survival analysis by the Kaplan and Meier method and the Cox proportional hazards model, the patients were divided into two groups with enhanced (+ + +/+ +) or normal/reduced (±) expression of individual CEACAM molecules, respectively. The group of patients displaying normal/reduced CEACAM6 expression had significantly better 5-year DFS than the group with enhanced CEACAM6 levels (Fig 3; Table 4). In univariate analysis, the HR for relapse associated with enhanced CEACAM6 expression was 2.44 (P < .0001). CEACAM6 overexpression was also associated with far worse OS (HR, 2.19; P < .0005). At a median follow-up of 5 years, 57% of patients with enhanced CEACAM6 expression, but 74% with low CEACAM6, expression were alive (Table 4; Fig 3). Multivariate Cox analysis including sex, age, tumor localization, tumor staging, differentiation grade, lymph node status, and actually applied treatment as covariates showed that CEACAM6 overexpression independently predicted for poor survival (Table 3; OS: HR, 1.86; P = .0100; DFS: HR, 2.00; P = .0028). There was no evidence that the effect of CEACAM6 on the survival end points differed according to treatment status (interaction terms for OS and DFS were P = .43 and P = .19, respectively), excluding CEACAM6 expression as a strong predictive marker of chemotherapy benefit in that situation.

View larger version (22K): [in this window] [in a new window]   Fig 3. (A) Overall and (B) disease-free survival. CEA, human carcinoembryonic antigen.

	DISCUSSION

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CRC, with its high incidence and significant morbidity and mortality, remains one of the leading causes of cancer-related deaths worldwide. Adjuvant chemotherapy may reduce the risk of recurrence and death in a proportion of patients with CRC after surgical resection.^36,46 Standard adjuvant chemotherapy of CRC currently comprises FU in combination with other drugs, such as levamisole or leucovorin. Recently, new drugs, including oxaliplatin, irinotecan (CPT-11), and capecitabine, have been introduced as second-line chemotherapy to improve efficacy of palliative treatment in patients who had already received FU, and some studies also show their superiority as first-line agents in CRC.^47,48 At present, tumor stage is the most significant prognostic indicator in early CRC and is also used to select patients for postoperative adjuvant chemotherapy.⁴⁹ Many molecular abnormalities have been identified in CRC, such as overexpression of P-glycoprotein or increased thymidylate synthase levels; mutations in DNA mismatch repair genes in hereditary nonpolyposis CRC; mutations in the APC gene in familial adenomatous polyposis; allelic losses, particularly on chromosomes 1, 5, 8, 17, and 18; deletions involving p53 and K-ras genes; gains of chromosomal arm 20q or deregulated expression of various CEACAM molecules.^7,8,11,50,51 Although some of the abnormalities have been suggested to be predictive factors,^52,53 thus far, none of these markers is used in clinical practice to better select patients for adjuvant treatments. A prognostic or predictive impact of other markers (eg, of deregulated expression of various CEACAM molecules^8,11,54) has not been proven until now.

We analyzed the expression of various CEACAM molecules in tissue microarrays (TMA) from 243 patients with CRC treated within a randomized study (SAKK 40/81) of perioperative, 5-FU-based chemotherapy. We found that CEACAM6, but not CEACAM1 or CEA, was of significant prognostic value in this group of patients. In a multivariate analysis, patients overexpressing CEACAM6 had far worse DFS and OS (OS: HR, 1.86; P = .0100; DFS: HR, 2.00; P = .0028), establishing CEACAM6 as a potentially important prognostic marker in CRC.

Conversely, there is no evidence that the effect of CEACAM6 on the survival end points differs according to treatment status (equivalently, there is no evidence that the effect of treatment on survival end points differs according to the presence or absence of CEACAM6 overexpression). The interaction terms were nonsignificant both for OS (P = .43) and DFS (P = .19). It is important to note that although this result provides no evidence of an interaction, it provides no evidence against such an effect (ie, the data are compatible with the hypothesis that the CEACAM6 effect is much smaller on treatment), and the hypothesis that the CEACAM6 effect is much larger on treatment.

Recently, it has been demonstrated that the increased cell surface levels of CEA and CEACAM6 in purified human colonocytes from freshly excised, well- to poorly differentiated colon carcinomas are inversely correlated with the degree of cellular differentiation.³⁵ That, in our study, there are so many moderate and well-differentiated CEACAM6 overexpressors and no observable tendency for poor differentiation to imply CEACAM6 overexpression, does suggest that CEACAM6 overexpression may not simply be a side effect of poor differentiation and that the apparent adverse effect of CEACAM6 on survival is not a confounded result due to an association with poor differentiation. Also, CEA expression in our patients supports this view. Whereas CEA expression was running in parallel to CEACAM6 overexpression in dedifferentiated colon carcinoma cells,²⁸ CEA was overexpressed in most of our patients (229 of 243), independent of their histological differentiation grade, and low CEA expression was found only in 14 patients who showed moderate (n = 13) or poor (n = 1) differentiation.

Tissue expression of CEACAM molecules shows striking variations in malignancies.^7–14 These molecules are coexpressed in normal colorectal epithelia,^6–8 but show deregulated cell surface expression in CRCs.^7–14 While CEACAM6 and CEA are overexpressed in many colorectal and other human cancers,^{7,9,12,14–18} CEACAM1 and CEACAM7 are usually downregulated in these tumors.^7,8,10,11 Recently, it has been shown that downregulation of CEACAM7 and upregulation of CEACAM6 expression in hyperplastic polyps and early adenomas represent some of the earliest observable molecular events leading to colorectal tumors.⁷ In this study, we have observed different tissue expression patterns with varying levels of CEA, CEACAM1, and CEACAM6 in the individual tumor tissue arrays with high levels of CEA in most patients (Fig 1C).

The frequently observed loss or reduction of CEACAM1 and/or CEACAM7 expression in tumor cells has indicated a functional role of these adhesion molecules in tumorigenesis and suggests a potential prognostic impact.^7,8,10,11 It has been demonstrated by transfection experiments that mouse and rat CEACAM1 homologues^27,30,31 and human CEACAM1⁵⁶ can display tumor suppressor function(s) in colonic or prostate cancer cells. Assuming a tumor suppressor function of CEACAM1, the outcome of patients whose tumors show a reduced expression of the molecule would be expected to be compromised. In contrast, two recent studies have shown that expression or overexpression of CEACAM1 has a negative prognostic impact in adenocarcinomas of the lung,²² and was associated with subsequent development of metastatic disease in cutaneous malignant melanoma.²³ In addition, CEACAM1 has been found to be one of the major effectors of vascular endothelial growth factor in early microvessel formation,²⁵ supporting the growth of invasive tumors.^57,58 Similarly, in two recent studies of non–small-cell lung cancer using complementary DNA (cDNA) microarrays for gene expression analysis, expression of CEACAM1 was associated with good prognosis in one,⁵⁹ while the other showed an association of CEACAM1 expression with lung metastases from colon cancer and not with primary lung adenocarcinoma.⁶⁰ It appears, therefore, that CEACAM1 might have both tumor growth stimulating and suppressing properties. This is in agreement with our study, in which we did not find any significant difference of OS or DFS between CRC patients with enhanced or reduced CEACAM1 expression. Similarly, patients with complete loss of CEACAM1 tissue protein expression (approximately 15% of the whole population) had the same prognosis as those whose tumors expressed CEACAM1 (data not shown).

CEACAM6 and CEA have been found to be upregulated in many tumor tissues.^{7,9,12,17,61–64} Positive CEACAM6 or CEA tissue reaction has been shown to be related to grade of differentiation and depth of tumor invasion.^{16,17,19,65,66} Upregulation of CEACAM6 expression in hyperplastic polyps and early adenomas was one of the earliest observable molecular events leading to colorectal tumors.⁷ The significance of tissue overexpression of CEA as a prognostic tool for colorectal and other carcinomas, however, is still uncertain.^{16,17,19–21} In our study, the expression of CEA was of no prognostic relevance because it was overexpressed in 94% of all investigated tumors (OS: HR, 1.96; P = .19; DFS: HR, 2.68; P = .092), compromising any chance to find statistically significant differences between the two populations. In contrast, tissue overexpression of CEACAM6 was significantly associated with worse prognosis (OS: HR, 1.89 [95% CI, 1.18 to 3.03], P = .0085; DFS: HR, 2.00 [95% CI, 1.27 to 3.15], P = .0027).

Recent studies showed that both molecules display functional properties promoting tumor growth. Ectopic expression of CEACAM6 and CEA was able to block differentiation^67,68 and disrupt cell polarization and tissue architecture of various types of cells.^{28,29,32,35,64,69} Transfecting cells with CEA reduced latency required to form tumors, and significantly enhanced experimental liver metastasis and implantation in liver in athymic nude mice.^26,34 Moreover, CEA cooperates with myc and bcl-2 in transformation, and CEACAM6 or CEA significantly inhibited anoikis (ie, apoptosis of cells deprived of their anchorage to the extracellular matrix).^32,34 Stable transfectants of human colonic cell lines constitutively expressing 10- to 30-fold higher cell surface levels of CEACAM6 or CEA, compared with endogenous levels, failed to polarize and differentiate into glandular structures in monolayer or three-dimensional culture, or to form colonic crypts in a tissue architecture assay in nude mice. In addition, these transfectants were found to exhibit increased tumorigenicity in nude mice.³⁵ These tumorigenic features are consistent with the observed upregulation of CEACAM6 and CEA in human tumors and make the strong association of CEACAM6 expression with patient outcome in our study biologically plausible. Preliminary investigations in breast cancer patients showed statistically significant correlation between the presence of CEACAM6 messenger RNA (mRNA) and larger tumor size (P = .008), presence of lymph node metastases (P = .016) or International Federation of Gynecology and Obstetrics stages greater than IIB (P = .001), suggesting that CEACAM6 expression might correlate with poor prognosis in these patients also (J. Thompson et al, unpublished data, December 2002).

In conclusion, our study is the first to demonstrate the prognostic significance of immunohistochemically detectable overexpression of CEACAM6 in patients with resectable CRC. Furthermore, this study is also the first to show that downregulation of the CEACAM1 protein has no prognostic impact in CRC patients.

If confirmed in large independent trials, CEACAM6 expression might become useful as a marker to identify patients who are at especially high-risk of relapse, and thus, likely to have a higher absolute benefit of adjuvant treatment and of intense follow-up protocols. Further studies are needed to evaluate the relationship between the expression of CEA family molecules and prognosis in other solid tumors or to evaluate whether these molecules might be useful as predictive markers of chemotherapy benefit.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicated no potential conflicts of interest.

	ACKNOWLEDGMENTS

 
We thank Drs C. Wagener (Hamburg, Germany), J.P. Johnson (Munich, Germany), and O. Bormer (Oslo, Norway) for providing monoclonal antibodies.

	NOTES

 
Supported by the Foundation for Clinical Cancer Research, Department of Oncology, Kantonsspital Basel, and by the Krebsliga Beider Basel, Basel, Switzerland. P.J., L.T., and A.L. contributed equally to this work.

	REFERENCES

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Submitted February 25, 2003; accepted July 17, 2003.

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