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Journal of Clinical Oncology, Vol 19, Issue 19 (October), 2001: 3951-3959
© 2001 American Society for Clinical Oncology

Ectopic Expression of Guanylyl Cyclase C in CD34+ Progenitor Cells in Peripheral Blood

By Tracy A. Fava, Rodwige Desnoyers, Stephanie Schulz, Jason Park, David Weinberg, Edith Mitchell, Scott A. Waldman

From the Divisions of Clinical Pharmacology, Medical Oncology and Medical Genetics, and Gastroenterology and Hepatology, and Departments of Medicine and Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, PA.

Address reprint requests to Scott A. Waldman, MD, PhD, 132 S 10th St, 1170 Main, Philadelphia, PA 19107; email: scott.waldman{at}mail.tju.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To examine the utility of guanylyl cyclase C (GC-C)–specific nested reverse transcriptase polymerase chain reaction (RT-PCR) to detect circulating tumor cells in patients with colorectal cancer.

PATIENTS AND METHODS: Peripheral-blood mononuclear cells from 24 patients with Dukes’ stage D colorectal cancer were analyzed by GC-C-specific nested RT-PCR using 1 µg of total RNA. Peripheral-blood mononuclear cells from 20 healthy volunteers served as controls. Additionally, peripheral-blood CD34+ progenitor cells were assayed for the expression of both GC-C and other epithelial cell–specific markers.

RESULTS: GC-C mRNA was detected in blood mononuclear cells from all 24 patients with colorectal cancer and all healthy volunteers. These unexpected positive results reflected low-level ectopic transcription of GC-C in CD34+ progenitor cells. Moreover, CD34+ progenitor cells expressed other epithelial cell–specific markers, including prostate-specific antigen, prostate-specific membrane antigen, carcinoembryonic antigen, CK-19, CK-20, mucin 1, and GA733.2. Limiting the quantity of mononuclear cell total RNA analyzed to <= 0.8 µg eliminated detection of GC-C and other tissue-specific transcripts in blood of healthy volunteers. However, under the same conditions, GC-C mRNA was detected in mononuclear cells from all 24 patients with metastatic colorectal cancer. Using 0.5 µg of total RNA and GC-C–specific primers, nested RT-PCR detected a single human colon carcinoma cell (approximately 20 to 200 GC-C transcripts/cell) in 106 to 107 mononuclear blood cells.

CONCLUSION: These data suggest that GC-C may be useful for detecting circulating colorectal cancer cells. They also demonstrate that CD34+ cells are a source of ectopically expressed epithelial cell–specific markers and that CD34+ cells may contribute to the high false-positive rate generally observed when those markers are used to detect rare circulating metastatic cancer cells by RT-PCR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
COLORECTAL CANCER is the third leading cause of cancer and cancer-related mortality worldwide.1-7 Forty percent of patients thought cured by surgery suffer disease recurrence within 3 years.1-7 At present, there are no effective blood-based methods to detect postoperative disease recurrence and reduce cancer-related mortality. Thus clinical outcomes in patients with colorectal cancer could be substantially improved by the availability of more sensitive and specific diagnostic markers for postoperative surveillance.8,9

Rare circulating tumor cells in blood may be detected by amplifying mRNA of tumor- or tissue-specific markers using reverse transcriptase polymerase chain reaction (RT-PCR). Although nested RT-PCR theoretically can amplify target-specific nucleic acids up to 1010-fold,10,11 enhanced detection is associated with a high false-positive rate.12-14 This has been especially true in RT-PCR studies examining the ability to detect rare tumor cells in blood using epithelial cell markers.12-15 The high false-positive rates seem to arise from ectopic transcription of epithelial cell markers.14-20

Guanylyl cyclase C (GC-C),7 a receptor that mediates fluid and electrolyte secretion, is expressed in brush border membranes of intestinal mucosa cells.21-24 GC-C is expressed only in mucosal cells lining the intestine, from the duodenum to the rectum, but not by other extraintestinal tissues.25-29 GC-C expression persists after intestinal mucosal cells undergo neoplastic transformation and is expressed by primary and metastatic colorectal tumors regardless of their anatomic location.30-34 However, GC-C is not expressed by tumors arising outside the gastrointestinal tract.25,27-29,34,35

These data suggest that GC-C may be a unique marker for detecting colorectal cancer cells in blood during postoperative surveillance.10,11,35 However, similar to other epithelial cell markers, the ectopic expression of GC-C in disease-free blood may undermine its utility for postoperative cancer surveillance. In an effort to better understand the potential utility of GC-C to detect circulating colorectal cancer cells, this study compared the expression of GC-C and other epithelial cell markers in the blood of healthy volunteers and patients with known metastatic colorectal cancer.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Clinical Specimens
Blood and tissue specimens were obtained from the hematology/oncology clinic under an institutional review board–approved protocol (control no. 98.0614) at Thomas Jefferson University Hospital (Philadelphia, PA) and the Cooperative Human Tissue Network (Philadelphia). Healthy volunteers and patients with Dukes’ stage D colorectal cancer were informed about the study and asked to participate. After informed consent was obtained, each participant received a unique identification number that was recorded on blood samples and any acquisition forms. Blood (approximately 16 mL), collected into Vacutainer CPT tubes (Becton Dickinson, Franklin Lakes, NJ) containing sodium heparin, was centrifuged at 25°C for 15 minutes at 1,700 rpm, and the resulting mononuclear cell, RBC, and granulocytes fractions were recovered for RNA extraction. In some experiments, whole blood was centrifuged at 1,300 rpm at 4°C for 10 minutes, the resulting supernatant containing the platelet-rich plasma was centrifuged at 3,000 rpm at 4°C for 10 minutes, and the platelet pellet was recovered for RNA extraction.

Peripheral-Blood Progenitor Cell (PBPC) Mobilization
Granulocyte colony-stimulating factor (G-CSF) increases the quantity of CD34+ stem cells in the peripheral circulation. To examine the relationship between the quantity of circulating CD34+ stem cells and the level of ectopic transcription of epithelial cell markers, blood was obtained from a patient with breast cancer undergoing PBPC mobilization in preparation for autologous transplantation. The patient received 10 µg/kg/d (total daily dose of 600 µg) of G-CSF (Neupogen; Amgen, Thousand Oaks, CA) as an intravenous bolus for 6 consecutive days. Blood samples were collected on day 5 of treatment and on the sixth day after the last dose of G-CSF.

Cell Culture
T84 and Caco2 human colon carcinoma cells obtained from the American Type Culture Collection (Rockville, MD) were grown to confluence and used as positive controls for GC-C mRNA in RT-PCR analyses.10 T84 and Caco2 cells were grown in media containing DMEM/F12 with 10% fetal bovine serum and 100 units of penicillin/100 µg of streptomycin per mL. Adherent cell lines were routinely passaged by trypsinization every 3 to 4 days.

Nucleic Acid Extraction
Total RNA was extracted with a modified version of the acid guanidinium thiocyanate/phenol/chloroform method using TRI-REAGENT (MRC, Cincinnati, OH). The concentration, purity, and amount of total RNA were determined by ultraviolet spectrophotometry. Only samples exhibiting intact 28S and 18S ribosomal RNA were subjected to RT-PCR. All RNA preparations were stored in RNase-free water (Promega, Madison, WI) at -70°C until analysis.

RT-PCR
The expression of epithelial cell markers in blood cells was examined by RT-PCR using transcript-specific primer sets (Table 1). Reverse transcription of total RNA (<=1 µg) was performed with 0.25 units/µL of AMV reverse transcriptase (Panvera, Madison, WI) and buffer containing 10 mmol/L of TrisHCl (pH 8.3); 50 mmol/L of KCl; 5 mmol/L of MgCl2; 1 mmol/L each of dATP, dCTP, dGTP, and dTTP; 1 unit/µL of RNase inhibitor (Panvera); and 1 µmol/L of the appropriate antisense primer in a total volume of 20 µL. Thermal cycling proceeded for one cycle at 50°C for 30 minutes, 99°C for 5 minutes (to inactivate reverse transcriptase), and 4°C for 5 minutes. The resultant cDNA was subjected to polymerase chain reaction (PCR) in the same reaction tube and included 2.5 units of TaKaRa Taq polymerase (Panvera) in 100 µL of 10 mmol/L of TrisHCl, 50 mmol/L of KCl, 2.5 mmol/L of MgCl2, and 0.2 µmol/L of the appropriate sense primer. Incubation and thermal cycling conditions were as follows: 95°C for 2 minutes, one cycle; 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 90 seconds, 35 cycles; 72°C for 5 minutes, one cycle. After RT-PCR, samples were stored at 4°C until analysis. Nested PCR (70 cycles) was performed using 5% of the PCR product (DNA) and 2.5 units of TaKaRa Taq polymerase (Panvera) in 100 µL of 10 mmol/L of TrisHCl, 50 mmol/L of KCl, 2.5 mmol/L of MgCl2, and 0.2 µmol/L of the appropriate sense and antisense primers. Incubation and thermal cycling conditions were 95°C for 2 minutes, one cycle; 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 90 seconds, 35 cycles; 72°C for 5 minutes, one cycle. Amplicons were separated by 2% Nusieve 3:1 agarose (FMC Bioproducts, Rockland, ME) and visualized by ethidium bromide. Amplicon identity was confirmed at least once by DNA sequencing. RT-PCR was performed using primers for beta-actin on all samples to confirm the integrity of RNA. RNA extracted from T84 human colon carcinoma cells was used as a positive control for GC-C mRNA. Negative controls included RT-PCR incubations that omitted RNA template. Primers used for GC-C amplification span predicted intron-exon junctions, reducing the probability that amplification products reflect contaminating DNA templates.


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  		Table 1.  Primer Sequences for RT-PCR Amplification of Different Transcripts
 
Isolation of Purified Cells From Blood Mononuclear Cells
Monocytes, NK cells, T cells, CD19+ B cells, and CD34+ progenitor cells were obtained commercially (Bio-Whittaker, Charlotte, NC). Also, purified NK and T cells were generously provided by Bice Perussia, MD, Kimmel Cancer Institute, Thomas Jefferson University. CD34+ progenitor cells were isolated from peripheral blood with the CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD34+ progenitor cells were indirectly magnetically labeled using hapten-conjugated primary monoclonal antibody directed to CD34 and an antihapten antibody, coupled to MACS microbeads (Bio-Whittaker). Magnetically labeled cells were purified and recovered in the magnetic field of a MACS separator (Bio-Whittaker).

Miscellaneous
All reagents were of analytic reagent grade. Results are representative of at least three experiments. Values representing the mean ± SD were calculated using Excel (Microsoft, Redmond, WA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Subject Characteristics
Volunteer and patient ages ranged from 20 to 51 years (32.9 ± 2.4 years) and 33 to 79 years (59.4 ± 2.7 years), respectively. Because there is an inverse relationship between age and the quantity of circulating CD34+ stem cells,38 it is reasonable to speculate that CD34+ cells contributed less to results obtained from older patients compared with younger volunteers. There were no significant differences between the ages of female (range, 23 to 51 years; 30.7 ± 3.3 years) and male (range, 20 to 48 years; 35.1 ± 3.8 years) volunteers or female (range, 33 to 79 years; 57.8 ± 2.7 years) and male (range, 40 to 78 years; 61.1 ± 3.0 years) patients, respectively. Four female patients and one male patient were black; all other patients were white. One female and three male volunteers were black; all other volunteers were white. Disease characteristics of patients are outlined in Table 2. Twenty-one of 24 patients had hepatic metastases, no patient had brain metastases, and five patients had bone metastases. All patients were receiving chemotherapy that included fluorouracil and leucovorin during this study.


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  		Table 2.  Patient Characteristics
 
Nested RT-PCR Detects GC-C Expression in Mononuclear Cells From Volunteers and Patients
Total RNA (1 µg) extracted from mononuclear cells of 20 healthy volunteers and 24 Dukes’ stage D patients was subjected to nested RT-PCR using GC-C–specific primers (Fig 1). Mononuclear cells were used because preliminary studies confirmed that human colorectal cancer cells, like other epithelial tumor cells, cosegregate with mononuclear cells rather than with red cells, granulocytes, or platelets (data not shown). GC-C mRNA was detected in mononuclear cells from all 24 patients. However, identical results were obtained with mononuclear cells from all 20 volunteers.



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  		Fig 1. GC-C mRNA expression in blood mononuclear cells analyzed by nested RT-PCR. Molecular weight markers are on the right. Arrow, size of the human GC-C (approximately 250 bp) amplicon. Representative samples of volunteers (n = 20) and stage D patients (n = 24) are shown.

 
Identification of the Source of GC-C mRNA in Blood From Healthy Volunteers
Blood from volunteers was separated into plasma, granulocytes, erythrocytes, platelets, and mononuclear cells, and 1 µg of total RNA from each of these components was analyzed by nested RT-PCR using GC-C–specific primers (Fig 2A). GC-C mRNA was detected specifically in mononuclear cells, but not in other components of blood. To determine which cell population in the mononuclear cell fraction expressed GC-C transcripts, 1 µg of total RNA was extracted from purified monocytes, T cells, NK cells, B cells, and CD34+ cells and analyzed by nested RT-PCR using GC-C–specific primers (Fig 2B). GC-C mRNA was detected specifically in CD34+ cells, but not in other purified cells.



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  		Fig 2. Expression of GC-C mRNA by purified blood mononuclear cells. Total RNA (1 µg) extracted from the indicated cells was analyzed by nested RT-PCR. Markers and symbols are similar to those in Fig 1.

 
Analysis of Mononuclear Cells From a Breast Cancer Patient Undergoing Treatment With G-CSF
Mononuclear cells were isolated from blood obtained on day 5 from a patient with breast cancer treated for 6 consecutive days with 600 µg of G-CSF (Neupogen). A second collection was obtained 6 days after the last dose of G-CSF. G-CSF stimulates the production of CD34+ progenitor cells and their mobilization from sites of hematopoiesis to the peripheral circulation and is used in stem cell collections in preparation for bone marrow ablation and autologous transplantation. The leukocyte count for this patient was 107,000/µL on the fifth day of treatment and 17,000/µL on the sixth day after the last dose of G-CSF. Total RNA (1 µg) extracted from mononuclear cells was subjected to RT-PCR, without nesting, using GC-C–specific primers (Fig 3). GC-C mRNA was detected in mononuclear cells obtained on day 5 of treatment, when the concentration of CD34+ stem cells would have been maximal.39 In contrast, GC-C mRNA was not detected in mononuclear cells obtained on the sixth day after the last dose of G-CSF, when the concentration of CD34+ stem cells presumably returned to baseline.39



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  		Fig 3. GC-C mRNA in blood from a patient undergoing progenitor cell collection. PBPC mobilization was performed using Neupogen (see Materials and Methods), blood was collected during and after treatment, and total RNA from mononuclear cells was analyzed by nested RT-PCR. Markers and symbols are similar to those in Fig 1.

 
Detection of GC-C mRNA After Depletion of CD34+ Cells From Mononuclear Cells
To further examine whether CD34+ progenitor cells were the source of GC-C mRNA in blood from healthy volunteers, mononuclear cells from these subjects were depleted of CD34+ cells, and GC-C expression was examined by nested RT-PCR. Total RNA (1 µg) extracted from mononuclear cells, CD34+-depleted mononuclear cells, and purified CD34+ cells was subjected to nested RT-PCR using GC-C specific primers (Fig 4). GC-C mRNA was detected using RNA from mononuclear cells and purified CD34+ cells, but not RNA from CD34+-depleted mononuclear cells.



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  		Fig 4. GC-C mRNA in mononuclear cells after depletion of CD34+ cells. Total RNA prepared from mononuclear cells depleted of CD34+ progenitor cells (see Materials and Methods) was subjected to nested RT-PCR. Markers and symbols are similar to those in Fig 1.

 
Expression of Epithelial Cell Markers in CD34+ Cells
The utility of epithelial cell markers to detect rare circulating tumor cells in peripheral blood has been limited by high false-positive rates reflecting ectopic transcription in unknown blood components.12-20 The present studies demonstrate that CD34+ progenitor cells are the source of GC-C mRNA in the blood of healthy volunteers. These data suggest that CD34+ cells also may be the source of other ectopically transcribed epithelial cell markers in blood. Thus the expression of epithelial cell-specific transcripts, including carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), prostate-specific membrane antigen (PSM), CK-19, CK-20, mucin 1 (MUC-1), and GA733.2, was examined in purified CD34+ cells (Fig 5). Total RNA (1 µg) extracted from purified CD34+ cells was subjected to nested RT-PCR using primers specific for those epithelial cell markers (Table 1). All epithelial cell–specific transcripts were expressed in RNA from CD34+ progenitor cells. As with GC-C, the expression of other epithelial cell markers was virtually eliminated by depleting mononuclear cells of CD34+ cells (Fig 6).



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  		Fig 5. Expression of epithelial cell biomarkers in CD34+ cells. RNA from CD34+ cells was subjected to nested RT-PCR using epithelial cell marker–specific primers as indicated. RNA from prostate served as a positive control for PSA and PSM RT-PCR analysis. Markers and symbols are similar to those in Fig 1.

 


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  		Fig 6. Expression of epithelial cell biomarkers in mononuclear cells depleted of CD34+ cells. Total RNA prepared from mononuclear cells depleted of CD34+ progenitor cells (see Materials and Methods) was subjected to nested RT-PCR using epithelial cell marker–specific primers as indicated. Sizes of molecular weight markers are indicated on the right.

 
Threshold for Detecting Ectopic Transcripts of GC-C and CEA in Blood
Total RNA from mononuclear cells of volunteers was serially diluted to define the threshold quantity of total RNA required to detect ectopic transcripts of GC-C and CEA using nested RT-PCR (Fig 7). GC-C and CEA transcripts were not detected using less than 1 µg of RNA in any sample obtained from healthy volunteers (n = 20). In contrast, GC-C mRNA was detected using 0.1 µg of RNA, and CEA amplicons were detected using 0.5 µg of RNA from samples obtained from all Dukes’ stage D patients (n = 24) (Fig 8A). These data establish a threshold of total mononuclear cell RNA in RT-PCR reactions of >= 1 µg for detecting GCC and CEA transcripts arising from ectopic transcription in CD34+ cells. Similarly, these data demonstrate that GC-C and CEA amplicons obtained in RT-PCR reactions using <= 0.8 µg of total mononuclear cell RNA reflect the presence of circulating metastatic colorectal cancer cells.



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  		Fig 7. Threshold for detecting GC-C and CEA mRNA in blood from healthy volunteers. Total RNA from mononuclear cells of healthy subjects (n = 20) was serially diluted and subjected to nested RT-PCR using both GC-C- and CEA-specific primers. Markers and symbols are similar to those in Fig 1.

 


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  		Fig 8. Threshold for detecting GC-C and CEA mRNA in blood from Dukes’ stage D patients. (A) RNA from mononuclear cells of stage D patients was diluted and subjected to nested RT-PCR using specific primers. (B) Sensitivity of RT-PCR using GC-C– and CEA-specific primers to detect circulating tumor cells in stage D patients.

 
Sensitivity of Detecting Circulating Metastatic Colorectal Cancer Cells Using GC-C Compared With CEA
Total RNA from mononuclear cells obtained from Dukes’ stage D patients was serially diluted and analyzed by RT-PCR using GC-C– and CEA-specific primers (Fig 8B). As demonstrated above, GC-C and CEA transcripts detected using less than 1 µg of RNA reflect circulating tumor cells in blood. CEA amplicons were detected in seven (approximately 30%) of 24 and five (approximately 21%) of 24 Dukes’ stage D patients using 0.8 µg or 0.5 µg of RNA, respectively. In contrast, all (n = 24) stage D patients yielded GC-C transcripts using >= 0.1 µg of RNA.

Sensitivity of Nested RT-PCR Using GC-C-Specific Primers for Detecting Circulating Tumor Cells
T84 or Caco2 human colon carcinoma cells (approximately 200 and 20 GC-C transcripts per cell, respectively) were serially diluted using excess mononuclear cells, as indicated (Fig 9). Total RNA extracted from these samples (0.5 µg) was used for nested RT-PCR employing GC-C-specific primers. A single T84 cell was detected in 107 mononuclear cells, whereas one Caco2 cell was detected in 106 mononuclear cells. This level of sensitivity for detecting human colorectal cancer cells by RT-PCR using GC-C-specific primers was highly reproducible and yielded identical results when 10 sequential analyses were performed.



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  		Fig 9. Sensitivity of RT-PCR using GC-C primers for detecting colorectal cancer cells in blood. RNA (0.5 µg) extracted from the indicated number of mononuclear cells spiked with one T84 (approximately 200 copies of GC-C mRNA) or Caco2 (approximately 20 copies of GC-C mRNA) colon carcinoma cell was subjected to nested RT-PCR. Markers and symbols are similar to those in Fig 1.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
GC-C is a cell surface receptor with a pattern of expression in humans that is restricted to brush border membranes of intestinal mucosa cells from the duodenum to the rectum.21-29 Of significance, GC-C continues to be expressed in intestinal mucosal cells after they have undergone neoplastic transformation. GC-C expression has been detected in all primary and metastatic colorectal tumors and cell lines, including those originating from poorly differentiated colorectal tumors, but not in any extraintestinal tissues, tumors, or cell lines examined.10,11,30-35 This restricted pattern of expression suggests that GC-C may be a marker for metastatic colorectal cancer cells in extraintestinal sites.

Previous studies in colorectal cancer patients have demonstrated that GC-C can identify micrometastatic foci in lymph nodes evaluated as free of disease by standard histopathology. Importantly, detection of micrometastases by GC-C RT-PCR was associated with a greatly enhanced risk of colorectal cancer–related mortality.10,11 GC-C analysis may be a sensitive and specific method for detecting clinically significant colorectal cancer micrometastases in lymph nodes and could improve the accuracy of staging.

Similarly, analyzing GC-C expression in blood to detect rare circulating colorectal tumor cells could improve the early detection of disease recurrence in patients undergoing postoperative surveillance. Current surveillance paradigms have not improved the overall survival of patients with recurrent colorectal cancer, in part reflecting their inability to detect recurrence at a point amenable to intervention.40-45 Development of a more effective surveillance marker would have significant impact on the management and outcome of colorectal cancer. A previous study detected GC-C mRNA in blood from colorectal cancer patients, although there was no obvious correlation between the detection of this transcript and disease stage.46 In addition, GC-C mRNA was detected in the blood of some healthy volunteers.46 The present study demonstrates that GC-C mRNA is expressed at low levels specifically in CD34+ progenitor cells in the blood of all healthy volunteers examined. Expression of this transcript in CD34+ cells is a potential explanation for the lack of correlation between GC-C expression in blood and colorectal cancer disease stage observed previously.46

Background GC-C expression was reliably and reproducibly eliminated by limiting the quantity of total RNA from blood mononuclear cells analyzed by RT-PCR to <= 0.8 µg. Indeed, GC-C mRNA expression was not detected in mononuclear cells from 20 healthy volunteers analyzing less than 1 µg of total RNA but was detected in mononuclear cells from 24 patients with metastatic colorectal cancer examined, analyzing as little as 0.1 µg of total RNA. Using this technique, a single colon cancer cell (approximately 20 to 200 copies of the GC-C transcript) was detected in 106 to 107 blood mononuclear cells, in the complete absence of a background signal. This corresponds to the detection of a single cancer cell in approximately 1 to 10 mL of whole blood, requiring a minimum of approximately 500 to 5,000 cancer cells in the circulation for detection. The performance characteristics of this assay, including the sensitivity to detect rare circulating cancer cells, high specificity of transcript detection, low background in normal individuals, and reproducibility, supports the suggestion that GC-C mRNA expression could improve the early detection of recurrent disease in patients undergoing postoperative surveillance.

The extremely high sensitivity of RT-PCR has revealed the ectopic transcription in blood of genes previously considered markers of specific epithelia.36,47-50 Transcripts for PSA and PSM (prostate cancer), CK-19 and CK-20 (gastric, colon, and breast cancer), CEA (colorectal cancer), CK-18 (breast cancer), CK-8 (breast cancer), MUC-1 (breast, ovary, colon, and lung cancer), and GA733.2, (breast cancer) have been detected in peripheral blood from healthy volunteers.12,15-18,36,49 Ectopic transcription and background mRNA expression in normal blood limit the sensitivity of detecting circulating cancer cells using PSM, CEA, and CK-18.51 Whether every cell has the ability to generate ectopic transcripts and whether ectopic transcripts have a biologic role are unknown.52,53 Because ectopically transcribed mRNA levels are extremely low, it is unlikely that a biologic role would involve protein synthesis. Ectopic transcription in CD34+ cells may reflect incomplete inactivation of thousands of leaky genes in pluripotent cells.53

Thus one dilemma associated with RT-PCR is an unacceptable high false-positive rate reflecting ectopic transcription of tissue-specific genes. Of significance, the present studies demonstrate that the expression of epithelial cell markers in blood of healthy volunteers, including GC-C, reflects ectopic transcription of those markers specifically by CD34+ cells. These results suggest that the high false-positive rate in blood observed in earlier studies of RT-PCR analysis and epithelial markers likely reflects ectopic transcription by CD34+ cells. Removing CD34+ cells from samples before RT-PCR analysis could reduce the high false-positive rate associated with RT-PCR of epithelial cell markers. Indeed, in the present study, depletion of CD34+ cells from mononuclear cells significantly reduced the detection of background ectopic transcripts of various epithelial markers in blood from healthy volunteers. Also, limiting the amount of total RNA analyzed eliminated the contribution of ectopic transcription of markers by CD34+ cells to the signal generated by RT-PCR. Indeed, expression of GC-C transcripts could not be detected by nested RT-PCR when <= 0.8 µg of total RNA from blood mononuclear cells from healthy volunteers was examined. In contrast, GC-C transcripts were reliably and reproducibly detected by nested RT-PCR when 0.08 to 0.8 µg of total RNA from blood mononuclear cells from patients with metastatic disease was examined. These observations suggest that the limitations to the utility of epithelial cell markers for detecting rare circulating tumor cells may be alleviated either by (1) separating CD34+ and tumor cells (positive or negative purification) before RNA extraction, (2) limiting the amount of total RNA analyzed to that below the limit of detection of ectopic transcripts before RT-PCR, or (3) setting the lower limit of detection of the assay to the level of ectopically expressed message defined in disease-free blood.

Most paradigms for postoperative surveillance include repeated measurements of serum CEA.54-57 Analysis of expression of GC-C by RT-PCR may be more sensitive and specific than CEA as a marker for metastatic colorectal cancer in blood. Whereas CEA is produced by less than 80% of colorectal tumors, GC-C has been detected in all primary and metastatic colorectal tumors examined.10,11,26,34,35,58 Similarly, although CEA is expressed by some extraintestinal tumors, GC-C is expressed only by colorectal tumors.10,11,26,34,35,58 CEA is expressed by tissues other than intestine that are involved in nonneoplastic conditions, whereas GC-C has been identified outside the intestine only in colorectal cancer cells.10,11,26,34,35 In a retrospective analysis, GC-C was identified in lymph nodes of all patients who were node-negative by histopathology and who developed recurrent disease, whereas CEA was identified in lymph nodes of only one of those patients.11 In the present study, CEA expression was specifically detected in blood of less than 30% of patients with metastatic colorectal cancer compared with GC-C, which was detected in blood from all of those patients. In addition, GC-C in blood from patients with metastatic colorectal cancer was detected by RT-PCR using quantities of total RNA as low as 0.08 µg, whereas CEA was detected with no less than 0.5 µg of RNA. Taken together, these data support the suggestion that GC-C is more frequently expressed and more abundant than CEA in colorectal cancer cells. As a result, GC-C may be a more sensitive and specific biomarker than CEA for detection of rare metastatic colorectal cancer cells in blood.

In summary, the present study demonstrates that, as with other epithelial cell markers, GC-C is ectopically expressed in blood mononuclear cells, resulting in a high false-positive rate in healthy volunteers. Ectopic expression of GC-C was localized specifically to CD34+ progenitor cells, and these cells also were a source of false-positive signals for seven other epithelial cell markers. Background signals reflect low-level transcription of these markers, and depletion of CD34+ cells or limiting the quantity of RNA analyzed can reliably eliminate false-positive results. Using this technique, GC-C expression was detected in the circulation of all patients with metastatic colorectal cancer, but not in any healthy volunteer examined. Thus, analysis of GC-C expression by RT-PCR may be a sensitive and specific diagnostic tool for early detection of disease recurrence in patients who have undergone resection for colorectal cancer. Similarly, other epithelial cell markers may be useful for detecting rare circulating tumor cells after elimination of ectopic transcription by separating CD34+ cells from tumor cells or limiting the quantity of RNA analyzed.


   ACKNOWLEDGMENTS
 
Supported by funding from the National Institutes of Health (NIH; grant nos. RO1 CA75123 and R21 CA79663), the American Cancer Society (grant no. EDT-106), and Targeted Diagnostics and Therapeutics, Inc, Exton, PA. J.P. was supported by NIH predoctoral training grant no. 5 T32 DK07705-05.

We thank Kenneth Chepenik, PhD, Thomas Jefferson University, for critical reading of the manuscript and Bice Perussia, MD, Kimmel Cancer Institute, Thomas Jefferson University, for generously providing purified human B and T cells.


   NOTES
 
S.A.W. is the Samuel M.V. Hamilton Professor of Medicine, Jefferson Medical College, Thomas Jefferson University.

T.A.F. and R.D. contributed equally to this work.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Submitted March 15, 2001; accepted May 22, 2001.




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