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10.1245/s10434-006-9039-3
Annals of Surgical Oncology 14:373-380 (2007)
© 2007 Society of Surgical Oncology
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Original Article

Prognostic Value of Cytokeratin-Positive Bone Marrow Cells of Gastric Cancer Patients

Stephan Gretschel, MD1, Christian Schick1, Ulrike Schneider, MD2, Lope Estevez-Schwarz, MD1, Andreas Bembenek, MD1 and Peter M. Schlag, MD, PhD1

1 Department of Surgery and Surgical Oncology, Charité-Universitätsmedizin Berlin, Campus Buch, Robert-Rössle-Klinik at the Helios Klinikum Berlin, Lindenberger Weg 80, 13125 Berlin, Germany
2 Institute of Pathology, Charité-Universitätsmedizin Berlin, Campus Buch, Robert-Rössle-Klinik at the Helios Klinikum Berlin, Lindenberger Weg 80, 13125 Berlin, Germany

Correspondence: Address correspondence and reprint requests to: Peter M. Schlag, MD, PhD; E-mail: pmschlag{at}charite.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Epithelial cells in the bone marrow of patients with gastric cancer suggest tumor dissemination; however, their prognostic implications are controversial. We prospectively evaluated the correlation of bone marrow findings, recurrence rate, and disease-free survival after long-term follow-up.

Methods: Bone marrow were aspirated from both iliac crests and stained with monoclonal cytokeratin (CK)-18 antibody in 209 patients before their initial operation. Patients were followed up for a median of 56 months.

Results: Overall, 39 (19%) of 209 patients and 15 (14%) of 109 R0-resected patients had CK-positive cells. CK-positive patients had more local, regional, and distant recurrence than CK-negative patients (P < .05). We found a significantly shorter disease-free survival (P < .05) in the patients with >2 CK-positive cells per 2 x 106 bone marrow cells (mean, 35 months) than in patients with ≤2 CK-positive cells per 2 x 106 bone marrow cells (mean, 70 months) or in patients with no CK-positive cells (mean, 86 months). Multivariate analysis confirmed that >2 CK-positive cells per 2 x 106 bone marrow cells was an independent prognostic factor for tumor-related death (P < .05).

Conclusions: Not only the mere presence of CK-positive epithelial cells in bone marrow, but also the cell number, correlates with prognosis. Our findings suggest that classifying CK-positive bone marrow cells in these patients will facilitate future studies.

Key Words: Gastric cancer • Bone marrow • Epithelial cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
More than 30% of gastric cancer patients undergoing potentially curative resection develop recurrence, even without evidence of tumor cell spreading at the initial operation. Presumably, minimal residual disease is responsible for cancer progression and recurrence.1–5 Isolated cytokeratin CK-positive cells, which can be found in lymphatics,6–10 ascites,11–14 blood, and bone marrow of cancer patients, were shown to influence the prognosis of patients with esophageal cancer,15 gastric cancer,16 pancreatic cancer,17 and colorectal cancer.18 Methods of identification of CK-positive cells are immunohistochemistry, flow cytometry, and reverse transcriptase-polymerase chain reaction.19,20 The identification rates of these cells by immunocytochemistry in the bone marrow of gastric cancer patients during primarily curative surgery vary. Different antibodies, varying cytological assessments from pathologists, and short follow-up times of too few patients may be responsible for these differences. We aimed to determine the correlation between CK-positive bone marrow cells and local or regional recurrence and disease-free survival in a large number of gastric cancer patients.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
We included 209 consecutive patients who were treated for gastric cancer from March 1994 through March 1999. After staging with endoscopy, endoscopic ultrasonography, abdominal ultrasonography, computed tomography, and chest radiography, all patients were scheduled for surgical resection or palliation. Written informed consent was obtained from all patients for all procedures on the day before intervention. Bone marrow aspiration was performed with patients under general anesthesia from both iliac crests. To avoid a contamination with dermal epithelial cells, a small skin incision was made before insertion of the 15-gauge aspiration needle (100 mm; Pflugbeil GmbH, Zorneding/München, Germany). Ten milliliters of bone marrow was aspirated from both sides with a syringe containing 5000 IU of heparin solution. A total of 165 of the 209 patients underwent gastric resection. There were 109 gastrectomies, 20 subtotal gastrectomies, 29 proximal gastric resections, and 7 gastric stump resections (after a partial gastrectomy of ulcer disease in 5 cases and in 2 cases of former gastric cancer). The same group of senior surgeons performed all operations. Aside from patients with proximal gastric resections, all other patients underwent D2 lymph node dissection. On average, 32 lymph nodes were analyzed. All specimens were assessed and classified according to the International Union Against Cancer classification system (tumor-node-metastasis).21–23 Forty of 165 patients underwent palliative resection because of distant metastases (pM1). From the remaining 125 patients, 16 patients were classified as R1 resections, and in 109 patients a R0 resection was achieved. None of the R0-resected patients received preoperative or postoperative chemotherapy or irradiation.

Bone Marrow Immunocytochemistry
The mononuclear cells were separated by Ficoll/Hypaque density graduation centrifugation. Subsequently, cytospins were prepared by using a centrifugal system Hettich Universal 16/16R (Andreas Hettich GmbH, Tuttlingen, Germany); 3.5 x 106 cells per patient were prepared for analysis. The assessment in terms of the presence or absence of epithelial cancer cells was performed by immunocytochemistry by using monoclonal anti–CK-18 antibody (Mab CK2; Boehringer Mannheim Pharma GmbH & Co.KG, Mannheim, Germany) and the alkaline phosphatase/anti–alkaline phosphatase technique for 2 x 106 cells, previously described in detail.16,24–27 For a positive control we used the colon cancer cell line SW 480 (consistent expression of CK-18). As isotype control, we replaced the Mab CK2 with an immunoglobulin G1 antibody. We performed a negative control without Mab CK2 and with the alkaline phosphatase/anti–alkaline phosphatase method in every bone marrow specimen.

Haemalaun counterstaining was routinely performed. An experienced pathologist assessed all CK-positive samples for morphological criteria of malignancy. Only those patients with unequivocal findings were assessed as positive. Unspecific positive results were excluded with May-Grünwald-Giemsa counterstaining. We expressed CK-positive cells in terms of 2 x 106 bone marrow cells.

Statistical Analysis
Statistical analyses were performed by using SPSS (version 12.0; SPSS Inc., Chicago, IL). To assess a correlation between bone marrow status and clinicopathologic parameters, we used the {chi}2 with log-rank test. Life-table curves considering disease-free survival were estimated by the Kaplan-Meier method (univariate test).28 To evaluate independent prognostic significance, multivariate analysis was performed by Cox logistic regression (proportional hazards model, stepwise forward method: likelihood ratio and 95% confidence interval) and included the results of the bone marrow immunocytochemistry and the established risk factors.29 The level of significance was P < .05.

Clinical Follow-Up
R0-resected patients were systematically followed up every 3 months for the first 2 years and every 6 months for the following 3 years. Thereafter, the patients were seen yearly. Follow-up examinations included physical examination, laboratory tests with tumor markers (carcinoembryonic antigen, CA19-9, and CA72-4), abdominal ultrasonography, and chest radiograms. If recurrence was suspected, additional imaging methods such as magnetic resonance imaging, multislice computed tomography, or endoscopy, including endoscopic ultrasonography and consecutive biopsy, were performed. Proven recurrence was classified as locoregional or distant. The median follow-up for all patients was 56 months; none was lost to follow-up.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Thirty-nine (19%) of 209 consecutive patients had CK-positive cells (Figs. 1Go and 2Go). Nineteen patients had 1 or 2 epithelial cells in 2 x 106 examined cells, whereas 20 patients had >2 cells. Table 1Go provides clinical and histopathologic characteristics from all 209 patients. Those patients with advanced disease stage according UICC (tendency) and primary distant metastasis (P < .05) more commonly had tumor cells in their bone marrow than patients with limited disease. The CK-positive patients survived a median of only 9 months, compared with 30 months for CK-negative patients (P < .05) for all 209 patients (Fig. 2Go). The limited survival in the CK-positive group was mainly driven by patients with 1–2 CK-positive cells per 2 x 106 bone marrow cells. They had a disease-free survival of only 6 months compared with patients with 1–2 cells, who enjoyed a 23-month disease-free survival (Fig. 3Go).


Figure 1
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  		FIG. 1. Shown is a cytokeratin-positive single cell (arrow on the left side) and small clustered cells (arrow on the right side) in bone marrow of gastric cancer patients by immunohistochemical staining with monoclonal anti–cytokeratin-18 antibody (stain, anti–cytokeratin-18; original magnification, x500).

 

Figure 2
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  		FIG. 2. Disease-free survival of all 209 gastric cancer patients is presented in terms of the identification of cytokeratin (CK)-positive cells in the bone marrow (Kaplan-Meier analysis with log-rank test; median, 9 vs. 30 months; P < .05).

 

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  		TABLE 1. Clinical and histopathologic characteristics of all 209 gastric cancer patients ({chi}2 analysis with Pearson test)
 

Figure 3
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  		FIG. 3. Disease-free survival of all 209 gastric cancer patients is shown in terms of the number of cytokeratin-positive cells per 2 x 106 bone marrow cells (Kaplan-Meier analysis; median, 6 vs. 23 vs. 30 months; P < .05).

 
Fifteen (14%) of the 109 patients who had undergone R0 resections had CK-positive cells in their bone marrow. Clinical and histopathologic features of CK-positive and CK-negative patients showed a homogenous distribution in both groups, as shown in Table 2Go. Nevertheless, bone marrow–positive patients had more local, regional, and systemic recurrences than patients without bone marrow involvement, as shown in Table 3Go. The site of systemic recurrence in CK-positive patients varied (3 peritoneal, 4 juxtare-gional lymph nodes, 1 bone, 1 lung, and 2 pleura).


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  		TABLE 2. Clinical and histopathologic characteristics of 109 curatively resected gastric cancer patients (R0; M0) given in terms of CK-positive cells
 

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  		TABLE 3. Local, regional, and distant recurrence during follow-up are reported in 109 curatively resected gastric cancer patients (R0; M0; {chi}2 analysis; Pearson test)
 
In the 109 R0-resected patients, we found a tendency toward a decreased survival in all CK-positive patients (Fig. 4Go). However, patients with more than two CK-positive cells revealed a disease free survival of 35 months, compared with 70 months for those with fewer than two CK-positive cells or 86 months for those without CK-positive cells (Fig. 5Go). In a multivariate Cox regression, shown in Table 4Go, lymphatic vessel invasion, depth of tumor infiltration, and the presence of >2 CK-positive cells per 2 x 106 bone marrow cells were identified as independent risk factors for tumor-related death.


Figure 4
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  		FIG. 4. Disease-free survival of 109 curatively resected gastric cancer patients (R0; M0) is presented in terms of the identification of cytokeratin CK-positive cells in the bone marrow (Kaplan-Meier analysis with log-rank test; mean, 57 vs. 86 months; P = .0824).

 

Figure 5
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  		FIG. 5. Disease-free survival of 109 curatively resected gastric cancer patients (R0; M0) is shown in terms of the number of cytokeratin CK-positive cells per 2 x 106 bone marrow cells (Kaplan-Meier analysis with log-rank test; mean, 35 vs. 70 vs. 86 months; P < .05).

 

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  		TABLE 4. Cox regression multivariate analysis of factors affecting disease-free survival after curative resection of gastric cancer patients (R0; M0) given in terms of CK-positive cells in 2 x 106 bone marrow cells
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We found that CK-positive bone marrow cells were significant markers of prognosis in this large cohort of gastric cancer patients. Similar to earlier reports,30,31 we observed that CK-positive bone marrow cells correlated with metastases. However, our patient cohort was substantially larger than those in earlier reports. We demonstrated a significantly shorter disease-free survival for patients with CK-positive bone marrow. The effect of a positive bone marrow was significant for all 209 patients, as well as for the 109 patients who could undergo R0 resections. More than two CK-positive cells (in the smaller R0-resected group) were a significant prognostic factor in the multivariate analysis. These findings are in accordance with earlier results reported by Jauch et al.32 The R0 resection is demonstrable as a strong positive prognostic factor. As a result, these 109 patients required more CK-positive cells to negatively influence the oncological outcome, compared with the total group of 209 patients.

The nodal status (pN) was a significant factor in the univariate analysis, even though pN was not significant in the multivariate analysis with regard to the R0-resected group. The influence of a radical lymphadenectomy is inconclusive at this point. We can conclude that according to the multivariate analysis, the significant effect of nodal status is lost when all other variables are considered. The same phenomenon was observed recently by Macadam and colleagues33 in their work on bone marrow micrometastases with esophageal and gastric carcinomas.

The frequency of CK-positive cells (19%) in the patients with gastric cancer of this study was within the lower scope of earlier reports (15%–88%) with similar immunocytological methods.16,30–32,34–41 The wide range of results may be explained by different aspiration sites with varying blood flow (ribs vs. iliac crest),42,43 differing antibodies, or different morphological criteria of CK-positive cells.11,17,33,44–46

Our results revealed a significant correlation of a positive bone marrow status to the presence of local and distant recurrence as well as to the time for distant relapse in the R0-resected patients. However, the site of systemic recurrences was variable. This state of affairs may indicate that CK-positive cells are not responsible for the location of metastases, but rather are a surrogate marker for the aggressiveness of any particular tumor.33,45 One approach to elucidate the relationship between the presence of CK-positive bone marrow cells and distant tumor relapse is the detailed characterization of the cell phenotype and its genetic alterations. The search for parameters to characterize attributes of malignancy in these cells revealed several molecular markers, including the loss of HLA class 1 antigens, expression of urokinase plasminogen activator receptor, overexpression of the HER-2 oncogene, or proliferation-associated molecules such as p120 and Ki-67.36,38,39,47–49 As a second step, the phenotypic profile of these cells will be characterized in detail to clarify the biological effect of CK-positive cells.

The perspective of our study is that CK-positive cells in the bone marrow indicate a worsened prognosis in gastric cancer patients. There is evidence that these cells are not influenced by current chemotherapy and are instead dormant. Alternatively, they might be accessible to an antibody-based therapy.50,51 Possibly, patients with CK-positive bone marrow cells who have no other overt metastases are candidates for novel adjuvant therapies. Further studies considering molecular cell characterization and new adjuvant therapeutic approaches will address this point.


   ACKNOWLEDGMENTS
 
The authors thank Markus Niederstrasser for his assistance in statistical analysis.

Received for publication November 2, 2005. Accepted for publication April 5, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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