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Immunomagnetic Enrichment and Detection of Micrometastases in Colorectal Cancer: Correlation With Established Clinical Parameters

From the Departments of Internal Medicine I and Cardiothoracic Surgery, University of Cologne, and Department of Surgery, St Elisabeth Krankenhaus, Cologne, Germany.

Address reprint requests to Martin R. Weihrauch, MD, Immunologisches Labor Haus 16, Uniklinik Koeln, Joseph-Stelzmann-Str 9, 50924 Koeln, Germany; email: martin.weihrauch{at}uni-koeln.de

	ABSTRACT

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PURPOSE: Micrometastatic disease in bone marrow is of prognostic significance in colorectal cancer patients. However, detection rates of standard immunocytology are relatively low. We used magnetic activated cell sorting (MACS), a highly sensitive method, to increase detection rates and correlated the presence of cytokeratin (CK)-expressing cells with clinical parameters.

PATIENTS AND METHODS: Bone marrow was obtained from 51 consecutive patients with newly diagnosed colorectal adenocarcinoma who underwent primary surgery and 18 control subjects. International Union Against Cancer (UICC) stage I disease was diagnosed in 11 patients, stage II disease was diagnosed in 14 patients, stage III disease was diagnosed in 12 patients, and stage IV disease was diagnosed in 14 patients. CK-positive cells were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to CK 7 and 8.

RESULTS: CK-positive cells were found in 33 (65%) patients and were absent in 18 (35%). Four of 11 (36%) patients with UICC stage I disease, nine of 14 (64%) with stage II diease, eight of 12 (67%) with stage III disease, and 12 of 14 (86%) with stage IV disease were CK-positive. Epithelial cells were more frequently found in pT3/4 (72%) than in pT1/2 (36%) tumors (P = .026), but there was no difference for lymph node status. CK-positive patients had a higher chance for elevated carcinoembryonic antigen (85% v 15%, P = NS) and CA 19-9 levels (92% v 8%, P = .019). There were no significant differences in CA 72-4, sex, age, tumor grading, or tumor localization regarding the presence of CK-positive cells. All control subjects were CK-negative.

CONCLUSION: In searching for micrometastases in colorectal cancer patients, we have achieved high detection rates by using MACS. The presence of these cells correlated significantly with tumor stage, tumor extension, and the tumor marker CA 19-9.

	INTRODUCTION

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COLORECTAL CANCER remains one of the leading causes of tumor-related deaths in developed countries despite screening for fecal occult blood¹ and colonoscopy,² application of "no-touch" surgery,³ successful adjuvant chemotherapy and radiotherapy,^4,5 and life-prolonging palliative cytostatic treatment.⁶ The 5-year survival for International Union Against Cancer (UICC) stage I patients is approximately 90%, but decreases to 75% and 50% for stages II and III, respectively, and drops below 5% for patients with stage IV (ie, metastatic) disease. Although fluorouracil-based adjuvant chemotherapy is effective in stage III colorectal cancer patients and different enhanced regimens are currently being tested, large trials failed to show an improved survival in stage I or II disease.⁷

The presence of micrometastases, also called minimal residual disease or isolated tumor cells, is an independent prognostic factor in colorectal cancer,^8,9 thus identifying patients at risk for relapse. The detection of these occult metastases has been described in numerous studies for most solid tumors.¹⁰ Usually, bone marrow is the organ of choice for taking samples because it is relatively easy to access and provides higher detection rates than peripheral blood. Autopsy studies have shown that although bone metastases are rare in stage IV colorectal cancer, micrometastatic disease can be detected quite frequently.¹¹ Current methods for evaluating micrometastases are immunocytology (usually conducted by the alkaline phosphatase–anti–alkaline phosphatase reaction),¹² polymerase chain reaction (PCR)-based techniques,^13,14 flow cytometry,¹⁵ and fluorescent in situ hybridization (FITC).¹⁶ To date, immunocytology with antibodies directed against epithelial epitopes (cytokeratins) on mononuclear cells is the most widely used technique because it is easy to perform and cost-effective, and has lower false-positive rates than flow cytometry or PCR.¹⁷ However, detection rates are relatively low at approximately 30% to 40% for all stages, even in metastatic disease,^18,19 and protocols are not standardized yet, which makes comparison of data from different study groups difficult.²⁰ One major weakness of methods using the alkaline phosphatase–anti–alkaline phosphatase reaction technique is that scanning large amounts of cells for cytokeratin-positive cells (ie, micrometastases) is rather tedious. Thus, most study groups agreed on sighting 0.5 to 2.0 x 10⁶ mononuclear bone marrow cells. However, 10 mL of bone marrow contains approximately 2.5 to 5 x 10⁷ mononuclear cells, and a complete preparation of all cells would require up to 1,000 microscopic slides. Magnetic activated cell sorting (MACS) is a possible solution to this problem, as it allows processing of large quantities of cells.²¹ Bone marrow cells are exposed to a specific, supramagnetic antibody and then passed through a magnetic field, selecting positive (ie, antibody-bound) cells. Micrometastatic cells can thus be labeled with a magnetic anticytokeratin antibody, singled out from the rest of the probe and finally detected by immunocytochemistry. In recovery experiments with cytokeratin (CK)-positive tumor cell lines and blood, it has been shown that enrichment rates are up to 10,000-fold²² and recovery rates are about 80%.^23,24

We hypothesized that we could increase detection rates of micrometastases in bone marrow of colorectal cancer patients by using MACS and that the occurrence of these cells would correlate with clinical parameters such as tumor stage, grading, tumor markers, and patient characteristics such as sex and age. Therefore, we conducted a prospective, clinical study in patients with newly diagnosed colorectal cancer.

	PATIENTS AND METHODS

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Patients
After informed consent, 51 consecutive patients with newly diagnosed colorectal adenocarcinoma who underwent primary surgery were enrolled onto our prospective trial between August and December 2000. Ten milliliters of bone marrow was obtained by a single puncture of the iliac crest under general anesthesia. Also, a complete oncologic staging was performed on all patients including histopathologic analysis of tumor and lymph nodes; computed tomographic scan of the abdomen; chest x-ray; and the tumor markers carcinoembryonic antigen (CEA), CA 19-9, and CA72-4. Tumors were staged according to the tumor-node-metastasis classification of the UICC.²⁵ UICC stage I disease was diagnosed in 11 patients, stage II disease was diagnosed in 14 patients, stage III disease was diagnosed in 12 patients, and stage IV disease was diagnosed in 14 patients. Two patients had a tumor in the colon and in the rectum. For data analysis, these patients were staged according to the more advanced stage. The sites of metastatic disease in 14 stage IV patients were liver (alone) in seven cases; peritoneum in two; uterus in one; bone and liver in two; and liver, lung, and peritoneum in two cases.

Controls
Bone marrow from 11 patients without any malignancies who underwent cardiothoracic surgery with median sternotomy and from seven patients with nonepithelial hematologic malignancies who received staging biopsies served as controls. All control subjects signed written informed consent. Table 1 lists the characteristics of 51 cancer patients and 18 controls.

Magnetic Labeling
The entire bone marrow was centrifuged for 10 minutes at 300 x g and the supernatant was removed. The cell pellet was resuspended in 40 mL of HEPES-buffered cell culture medium (PAA Laboratories, Inc, Coelbe, Germany) containing 50 to 100 U/mL DNAse I (Sigma-Aldrich, Munich, Germany) and incubated for 30 minutes at room temperature (RT). Afterward, cells were permeabilized for 5 minutes at RT with 5 mL of 0.1% saponin and fixed on addition of 5 mL of 37% formaldehyde (Merck, Haar, Germany) for 45 minutes at RT. Cells were washed again, centrifuged at 300 x g for 10 minutes, and resuspended in a final volume of 600 µL of MACS Cell Stain buffer (Miltenyi Biotec, Bergisch Gladbach, Germany). To block Fc receptors, 200 µL of FcR Blocking Reagent (Miltenyi Biotec) was added. For magnetic enrichment, 200 µL of MACS Cytokeratin MicroBeads (Miltenyi Biotec), containing the magnetically labeled CAM 5.2 antibody directed against cytoplasmatic CK 7 and 8, was added and incubated for 45 minutes at RT. For analysis of CK-expressing tumor cells by immunocytology, 40 µL of FITC-labeled CAM 5.2 antibody (Becton Dickinson, Germany) was added and incubated for 10 minutes at RT. Afterwards, cells were washed and resuspended in 500 µL of buffer. To enable the enzymatic staining reaction later, 5 µL of anti-FITC alkaline phosphatase conjugate (Sigma-Aldrich) was added and incubated for 10 minutes at RT.

Magnetic Enrichment
To remove cell clusters, the suspension was passed through a 30-µm filter (Miltenyi Biotec) and then applied on a MACS MS⁺ column (Miltenyi Biotec) in order to separate CK-positive cells from unlabeled cells. The negative cells were washed off the column with 1.5 mL of buffer solution. Cells within the column were eluted outside the magnetic field with phosphate-buffered saline and counted.

Detection of CK-Positive Cells
The magnetically enriched cell fraction with an average yield of 0.86 x 10⁵ ± 0.3 cells was applied onto glass slides with a cytocentrifuge (Universal 16A, Hettich Zentrifugen, Tuttlingen, Germany). After air-drying overnight, slides were washed once in phosphate-buffered saline for 1 minute and incubated with 50 µL of freshly prepared Sigma Fast Red TR/Naphthol AS-MX substrate solution (Sigma-Aldrich) for 30 minutes at RT. Finally, slides were washed twice in double-distilled water and air-dried.

Statistical Analysis
Slides with the enriched fraction were scanned under light microscopy by two independent observers for CK-positive cells. In case of discrepancy, a consensus was established between the observers by a joined evaluation. The observers (from the Department of Hemato-Oncology) were blinded to all patient characteristics without receiving any information before they had sent their results to the department of surgery to exclude any possible bias. Data from patient characteristics (age and sex), tumor stages, tumor grading, and positivity of serum tumor markers were entered into contingency tables, and comparison between groups for the probability of CK-positive cells were calculated with ² tests with the statistical software SPSS Version 10.0 for Windows (SPSS, Inc, Chicago, IL). For comparison of groups with smaller numbers, Fisher’s exact test was used. The correlations of tumor stages and the presence of CK-positive cells were calculated with a linear trend test (² test, linear-by-linear association), which evaluates the differences of a parameter between groups that follows a hierarchical order. Probabilities less than 5% (P < .05) were considered as statistically significant. To assess significant relationships between tumor markers and CK-positive cells, we performed qualitative statistical analysis with contingency tables (elevated v normal), and quantitative, bivariate correlations procedures for Pearson’s correlation coefficient, Spearman’s rho, and Kendall’s tau-b with their significance levels.

	RESULTS

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We obtained 10 mL of bone marrow from 51 colorectal cancer patients during primary surgery. The average amount of bone marrow cells of the leukocyte lineage was 8.3 x 10⁷ ± 5.5 x 10⁷. CK 7 and 8 was found in 33 (65%, CK-positive) patients and was absent in 18 (35%, CK-negative). In CK-positive patients, the absolute number of CK-expressing cells varied from one to 49, with a mean value of 8.1 ± 10.8 cells and a mean frequency of 3.3 ± 8.0 cells per 10⁷ bone marrow cells (Fig 1).

View larger version (13K): [in this window] [in a new window]   Fig 1. Frequency of CK-positive cells in bone marrow of patients with micrometastatic disease.

Micrometastases and Stage
CK-expressing cells were found in four of 11 (36%) patients with UICC stage I disease, in nine of 14 (64%) patients with stage II disease, in eight of 12 (67%) patients with stage III disease, and in 12 of 14 (86%) patients with stage IV disease, as shown in Fig 2. The increasing occurrence of CK-positive cells with increasing number of tumor stage was calculated with a linear trend test and was statistically significant (P = .016), although the differences between stage II and stage III were not (64% v 67%). CK-positive cells were more frequently found in tumors of higher pT stages than in those of lower stages (linear correlation, P = .045). As stages II and III only differ by the lymph node status (pN0 v pN1) and not by tumor extension (pT), it is obvious that the involvement of lymph nodes does not have any significant effect on the presence of CK-positive cells. The mean values of the absolute numbers of CK-positive cells, which were detected in tumor stages I through IV, were 9.5 ± 11.3 for UICC stage I, 10.8 ± 15.4 for stage II, 9.9 ± 8.9 for stage III, and 4.3 ± 4.9 for stage IV. However, the difference between stage IV and stages I through III was not statistically significant. There was no relationship between CK-positivity and tumor grading (grade 1/2, grade 2, grade 2/3, and grade 3).

View larger version (22K): [in this window] [in a new window]   Fig 2. Percentage of patients with CK-positive bone marrow in correlation with UICC stage. The differences between tumor localizations "colon" and "rectum" were statistically not significant.

View larger version (19K): [in this window] [in a new window]   Fig 3. Number of patients with CK-positive and CK-negative bone marrow in correlation with normal (low) or elevated (high) tumor markers.

	DISCUSSION

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We found that CA 19-9 correlated significantly with the presence of micrometastatic cells. CEA was also more frequently elevated in CK-positive patients than in CK-negative patients, although it did not reach significance (P = .051). There were no differences in the CK groups for CA 72-4. Tumor markers have not been investigated before in micrometastatic colorectal cancer. However, there is one report about prostate-specific antigen, which was evaluated together with CK18-positive cells in bone marrow of 287 prostate cancer patients.³⁰ Micrometastatic disease did not correlate with elevated prostate-specific antigen levels or with other established prognostic factors such as pathologic stage, lymph node status, or perineural infiltration.

Interestingly, data from previous trials differ regarding the presence of micrometastases in various staging attributes. Whereas Schlimok et al²⁶ found significant correlations between micrometastases and tumor extension (pT), lymph node involvement (pN), and stage IV disease (M1), Juhl et al,¹⁹ Lindemann et al,⁸ and Leinung et al⁹ did not. According to our data, the occurrence of epithelial cells in bone marrow correlates with the tumor extension (pT1/2 v pT3/4). This demonstrates that a growing tumor extension increases the chance that single tumor cells are detached from the primary colorectal tumor compound and spread to distant organs like the bone marrow. The lymph node status did not influence the presence of bone marrow micrometastases, because almost as many stage II patients showed CK-expressing cells as stage III patients. However, this could also be because some stage II patients also harbored nodal micrometastases that escaped the attention of routine hematoxylin and eosin staining. Other groups have evaluated the occurrence of isolated tumor cells in lymph nodes of colorectal cancer patients who were free of lymph node metastases according to standard histopathology. Micrometastases were found in 30% to 60% of all patients, but results are contradictory. Whereas Liefers et al³¹ found that the 5-year survival was lowered, when micrometastases were detected by a highly specific nested CEA-specific reverse-transcriptase PCR, two other groups could not show any differences between CK-positive and CK-negative patients with standard immunocytochemistry.^32,33

We found more stage I and II patients positive for micrometastases than we would have anticipated, as only 10% and 20%, respectively, will eventually relapse. Previous studies reported that 43%,⁸ 32%,²⁶ and 40%⁹ of patients with bone marrow tumor cells stayed in remission. Apparently, a great part of micrometastases do not have any influence on the patient’s health. This is not a problem of specificity, because CK-expressing cells were not found in the samples of our control patients. Studies suggest that micrometastatic cells are eventually cleared by immune response or remain in a dormant, nonproliferating stage (G₀ or early G₁ phase), with downregulated or absent proliferation markers such as Ki-67 or p120.^18,34 The status of micrometastases in our study appears to contribute little to the prognostic information derived from clinical stage. However, we have increased the detection sensitivity compared with standard immunocytology while keeping specificity at 100%. In other words, MACS improves current methods to single out and examine micrometastatic cells in colorectal cancer patients, which will help to evaluate intrastage variations in disease-free survival and overall survival of stages I to III independent of the status of micrometastases. With immunomagnetic techniques, it is possible not only to detect CK-expressing cells but to increase their yield for further processing. In combination with cell cultivation methods, as described before,⁹ tumor cell lines can be established from micrometastases, which may help us to understand their biology.

The presence of isolated epithelial cells in bone marrow aspirates has been shown to be an independent prognostic factor for the relapse rate and the survival of many cancer entities.^8,35-39 The origin of these cells has been examined by double-staining cells on coexpressed markers such as the proliferation-associated molecule erbB2,^18,40 K-ras mutations,^41-43 or the epithelial cell adhesion molecule 17-1A,⁴⁴ and were thus associated with the primary tumors. However, for more than 15 years, there has been ongoing, dynamic research in the field of micrometastases, which still receives tremendous scientific attention; nevertheless, its insights have not helped a single cancer patient yet. In most common carcinomas, the information about the micrometastatic status in nonmetastatic patients is quite academic, because there are no possibilities for escalating adjuvant treatment. In breast cancer, for instance, Braun et al⁴⁵ have shown that the effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in the bone marrow of high-risk patients is minimal. High-dose chemotherapy in breast cancer did not fulfil expectations, and it is doubtful that it would have a great effect on less chemosensitive malignancies such as adenocarcinomas.⁴⁶ However, by detecting micrometastatic disease in colorectal cancer, we could identify stage I and II high-risk patients and may be able to improve their prognosis by adjuvant therapies such as fluorouracil and leucovorin. Also, immunotherapeutic approaches will play an important role in the future. It has already been demonstrated that the monoclonal antibody 17-1A (edrecolomab) is capable of reducing or eliminating micrometastases in vivo,⁴⁷ although it failed to prove the promising effects in adjuvant treatment of colorectal cancer patients that had been published earlier⁴⁸ in a large randomized trial. There is a growing necessity for monitoring success in oncologic treatments. Numerous new drugs and protocols are tested every year. Evaluating micrometastatic disease will help us to understand the spread of cancer and the mechanisms behind (sometimes puzzling) relapses. Immunomagnetic enrichment of isolated tumor cells may contribute to diagnostics and improve future staging concepts by its high sensitivity and may support the choice of treatment strategies.

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TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted February 28, 2002; accepted July 22, 2002.

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