Annals of Surgical Oncology Cite Track
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10.1245/s10434-006-9051-7
Annals of Surgical Oncology 14:810-817 (2007)
© 2007 Society of Surgical Oncology
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Original Article

Detection of Disseminated Tumor Cells in Liver Biopsies of Colorectal Cancer Patients Is not Associated with a Worse Prognosis

Moritz Koch, MD, Peter Kienle, MD, Emily Logan, Dalibor Antolovic, MD, Luis Galindo, MD, Friedrich Hubertus Schmitz-Winnenthal, MD, Jan Schmidt, MD, Christian Herfarth, MD and Jürgen Weitz, MD, PhD

Department of Surgery, University of Heidelberg, INF 110, 69120, Heidelberg, Germany

Correspondence: Address correspondence and reprint requests to: Jürgen Weitz, MD, PhD; E-mail: juergen.weitz{at}med.uni-heidelberg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Liver metastases occur frequently in colorectal cancer and are probably caused by disseminated tumor cells having been trapped in the liver. The prognostic significance of hematogenous tumor cell dissemination has already been demonstrated for blood and bone marrow of patients with colorectal cancer. The aim of this study was to investigate the frequency and prognostic significance of disseminated tumor cells in liver biopsies of colorectal cancer patients.

Methods: Liver biopsies from 100 patients with UICC stage I–III colorectal cancer were taken prospectively during resection of the primary tumor. Liver biopsies obtained from 16 patients with benign gastrointestinal diseases served as negative controls. Liver samples from seven patients with liver cirrhosis were additionally taken. Liver biopsies were examined using a reverse transcriptase (RT)-PCR assay to amplify cytokeratin (CK) 20 transcripts. The median follow-up of the patients was 55 months.

Results: Disseminated tumor cells were detected in liver samples of 10/100 (10%) patients with UICC stage I–III colorectal cancer. Liver specimens from all seven patients with liver cirrhosis were CK 20-positive, whereas 16 patients with other benign gastrointestinal diseases were all CK 20-negative. There was no correlation between tumor cell detection in liver biopsies and survival of the patients. The only significant prognostic factor on uni- and multivariate analysis was the UICC stage.

Conclusions: This study demonstrates that detection of disseminated tumor cells in liver samples from patients with UICC stage I–III colorectal cancer has no prognostic influence. UICC classification was the strongest prognostic factor in this patient series.

Key Words: Disseminated tumor cells • Liver biopsy • Colorectal cancer • Prognosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Distant metastasis is a frequent clinical phenomenon in colorectal cancer and occurs predominantly in the liver.1 Approximately 15–30% of colorectal cancer patients will develop liver metastases after curative resection of the primary tumor.2 Metachronous liver metastasis is probably caused by hematogenous tumor cell dissemination. The detection of disseminated tumor cells with newly developed molecular assays is increasingly being used for predicting the individual prognosis of patients with gastrointestinal cancer.3,4 The presence of disseminated tumor cells has been investigated in various organs and body compartments – for example, bone marrow, blood, and lymph nodes – in patients with colorectal cancer,1 and several studies using different molecular detection methods have confirmed their prognostic significance in different body compartments.4 Cytokeratin 20 (CK 20) is ubiquitously expressed in colorectal cancer,5 and we have previously shown that CK 20 reverse transcriptase (RT)-PCR is a reliable assay to detect disseminated colorectal cancer cells.6,7 Furthermore, our group has demonstrated that the detection of disseminated tumor cells by this assay in central venous blood and bone marrow samples of patients undergoing surgery for primary tumor or liver metastases from colorectal cancer is associated with a worse prognosis.8,9

Although the liver is the most common and primary site of metastases in patients with colorectal cancer, the incidence and prognostic relevance of disseminated tumor cells in this organ have not yet been adequately investigated.10 The results of human and animal studies suggest that circulating colorectal cancer cells being shed into the mesenteric venous blood from the primary tumor could be retained by filter effects in the liver.11,12 In another study, liver biopsies from colorectal cancer patients with and without clinically detectable liver metastasis were examined for the presence of disseminated tumor cells using different molecular detection assays.13 However, due to the heterogeneity within the examined patient groups and the wide variety of detection methods used no definitive conclusion regarding the clinical significance of disseminated colorectal cancer cells in the liver could be drawn.10 To date, there has been no published study of the long-term prognostic significance of disseminated tumor cells in the liver of colorectal cancer patients.

The aim of this prospective study was to clarify the frequency and prognostic significance of disseminated tumor cells in liver specimens of colorectal cancer patients without clinically detectable metastasis (i.e. UICC stage I–III).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
A total of 100 patients with colorectal cancer were prospectively enrolled into our study between October 1996 and October 1998. Patients with colorectal cancer and without distant metastases (colorectal cancer UICC stage I–III) undergoing subsequent surgical resection of the primary tumor at the Department of Surgery, University of Heidelberg were eligible for this study. Only patients with a curative resection (R0) were included. Informed consent was obtained from all patients. The study protocol was approved by the ethics committee of the University of Heidelberg. Participation did not delay or change the treatment of the patients. Patients with liver cirrhosis (due to known CK 20 expression in liver biopsies from cirrhotic liver tissue) and patients with other malignant diseases in their medical history were excluded, as were patients with neoadjuvant therapy as we could demonstrate a significantly reduced detection rate of disseminated tumor cells in blood and bone marrow samples of rectal cancer patients after preoperative chemoradiation.14 Tumor stage and grading were classified according to the fifth edition of the TNM classification of the International Union Against Cancer (UICC).15 According to recommendations for colorectal cancer valid at the time patients were included in the study, adjuvant chemotherapy was recommended for patients with stage III colon cancer only and not for patients with lower tumor stages. For patients with stage II and III rectal cancer adjuvant chemoradiation was generally recommended. Adjuvant therapy was for the most part performed at the discretion of the local oncologist. Altogether, 16 patients with rectal cancer underwent adjuvant chemoradiation, and 25 patients received adjuvant chemotherapy.

The routine preoperative workup of the patients for examining distant metastasis included a chest X-ray [and, in the case of suspicious lesions, an additional computed tomography (CT) scan], abdominal ultrasound, and/or CT scan of the abdomen. The liver was inspected and palpated intraoperatively. Intraoperative hepatic ultrasound was performed in the case of a suspicious lesion on the preoperative images or a pathologic finding intraoperatively.

Control Group
Liver tissue biopsies from 16 patients undergoing abdominal surgery for benign gastrointestinal diseases (e.g., liver adenoma, colorectal adenoma, sigmoid diverticulitis) served as negative controls. Additionally, liver biopsies from seven patients with liver cirrhosis undergoing laparotomy were examined.

Tissue Sampling
During the operation liver specimens were taken from the right and left liver lobe (segments 4b and 5) using a commercially available biopsy needle system (Tru-Cut; Allegiance., McGaw Park, Illinois). The weight of the liver specimens was about 20–30 mg. All tissues were immediately stored in cryotubes under sterile conditions in the operating room, shock frozen in liquid nitrogen, and stored at –80°C.

RNA Isolation
RNA extraction was done using the commercially available Qiagen RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Liver tissue was mechanically minced with a scalpel under sterile conditions and further homogenized with lysis buffer before starting the RNA extraction.

CK 20-RT-PCR
CK 20 RT-PCR was performed as previously described.7 PCR products were analyzed by electrophoresis on 2% agarose gels. The sensitivity of the CK 20-RT-PCR assay was determined in preliminary cell spiking experiments and was found to be able to detect ten HT 29 cells in 10 ml of blood.6 Further data regarding the sensitivity of the CK 20 RT-PCR assay to detect tumor cells in liver homogenates are not available as there is currently no experimental protocol available by which to precisely calculate the absolute number of cells in a defined amount of liver tissue without destroying the cells.

The quality and performance of the reverse transcription of all RNA samples analyzed were con-firmed by RT-PCR amplification of glyceraldehyde phosphate dehydrogenase (GAPDH) transcripts, as previously described.6

Follow-up and Statistical Analysis
All patients were primarily included into the follow-up. Statistical computations were performed with the software package JMP (JMP, Cary, North Carolina). The length of the follow-up was calculated using the date of surgery at our institution as the starting point. Survival was estimated according to the Kaplan-Meier method and compared using the log-rank and Wilcoxon tests.16 A multivariate proportional hazards model was built using the variables that had prognostic potential as suggested by univariate analysis (i.e., P < 0.05).17 Statistical significance was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Control Group
All liver samples from the 16 patients undergoing surgery for benign gastrointestinal diseases (e.g., liver adenoma, colorectal adenoma, sigmoid diverticulitis) consistently tested negative for CK 20 expression. The GAPDH RT-PCR was positive in every sample, thereby confirming adequate RNA quality and reverse transcription.

Liver Cirrhosis Group
Of the seven patients with liver cirrhosis, all seven (100%) tested positive for CK 20 expression in their liver biopsies, indicating a de novo expression of CK 20 in liver cirrhosis, as previously described.18

Study Group
One hundred patients undergoing curative (R0) resection for UICC stage I–III colorectal cancer at the Department of Surgery, University of Heidelberg (59 males, 41 females) were included in this analysis. Median age of the patients was 63 years (interquartile range: 57–71 years). Routine histopathology confirmed the presence of colorectal adenocarcinoma in all patients. According to the UICC classification for the 100 patients, 32 were in stage I, 40 in stage II, and 28 in stage III. Primary tumors were located in the rectum in 49 patients and in the colon in 51 patients. A summary of the patients‘ characteristics is given in Table 1Go.


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  		TABLE 1. Distribution of clinical, pathological, and treatment factors in 100 patients with colorectal cancer UICC stage I–III having undergone intraoperative liver biopsy for detection of disseminated tumor cells
 
Tumor cells were detected in liver samples of ten of the 100 patients: four patients were in UICC stage I (40%), five were in stage II (50%), and one patient was in stage III (10%). Table 2Go summarizes all data in regard to tumor cell detection in the analyzed liver biopsies.


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  		TABLE 2. Correlation of tumor cell detection in liver biopsies with UICC stage
 
Follow-up
The median follow-up of all patients was 55 months (interquartile range: 46–61 months), and for surviving patients 58 months (interquartile range: 50–62 months).

For all of the 100 patients (with and without tumor cells in liver specimens) the 5- year overall survival (OS) rate was 78%, the 5-year disease-specific survival (DSS) rate was 84%, the 5-year recurrence-free survival (RFS) rate was 74%, the 5-year distant recurrence-free survival (DRFS) rate was 78%, and 5-year liver metastases-free survival (LMFS) rate was 87%.

The 5-year OS of patients with tumor cells in the liver biopsies was 80% compared to 77% in patients without tumor cell detection in liver specimens. This difference was statistically not significant. All other survival rates (DSS, RFS, DRFS, and LMFS) did also not differ significantly when patients with CK 20-positive liver biopsies were compared to those with negative-liver biopsies. Figure 1Go shows the effect of tumor cell detection in liver biopsies on DSS [P = not significant (N.S.)].


Figure 1
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  		FIG. 1. Influence of tumor cell detection in liver biopsies on disease-specific survival in 100 colorectal cancer patients (P = N.S.).

 
The univariate analysis of all prognostic factors in our patient cohort is depicted in Table 3Go. Only the T-category, N-Category, and adjuvant chemotherapy were revealed to be significant prognostic factors. The significant influence of UICC stage on DSS is shown in Fig. 2Go (P < 0.001). Multivariate analysis of prognostic factors was performed for RFS, DRFS, and LMFS because only here was more than one factor significant on univariate analysis. As both the T- and N-categories are components of the UICC stage classification, UICC tumor stage was used in the multivariate analysis (see Table 4Go). UICC tumor stage remained the only significant prognostic factor on multivariate analysis.


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  		TABLE 3. Univariate analysis of prognostic factors in 100 patients with colorectal cancer UICC stage I–III having undergone intraoperative liver biopsy for detection of disseminated tumor cells
 

Figure 2
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  		FIG. 2. Influence of UICC stage on disease-specific survival (P < 0.001).

 

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  		TABLE 4. Multivariate analysis of prognostic factors in 100 patients with colorectal cancer UICC stage I–III. Only variables with P < 0.05 on univariate analysis were entered into multivariate analysis
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Metachronous liver metastasis is probably caused by disseminated tumor cells being spread from colorectal cancer into the circulatory system and subsequently being entrapped in the liver. Disseminated tumor cells could also reside in the bone marrow as a reservoir from where they may later disseminate to distant organs.19 It therefore seems logical to examine blood and bone marrow for the presence of disseminated tumor cells as a means of predicting the individual risk of a colorectal cancer patient for developing tumor recurrence. In support of this hypothesis, several studies using sensitive and specific molecular detection methods have already shown that the presence of disseminated tumor cells [also called minimal residual disease (MRD)] in blood and bone marrow of colorectal cancer patients may have a prognostic value.8,9,20–23 Despite the very encouraging results to date, further studies, including larger patient cohorts and methodological standardization of the different detection methods, are needed before these diagnostic techniques and the concept of MRD can be implemented into clinical routine.4

In addition to the above-mentioned body compartments, the liver seems to be an ideal organ for detecting MRD in colorectal cancer as all tumor-draining portal venous blood flows through the liver. In a previous work we demonstrated a possible filtering function of the liver for circulating tumor cells in mesenteric venous blood.12 Interestingly, only a few studies have to date analyzed the incidence and prognostic significance of disseminated tumor cells in liver specimens from patients with colorectal cancer.10

In this study, we investigated the extent and prognostic significance of disseminated tumor cells in liver biopsies taken intraoperatively from colorectal cancer patients (UICC stage I–III) without clinically overt metastasis. Disseminated tumor cells were found in ten of the 100 patients examined by CK 20 RT-PCR. Our detection rate is relatively low compared to previously published data by other groups where the detection rates of disseminated tumor cells ranged between 5 and 69%.10 The low detection rate of disseminated tumor cells in the liver found in our analysis may partly be explained by the fact that in the present study only patients without overt liver metastasis (UICC stage I–III) were included. In contrast to this, all other published studies also included patients with liver metastases (UICC stage IV), which significantly increased the detection rate of disseminated tumor cells in the liver. As we were only interested in the question of whether the detection of disseminated tumor cells in the liver by means of CK 20 RT-PCR is a potential prognostic predictor for later tumor recurrence and/or metastasis, we did not include patients with overt liver metastasis. Additionally, 72% of our patient cohort comprised patients with an early tumor stage (UICC stages I and II). Therefore, our study is the first larger homogenous analysis of liver biopsies from colorectal cancer patients without clinically detectable liver metastasis. Another possible reason for a low detection rate may be the lack of sensitivity of our detection method, although this is quite unlikely as we have already demonstrated the high sensitivity of our CK 20 RT-PCR method in previous studies.6–8,12,24 The low detection rate may also be explained by a sampling error as we obtained only a very small portion of liver tissue for the detection of disseminated tumor cells. Since the liver weighs approximately 1500 g and our liver biopsies were only about 30 mg, we essentially examined only 0.002% of the whole liver tissue for the presence of disseminated tumor cells. In order to further enhance the detection rate of disseminated tumor cells in liver biopsies, we therefore combined several detection methods. However, the utility of the data available on the prognostic impact of this multiple marker approach remains inconclusive.25

The specificity of CK 20 RT-PCR for detecting disseminated tumor cells in liver tissue is an important issue as a previous study showed frequent expression of CK 20 in liver biopsies taken from patients without malignant disease.26 On the other hand, several immunohistological analyses showed no or negligible levels of CK 20 expression in normal liver tissue.5,18,27 A possible explanation for these conflicting results may lie in the fact that there is a known de novo expression of nonhepatocellular cytokeratins – for example, CK 20 – in Mallory body formation observed predominantly in alcoholic liver disease.18 Indeed, we found CK 20 expression in all of the liver specimens we examined from patients with liver cirrhosis and in none of the liver samples from patients with other benign gastrointestinal diseases.

During the follow-up of our patient cohort we did not find that the presence of disseminated tumor cells in liver biopsies taken intraoperatively from patients with colorectal cancer UICC stage I–III were prognostic. Multivariate analysis revealed only a single independent prognostic marker in our patient cohort – the tumor (UICC) stage of the primary tumor. As UICC stage is one of the strongest and best-established prognostic factors in patients with colorectal cancer, this result underlines the validity and reliability of our follow-up data. Data from previous studies regarding the prognostic influence of disseminated tumor cells in the liver of colorectal cancer patients are controversial as all of these analyses included patients with overt liver metastasis.10 Since liver metastasis per se is a major prognostic factor for poor outcome in colorectal cancer, it is difficult to examine the prognostic value of disseminated tumor cells in normal liver samples taken from patients with liver metastasis. Two studies aimed at determining the prognostic significance of disseminated tumor cells in normal liver biopsies taken solely from colorectal cancer patients with clinically overt liver metastasis (UICC stage IV) obtained contradictory results. Topal et al.28 did not find a prognostic influence when using CK 20 and carcinoembryonic antigen (CEA) as detection markers, whereas Yokoyama et al.29 demonstrated a significantly poorer prognosis and increased risk of intrahepatic recurrence for patients with immunohistochemically detected hepatic micrometastases surrounding liver metastases. Linnemann et al. examined 54 colorectal cancer patients with tumor(s) in different stages (UICC stage I–IV) for the presence and prognostic significance of disseminated tumor cells in the liver and was also able to demonstrate a prognostic significance for the whole patient cohort.30 However, after the exclusion of patients with overt liver metastasis (UICC stage IV), these researchers found that the detection of disseminated colorectal cancer cells was no longer a significant prognostic factor. Multivariate analysis of prognostic factors has not been included in any of the studies published to date, but this is an essential step in demonstrating the independence of the new prognostic marker being evaluated from other prognostic parameters.

We did not perform a power calculation for the number of subjects that would have been required to demonstrate a survival difference between the two groups (with or without tumor cells in liver biopsy). The reason for this is that there are no reliable or comparable data in the literature relating to the incidence and prognostic relevance of disseminated tumor cells in the liver on which an adequate sample size calculation could be based. Therefore, we cannot exclude the possibility that our study was underpowered.

In conclusion, the present study is the first analysis on the prognostic significance of disseminated colorectal cancer cells in the liver to include long-term-follow-up data and multivariate analysis of prognostic factors. Our data indicate that the detection of disseminated tumor cells in the liver is not of general prognostic value in colorectal cancer patients. The detection of hematogenous tumor cell dissemination in the blood and bone marrow of patients with colorectal cancer is probably better suited for the prediction of an individual patient’s prognosis.


   FOOTNOTES
 
The first two authors contributed equally to this study.

Received for publication February 6, 2006. Accepted for publication May 2, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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