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Discordance Between K-ras Mutations in Bone Marrow Micrometastases and the Primary Tumor in Colorectal Cancer

From the Fondation Pour Recherches Médicales, Geneva, Switzerland; Departments of Surgery and Pathology, Carl-Thiem-Klinikum, Cottbus, and Department of Surgery, Otto-von-Guericke University, Magdeburg, Germany; and Institut de Recerca Oncològica, Hospital Duran i Reynals, Barcelona, Spain.

Address reprint requests to Marc A. Reymond, MD, Department of Surgery, Otto-von-Guericke University, Leipziger Str 44, D-39120 Magdeburg, Germany; email: marc.reymond{at}medizin.uni-magdeburg.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To study bone marrow micrometastases from colorectal cancer patients for the presence of K-ras mutations and to compare their genotype with that of the corresponding primary tumor.

PATIENTS AND METHODS: Bilateral iliac crest aspiration was performed in 51 patients undergoing surgery for colorectal cancer, and bone marrow micrometastases were detected by immunohistochemistry. The presence of K-ras mutations was determined by single-strand conformation polymorphism analysis on both primary tumors and paired bone marrow samples and was confirmed by sequencing.

RESULTS: In six patients with primary tumor mutations, it was possible to amplify a mutated K-ras gene also from the bone marrow sample. In three of those patients the pattern of K-ras mutations differed between both samples, in two patients the mutation was identical between the bone marrow and its primary tumor, and in one patient the same mutation plus a different one were found. Fifteen of 17 K-ras mutations found in primary tumors were located in codon 12, whereas in bone marrow, five of seven mutations were found in codon 13 (P = .003).

CONCLUSION: Our results demonstrate that, at least for K-ras mutations, disseminated epithelial cells are not always clonal with the primary tumor and they question the malignant genotype of bone marrow micrometastases. They also indicate that different tumoral clones may be circulating simultaneously or sequentially in the same patient. Analysis of the type of mutations suggests that cell dissemination might be an early event in colorectal carcinogenesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
COLORECTAL CANCER is one of the leading causes of death in developed countries. Most deaths related to colorectal cancer are caused by metastasis, a complex multistage process by which tumor cells escape from the primary tumor and establish secondary foci at distant sites.¹ The identification of patients at risk of developing distant metastases is fundamental for the management and prognosis of the disease, but it is not always easy. For instance, a significant proportion of patients undergoing curative (R0) surgery for early-stage rectal cancer—eg, 8% in stage I and 16% in stage II—develop distant metastases in the absence of local recurrence.² This suggests that, at least in those cases, metastases are caused by tumor cells that had already disseminated but could not be detected at the time of diagnosis and surgical removal of the primary tumor.

Current clinical detection of metastases by standard imaging techniques or serum marker assays is limited to those with a minimal number of cells. Thus in recent years many studies have approached the detection of micrometastases in the lymph nodes,³ the blood, and the bone marrow of colorectal cancer patients using more sensitive techniques.^4-8 The great majority have used immunochemistry or reverse transcriptase polymerase chain reaction to detect either specific epithelial markers (eg, cytokeratins) or nonspecific tumor markers (eg, carcinoembryonic antigen, CD44, and so on) as an indication of tumoral dissemination. However, because of the clonal origin of colorectal cancer,⁹ the best confirmation that disseminated cells are indeed derived from the primary tumor and can potentially develop into distant metastases is to demonstrate their clonality, ie, that they show the same mutations as the tumor of origin.

The bone marrow is generally viewed as an optimal site to look for metastases because it is an accessible and rich cellular site with a large blood supply and because epithelial deposits can be easily distinguishable from mesenchymal tissues.¹⁰ However, neither the bone nor the bone marrow are characteristic sites of metastases in colorectal cancer. Therefore, the demonstration that putative bone-marrow micrometastases are clonal with regard to the primary tumor is fundamental to decide on the usefulness of bone marrow material for prognosis.

We have for the first time assessed the clonality of the epithelial cells found in bone marrow aspirates from colorectal cancer patients based on the somatic mutations of K-ras present in the corresponding primary tumors. We based our study on the analysis of K-ras because this gene is mutated in a significant proportion of colorectal tumors and has the advantage that mutations occur mainly in two of its codons,^11,12 a fact that greatly simplifies the analysis. Mutations at codons 12 and 13 of the human K-ras gene were analyzed by the single-strand conformation polymorphism (SSCP) method¹³ and confirmed by sequencing. When necessary, an enriched SSCP (E-SSCP) approach¹⁴ was used to increase the sensitivity of detection of mutated alleles.

	PATIENTS AND METHODS

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Patients
Fifty-one patients underwent operations for colorectal cancer at the Carl-Thiem Academic Hospital in Cottbus, Germany, between October 1997 and June 1998 and were consecutively entered onto this trial. Diagnosis was confirmed in all cases by standard histopathologic analysis, and tumors were staged according to the tumor-node-metastasis classification of the International Union Against Cancer.¹⁵ Written informed consent was obtained from each patient. Data were collected prospectively and regularly entered into a computer database. Characteristics of the patients are listed in Table 1.

Epithelial Cell Purification and K-ras Analysis
Epithelial cells from all normal mucosa, tumors, and bone marrow samples were purified using immunomagnetic beads (anti-Epithelial Cell Dynabeads) coated with the epithelial-specific monoclonal antibody Ber-EP4 (Deutsche Dynal GmbH, Hamburg, Germany), as previously described.¹⁷ Genomic DNA was extracted using conventional methods and a 241–base pair fragment of K-ras including codons 12 and 13 was amplified from each sample in a single polymerase chain reaction (PCR) using primers K1-U-SO (5'-GGT GGA GTA TTT GAT AGT GTA-3') and K1-D-SO (5'-GGT CCT GCA CCA GTA ATA TGC A-3'). For that purpose, 1 to 10 µL of the isolated genomic DNA was added to a tube containing 1 unit of Taq DNA polymerase (Boehringer Mannheim Gmbh, Mannheim, Germany), 4 µmol/L of each primer, 100 µmol/L of each dNTP, 2 µCi of alpha phosphorus-32 dCTP and PCR buffer (10 mmol/L of Tris-HCl, pH 7.8, 50 mmol/L of KCl, 1.5 mmol/L of MgCl₂, and 0.01% gelatin) in a final volume of 25 µL. The reaction was carried out for 40 to 45 cycles in a thermal cycler (Gene Amp 2400 PCR System; Perkin-Elmer Europe, Rotkreuz, Switzerland) at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 45 seconds. For the SSCP analysis, the resulting radioactive PCR products were diluted five-fold in a formamide-dye loading buffer¹⁸ and denatured for 10 minutes at 95°C. Tubes were cooled on ice and 1.5 to 3 µL each loaded on a 8% polyacrylamide, nondenaturing sequencing gel. Electrophoresis was carried out at room temperature for 12 minutes at 30 W and 18 hours at 6 W. Gels were dried under vacuum at 80°C and exposed to an x-ray film at room temperature without intensifier screen. In the case of bone marrow samples, DNA fragments corresponding to the different mutated bands (as seen in the tumors) were isolated from the SSCP gels, eluted in water, and used in a second round of PCR amplification and SSCP analysis, similar to previously described.¹⁴ Correct isolation of the SSCP bands was always verified by re-exposure of the gels.

To confirm the SSCP results, a volume corresponding to 1% to 2% of the PCR products, previously purified from primers and salts using the Qiaquick PCR Purification Kit (Qiagen, Basel, Switzerland), was used in each case as a template for sequencing. Reactions were performed with primer K1-U-SO using the Thermo Sequenase radiolabeled terminator cycle sequencing kit and alpha phosphorus-33 ddNTPs from Amersham-Pharmacia Biotech (Cleveland, OH) following the manufacturer’s instructions. Amplification was performed for 30 cycles under the same conditions as the PCR. The final product was diluted two-fold in formamide-dye loading buffer, denatured for 10 minutes at 95°C, and cooled on ice. Three microliters of each diluted product were analyzed in a denaturing 6% polyacrylamide, 8 mol/L of urea, glycerol-tolerant sequencing gel for 3.5 hours at 60 W. Finally, gels were dried under vacuum at 80°C and exposed to an x-ray film at room temperature without intensifier screen.

Statistical Analysis
Comparison of the type of mutations between groups (primary tumors v bone marrow) was performed using the ² test, the H0 hypothesis being that if bone marrow micrometastases were clonal with the primary tumor, the proportion of mutations in codon 12 versus codon 13 would be the same in both groups. A P value less than .05 was considered significant.

	RESULTS

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Using Ber-EP4 and pan-cytokeratin A45-B/B3 antibodies, epithelial cells were demonstrated in the bone marrow of 24 patients (47%) (Fig 1). Mutations in K-ras were detected in 17 of 51 primary tumors analyzed (33%). In 13 of 17 patients with primary tumor mutations, a fragment of K-ras could also be amplified from the paired bone marrow sample. Six of these samples depicted a mutation by E-SSCP (examples in Fig 2).

View larger version (118K): [in this window] [in a new window]   Fig 1. Ber-EP4 immunohistochemistry of a bone marrow aspirate showing the presence of a circulating tumor cell. Alkaline phosphatase anti–alkaline phosphatase technique, original magnification x400.

View larger version (157K): [in this window] [in a new window]   Fig 2. SSCP and E-SSCP analysis of K-ras mutations in normal mucosa (N), tumor (T), and bone marrow (BM) samples from four different patients. E-BM corresponds to the previous bone marrow sample after enrichment in the potentially mutated fragments.

View larger version (48K): [in this window] [in a new window]   Fig 3. Sequencing of mutations in tumor (T) and enriched bone marrow (EBM) samples from two cases showing (3a) the same mutations and (3b) different mutations.

In all the cases with K-ras mutations in the bone marrow, the immunocytochemical analysis of bone marrow aspirates showed the presence of epithelial cells, whereas two more cases with primary tumor mutations and positive by immunocytochemistry showed no K-ras mutations in the bone marrow (Table 2). None of the cases with primary tumor mutations but negative by immunocytochemistry appeared positive by SSCP or E-SSCP.

Of 17 primary tumor mutations, 15 (88%) were located in codon 12 versus two in codon 13, which significantly differs from the proportion observed in bone marrow, where only two of seven mutations (29%) were located in codon 12 versus five in codon 13 (Pearson ²₁ = 8.54, P = .003).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
In 1992, a group of German surgeons and pathologists claimed that the presence of immunohistochemically defined epithelial cells in bone marrow constitutes an independent, significant determinant of relapse in colorectal cancer.¹⁹ In the meantime, tumor cells have been detected at a molecular level not only in bone marrow, but also in blood and lymph nodes,^4-8 calling for a new definition of local and systemic disease in colorectal cancer.^20,21

In clinical practice, the independent prognostic significance of disseminated tumor cells is still debated.²² In a meta-analysis performed on different types of cancer, positive bone marrow micrometastases status was found to be an independent predictor of short disease-free survival in five of 11 studies and an independent prognostic factor of poor overall survival in only two studies.²³ These results are largely due to problems of definition between disseminated tumor cells and micrometastases,²⁴ although the heterogeneity of the studies greatly contributes to the controversy. To enable comparison of results and avoid variations in staging, the International Union Against Cancer proposed recently that any finding of disseminated tumor cells should be documented, but according to uniform criteria and only for study purposes.²²

In colorectal cancer, the true metastatic potential of tumor cells in bone marrow should be questioned, as the skeleton is a rare site of overt metastasis in those patients.²⁵ To resolve the clinically relevant questions of metastatic potential and prognostic value, molecular studies addressing the origin and clonality of disseminated tumor cells are necessary.

Colorectal cancers are clonal tumors,⁹ their major clonal expansion occurring after the acquisition of mutations in genes such as K-ras and p53.²⁶ Metastases from colorectal cancer patients are also clonal, and they carry the same K-ras and p53 mutations as the primary tumor.^26-30 Only few studies have described the presence of de novo mutations in metastases derived from colorectal carcinomas or the absence in the distant recurrence of mutations present in the primary tumor.^27,30 It thus seems that K-ras mutations are stable throughout the progression of human colorectal cancer,²⁸ making them useful molecular markers in clonality studies.

Here we report for the first time—to our knowledge—the clonal analysis of bone marrow micrometastases from colorectal cancer patients based on the somatic mutations of K-ras found in paired primary tumors. To our surprise, the experimental results show clearly that tumor cells disseminated to the bone marrow do not always carry the same mutation as the primary tumor. This means that their clonal origin has to be questioned, at least in a subset of patients. Effectively, although we cannot guarantee the homogeneous distribution of the K-ras mutation in the primary tumor, the relative abundance of the mutated allele versus the normal one, as assessed in the SSCP gels, suggests that most of the tumor cells contain a mutated K-ras. This implies that, at least in some cases, bone marrow micrometastases are not derived from the main population of the primary tumor.

Our results are puzzling because not only do they differ from data on the clonal nature of established visceral metastases (see above), but they also differ from data on the clonal nature of micrometastases. Previous studies of K-ras and/or p53 mutations in disseminated cells in blood or lymph nodes from colorectal cancer patients have never shown any differences with regard to the mutations present in the primary tumors^31-34 or hepatic metastases.³⁵ This may be due to the fact that the techniques engaged—the mutant allele–specific amplification and the mismatch ligation assay—were designed to detect in the micrometastases specifically the mutations found in the primary carcinomas, but not de novo mutations. In addition, except for one study,³² in which only codon 12 of K-ras was analyzed by restriction fragment length polymorphism without confirmation of the type of mutation by sequencing, none of the previous work has taken the precaution to look only at the DNA of isolated epithelial cells as we did here, despite the fact that DNA is a rather stable molecule and that the detection of DNA-based markers does not necessarily indicate the presence of viable disseminated tumor cells. Indeed, investigations performed with DNA extracted from whole blood or lymph nodes are prone to an error from the interference of tumor-derived DNA, obscuring the contribution of micrometastases.³⁶

In all, our finding raises important questions with regard to the origin of disseminated cells, the time point of cell dissemination along the tumorigenic process, the possible role of the bone marrow as an organ of clearance of circulating tumor cells, and the prognostic significance of disseminated cells, clonal or not, to the primary tumor. The main question that arises from our study concerns the origin of the epithelial cells found in the bone marrow of some colorectal cancer patients, as in two patients with primary tumor mutations. The epithelial cells in the bone marrow showed no mutations, even by E-SSCP, whereas in three cases they showed a mutation different from that found in the primary carcinoma. Interestingly, one patient depicted two different mutations in the bone marrow (one in codon 12 and another one in codon 13), whereas only one (codon 12) was present in the tumor.

In 1997, Little et al³⁷ documented that certain cytogenetic alterations found in late-stage colorectal carcinomas and/or hepatic metastases were rarely present in the epithelial cells disseminated to the bone marrow. The authors concluded that "it was unclear whether CK20-labeled cells in bone marrow aspirates derived from the primary tumor and/or hepatic metastases." We think, however, that, as in two cases of our study, these cells could have migrated from the primary carcinoma at an early stage of its development, before the acquisition and/or clonal expansion of the alterations that would finally lead to the establishment of visceral metastases.^38,39 Similarly, tumor cells in the bone marrow that depict mutations different from those found in primary carcinomas could have derived from an early subclone within the primary, showing a mutated K-ras that was later overgrown by a sibling subclone harboring a different K-ras mutation with a higher malignant potential. The fact that we found most bone marrow K-ras mutations in codon 13, a codon barely mutated in invasive colorectal cancer^35,40,41 but frequently mutated in aberrant crypt foci, the early lesion of colorectal tumors,^41,42 supports this idea. Alternatively, cells with codon 13 K-ras mutations could have been shed to the bone marrow from colorectal lesions that did not necessarily progress to carcinomas, consistent with a recent report showing that colonic mucosal cells may be shed spontaneously into the circulation in cases of adenoma and inflammatory bowel disease.⁴³ Thus the presence of codon 13 mutations in bone marrow might be an indirect indicator of early, nonmalignant cell dissemination.

K-ras genetic heterogeneity, ie, the nonhomogeneous distribution of K-ras mutations among intratumoral subclones, is well established^38,44,45 and reflects the clonal evolution during tumor progression. The presence of two different K-ras mutations within a single tumor, although much less frequent, has also been described.^12,44,46 In our study, a double mutation affecting the same allele of K-ras was detected in case 12 (Table 2), but no more than one mutation per tumor was detected in the other cases. Presumably, overgrowth of the major subclone resulted in the dilution or even extinction of other subclones carrying different or no K-ras mutations. Unfortunately, only major cell populations within an heterogeneous mixture can be detected by SSCP, and the E-SSCP analysis on tumor samples was not performed, as contamination with the main mutated allele would have interfered with the amplification of any potential minor mutant allele.

The rarity of bone marrow metastases in colorectal cancer implies some ability of this organ, in contrast to the liver and the lungs, to clear disseminated tumor cells or to prevent their proliferation.^25,47 Clearance of disseminated cells from the bone marrow has been documented after curative surgery for gastrointestinal cancer in a subset of patients, whereas in other patients bone marrow deposits persisted 6 months after resection.⁴⁸ Our finding that not all tumor cells disseminated to the bone marrow are clonal with the primary tumor would support this idea.

It is not illogical to think that the prognosis of patients in whom deposits of tumor cells clonal to the primary tumor are present in bone marrow could be worse than the prognosis of patients with cells originating from early tumor stages (previous to the clonal expansion of malignant cells). Confirmation of this hypothesis in a well-designed prospective clinical trial integrating phenotypic and genotypic analysis of bone marrow micrometastases using different molecular markers is urgently needed. Currently available data suggest that bone marrow micrometastases represent a selected population of cancer cells that express a considerable degree of heterogeneity with regard to the expression of major histocompatibility complex class I antigens, adhesion molecules (EpCAM), growth factor receptors (eg, epidermal growth factor receptor, erb-B2, and transferrin receptor) or proliferation-associated markers (Ki-67, p120, and so on).⁴⁹ Thus molecular analysis of micrometastatic cells is necessary to open new insights into the determinants of early tumor cell dissemination and subsequent outgrowth into overt metastases. Monitoring the elimination of bone marrow micrometastases and identification of treatment-resistant tumor cell clones may contribute to increase the efficacy of adjuvant therapy.⁴⁹

In summary, our results document that disseminated tumor cells are not always clonal with the primary carcinoma and reveal a heterogeneous character. These results add further evidence that tumor cell dissemination can occur at early stages in colorectal carcinogenesis and support the idea that the presence of tumor cells in the bone marrow may reflect either the shedding of cells without metastatic potential or true residual disease.

	ACKNOWLEDGMENTS

 
Supported by the Fondation Pour Recherches Médicales (Geneva, Switzerland), the Swiss National Science Foundation (grant no. 3200.050879.97/1), Glaxo Wellcome (Hamburg, Germany), and Europroteome SA (Geneva, Switzerland).

We thank Isabel González (Barcelona, Spain) and Dr Schwanitz (Cottbus, Germany) for technical support, and Dr Karstens (Berlin, Germany) for kindly providing the A45-B/B3 antibody.

	NOTES

 
S.T. and R.S. contributed equally to this study.

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Submitted November 30, 2001; accepted March 5, 2001.

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