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Articles |
Departments of
1
Clinical Biochemistry and
2
Oncology, AKH, University Hospital in Aarhus, DK-8000 Aarhus, Denmark.
3
Department of Tumor Cell Biology, Institute of Cancer
Biology, Danish Cancer Society, DK-2100 Copenhagen, Denmark.
a Address correspondence to this author at: Department of Clinical Biochemistry, AKH, Aarhus University Hospital, Nørrebrogade 44, DK-8000 Århus C, Denmark. Fax 45-89493060; e-mail
AKH.grp02s.bsl{at}aaa.dk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: To establish a calibration curve, we measured the concentration of MART-1 mRNA in SK-MEL-28 melanoma cells grown in vitro by competitive RT-PCR. Serial dilutions of these cells were used as calibrators in the assay. The assay was conducted by adding a fixed amount of a RNA internal standard to RNA isolated from either peripheral blood or the calibrators before RT-PCR amplification with MART-1 primers in a nested PCR design. The amount of MART-1 mRNA in blood samples was calculated from the calibration curve.
Results: Addition of melanoma cells grown in vitro to blood from healthy donors demonstrated that the method can detect a single SK-MEL-28 melanoma cell in 1 mL of blood (1.5 x 10-21 mol MART-1 mRNA/mL). MART-1 mRNA was observed in 4 of 12 blood samples from patients with malignant melanoma, at concentrations of 3–18 x 10-21 mol MART-1 mRNA/mL of blood. No MART-1 mRNA was detected in blood samples from 25 controls without malignant melanoma. Intra- and interassay CVs were 15% (n = 12; mean = 44 x 10-21 mol MART-1 mRNA/mL) and 33% (15 samples analyzed in two different analytical runs; mean = 30 x 10-21 mol MART-1 mRNA/mL), respectively.
Conclusions: Our method is the first competitive RT-PCR assay for quantification of melanoma cells in blood samples that compensates for the variation of both the reverse transcription and PCR reactions. The method allows the inclusion of control samples for continuous quality assessment.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2% of malignant tumors and
represents one of the cancer forms with the highest increase in
incidence rate. The prognosis of the primary tumor usually is
determined by histopathological examinations, but there is increasing
interest in the development of additional prognostic markers. New
methods that detect circulating cancer cells by reverse
transcription-PCR (RT-PCR) have emerged for several cancer forms,
including malignant melanoma (1), prostate cancer
(2), breast cancer (3), and hematopoietic
malignancies (4). The basis of the technology is the
identification of specific mRNA molecules, which are present in the
circulating tumor cells but absent in nonmalignant cells of the
peripheral blood. Tyrosinase mRNA is the most extensively studied
marker for detection of malignant melanoma cells in peripheral blood,
although other markers have been described (5). Recently,
melanoma antigen recognized by T cells 1 (MART-1) has been suggested
as a potential marker for malignant melanoma cells in the blood
(6)(7)(8). MART-1 was identified as the target of cytotoxic T
lymphocytes from patients with malignant melanoma (9). The
gene for MART-1 has been cloned (10)(11), but
the cellular function of the protein is still unknown. A major problem in detecting circulating cancer cells is the difficulty in performing reliable quantitative assays for mRNA by nested RT-PCR, because of the high degree of variation in both the reverse transcription and the PCR. We have developed a quantitative nested RT-PCR for measurement of MART-1 in peripheral blood. By combining the use of a RNA internal standard with the use of a calibration curve, we have obtained an assay that allows us to compensate for variations in both the reverse transcription and the PCR. At the same time, only minute amounts of RNA are required for quantification of MART-1 mRNA in peripheral blood.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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patients and blood samples
Blood samples were drawn from 12 patients with biopsy-verified
metastatic malignant melanoma. The patients had stage IV disease
according to American Joint Committee on Cancer criteria
(13) and were staged at the time of blood sampling with a
physical examination, a computed tomography scan of the brain and
abdomen, and a chest x-ray. The project was accepted by the ethics
committee, and written informed consent was obtained from all patients.
The first 10 mL of blood obtained was not used for RNA extraction to avoid contamination with melanocytes. Thereafter, 10 mL of blood was collected, and 1 mL was used for RNA isolation. Total RNA was isolated by the Qiagen total RNA isolation method (Qiagen) according to the manufacturer’s instructions. Purified RNA was dissolved in RN'ase-free water, and the concentration and purity were determined by spectrophotometry (GeneQuant; Pharmacia) (12). A group of 25 patients without a history of malignant melanoma was used as controls.
generation of rna internal standard
The RNA internal standard was generated by PCR amplification of a
spacer DNA molecule [0.6-kb EcoRI-BamHI fragment
of the v-erbB oncogene, available commercially from Clontech
(MIMIC)]. Amplification was performed with two composite primers
(Std.1 and Std.2). The composition of the Std.1 and Std.2 primers and
their annealing to the spacer DNA sequence are shown in Fig. 1A.
The Std.1 primer consists of a region at its 3' end that
hybridizes to the spacer DNA, followed by regions containing the
sequences of the MART-1 sense primers M1 and nested sense primer M3. At
the 5' end of the Std.1 primer, there is a region containing the
sequence of the T7 RNA polymerase promoter. The Std.2 primer consists
of a 3' end that hybridizes to the spacer DNA, followed by the MART-1
antisense primers M2 and nested antisense primer M4, and a stretch of
20 (dT) nucleotides at the 5' end.
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The spacer DNA fragment (1 ng) was incubated with 10 pmol of each of the Std.1 and Std.2 primers in a reaction composed of 1.5 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 9.0), 1 mmol/L each of the four deoxynucleotides, and 1.25 U of Taq DNA polymerase (all reagents from Perkin-Elmer) in a total reaction volume of 25 µL. The PCR was performed in a Perkin-Elmer 9700 Thermal Cycler with the following cycle conditions: 94 °C for 1 min, 57 °C for 30 s, 72 °C for 90 s. Initially, the PCR reaction was heated to 94 °C for 3 min. After 25 cycles of PCR amplification, the reaction was incubated at 72 °C for 7 min. The PCR products were checked by agarose gel electrophoresis, and the presence of a single band of the correct size was demonstrated. RNA internal standard was generated by incubation of the obtained PCR product (1 µg) with 40 U of T7 RNA polymerase at 37 °C for 2 h in the buffer supplied by the manufacturer (Boehringer Mannheim). The DNA was removed by treatment with 10 U of RN'ase-free DN'ase I (Promega) for 15 min at 37 °C in Multicore buffer (Promega). The reaction was extracted with 1 volume of water-saturated phenol:chloroform (99:1 by volume), and the water phase containing the RNA was precipitated by the addition of 2.5 volumes of 960 mL/L ethanol and 1/10 volume of 4 mol/L NaCl. The sample was placed at -20 °C for 30 min and centrifuged for 15 min at 20 800g in an Eppendorf centrifuge. The pellet was washed with 1 mL of 700 mL/L ethanol, dried, and redissolved in 100 µL of distilled water. The RNA was quantified by absorbance at 260 nm (12). The absence of DNA in the RNA internal standard was demonstrated by the inability to produce PCR products in the absence of reverse transcription.
competitive rt-pcr quantification of mart-1 mRNA IN
CALIBRATORS
Calibrators for MART-1 mRNA quantification were obtained from a
RNA stock solution isolated from 10 000 SK-MEL-28 cells added to 1 mL
of MART-1-negative blood from a healthy individual. The concentrations
of MART-1 mRNA in the calibrators were determined by competitive
RT-PCR. This was performed by combining serial dilutions of the RNA
internal standard with the calibrator RNA followed by RT-PCR
amplification under the conditions described below. Plotting the ratio
of the MART-1:internal standard band intensities against the
concentration of the internal standard added made it possible to
determine the MART-1 concentration in the calibrator as the internal
standard concentration where equal amounts of RT-PCR products were
generated from the internal standard and the calibrator
(14)(15).
quantitative rt-pcr
The calibrated RT-PCR method for MART-1 mRNA quantification
was performed by a modification of the procedure described previously
for the epidermal growth factor receptor (14).
Briefly, a fixed amount (4.3 x 10-22 mol)
of RNA internal standard was added to the RNA samples and to each of
the calibrators in a reverse transcription reaction consisting of 10
mmol/L Tris-HCl (pH 8.3), 6.25 mmol/L MgCl2, 50
mmol/L KCl, 1 unit/L RN'ase inhibitor, 1 mmol/L deoxyribonucleoside
triphosphates (dATP, dCTP, dTTP, and dGTP), 2.5 mmol/L 16-mer oligo(dT)
primer, and 2.5 U of reverse transcriptase (all reaction components
were from Perkin-Elmer) in a total volume of 20 µL. The reaction
mixture was incubated at 42 °C for 30 min in a Perkin-Elmer 9700
thermocycler, and 2.5 µL of the resulting cDNA product was
PCR-amplified with 1.25 U of Taq DNA polymerase (Pharmacia) and 10 pmol
of each of the M1 and M2 primers in a buffer containing 10 mmol/L
Tris-HCl (pH 9.0), 1.5 mmol/L MgCl2, 50 mmol/L
KCl, and 0.2 mmol/L deoxyribonucleoside triphosphates (dATP, dCTP,
dTTP, and dGTP). The sequence of the M1 and M2 primers were
5'-GAAGGTGTCCTGTGCCCTGACCC-3' and 5'-GGCTTGCATTTTTCCTACACCATTCC-3',
respectively. The primers spanned intron sequences, thereby ensuring
that no genomic DNA was amplified. Samples were subjected to PCR
amplification in a Perkin-Elmer 9700 thermocycler with the following
profile: 94 °C for 1 min, 57 °C for 30 s, and 72 °C for
90 s. After 40 cycles, the PCR products were extended at 72 °C
for 7 min. All reactions were initially denatured at 94 °C for 3 min
before PCR amplification. After amplification, 2.5 µL of the PCR
product was used as template in the nested PCR reaction using the M3
and M4 primers (10 pmol each), which had the sequences
5'-ATGCCAAGAGAAGATGCT-3' and 5'-GGAGAACATTAGATGTCTG-3', respectively.
The nested-PCR buffer and other conditions were the same as for the
primary PCR. A PCR reaction of 30 cycles was performed with an
amplification profile of 99 °C for 1 min, 57 °C for 30 s,
and 72 °C for 90 s. Finally. the PCR products were extended at
72 °C for 7 min. The reactions were analyzed on a 2% agarose gel
stained with ethidium bromide. The intensity of the bands was
determined by computer scanning (Gel doc 1000; Bio-Rad), and the
intensities of the bands were corrected for the difference in size
between the PCR products originating from the RNA internal standard
(317 bp) and the MART-1 mRNA (440 bp). The identity of the PCR products
was verified by sequencing (BigDye terminator chemistry and an ABI
Prism 310 Genetic Analyzer; Perkin-Elmer).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. MART-1 mRNA and the RNA internal standard were amplified by reverse
transcription followed by two rounds of PCR amplification using two
sets of MART-1-specific primers as outlined in Fig. 1B
. Two rounds of
PCR were necessary because no MART-1 RT-PCR product could be detected
when only a single round of PCR was performed. The RNA internal
standard was RT-PCR-amplified with the same primer sets and in the same
reaction tube as the MART-1 mRNA (Fig. 1B
). Nested RT-PCR amplification
of MART-1 mRNA from RNA corresponding to 10 SK-MEL-28 cells produced a
single band with a size of 440 bp (Fig. 2A,
lane 1). When the RNA internal standard was used as template,
a PCR product migrating to the expected position (317 bp) was generated
(Fig. 2A
, lane 2).
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generation of calibrator rna
SK-MEL-28 cells (10 000) diluted in 1 mL of MART-1 mRNA-negative
blood were used to prepare the calibrators. The concentration of MART-1
mRNA in the calibrator was determined by competitive RT-PCR. Calibrator
RNA diluted to a concentration corresponding to 10 SK-MEL-28 cells was
mixed with serial dilutions of RNA internal standard followed by
reverse transcription and MART-1-specific nested PCR (Fig. 2B
, lanes
1–6). The intensities of the bands were determined, and the ratio
between the PCR products from the MART-1 mRNA and RNA internal standard
in each lane was plotted against the concentration of the RNA internal
standard added (Fig. 2C
; mean value of four independent experiments).
The MART-1 mRNA in the calibrator RNA was calculated as the RNA
internal standard concentration, where equal amounts of PCR products of
the two types were generated (ratio equal to 1, corresponding to a log
ratio equal to 0). The amount of MART-1 mRNA corresponding to 10
SK-MEL-28 cells was 30 x 10-23 mol in 1
µL of the 50 µL in which RNA from 1 mL of blood was dissolved,
corresponding to 15 x 10-21 mol MART-1 RNA/mL. Equal
amplification kinetics of the MART-1 mRNA and the RNA internal standard
are a requirement for quantification. The amplification efficiency of
the MART-1 mRNA and the internal standard RNA was investigated by a
series of RT-PCR amplifications in which the number of PCR cycles in
either the first or second round of the nested PCR reaction was
gradually reduced. The amplification efficiencies of the MART-1 mRNA
and the RNA internal standard were equal (data not shown).
quantification of mart-1 mRNA
The unknown samples and a dilution series of the calibrator RNA
covering the range 1.5–150 x 10-21 mol of MART-1 mRNA/mL
were incubated with RNA internal standard, and the nested MART-1 RT-PCR
reaction was performed. In our assay design, RNA from 1 mL of
calibrator or sample was dissolved in 50 µL of buffer, and 1 µL was
used for RT-PCR. Thus, in absolute amounts the calibrators ranged from
1.5 x 10-21 to 150 x
10-21 mol of MART-1 mRNA/mL. Reaction products
were separated by gel electrophoresis, and the intensities of the DNA
bands originating from the MART-1 mRNA and the RNA internal standard
were quantified. Fig. 3
shows the resulting calibration curve. The amount of MART-1
mRNA present in the individual unknown samples could be determined from
the calibration curve. The point on the calibration curve with the
lowest amount of SK-MEL-28 RNA added corresponded to 1 cell/mL of
blood. The intraassay imprecision (CV) was 15% (n = 12;
mean, 44 x 10-21 mol MART-1 mRNA/mL), and the
interassay CV was 33% (15 samples analyzed on two occasions; mean,
30 x 10-21 mol MART-1 mRNA/mL).
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quantification of mart-1 mRNA IN BLOOD FROM PATIENTS
WITH MALIGNANT MELANOMA AND CONTROLS
RNA was isolated from blood samples obtained from 12 patients with
metastatic melanoma and from the 25 controls. The purified RNA was
subjected to the quantitative MART-1 mRNA assay. MART-1 mRNA was found
in 4 of the 12 patients (33%). The MART-1 mRNA concentrations observed
in the four patients were 3, 3, 12, and 18 x 10-21 mol
MART-1 mRNA/mL of blood. Table 1
presents the clinical data of the patients. No
MART-1 mRNA was found in any of the 25 controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we developed a technique for quantification of MART-1 mRNA in RNA from peripheral blood samples. In this method, RNA is purified from the blood sample, and a known concentration of a RNA internal standard is added. The RNA internal standard is coamplified with the same kinetics as the MART-1 mRNA present in the blood sample. The concentration of MART-1 mRNA in the melanoma cell line SK-MEL-28 was determined, which enabled us to use serial dilutions of this RNA as calibrators for the generation of a calibration curve. Compared with competitive RT-PCR, where several concentrations of RNA standard are used for each unknown RNA sample, this method has the advantage of requiring only a single RT-PCR for each sample, thereby saving time and sample material. In addition, the method allows the inclusion of RNA samples with known amounts of MART-1 mRNA for quality control. A linear calibration curve was observed for 1.5–150 x 10-21 mol of MART-1 mRNA (1–100 SK-MEL-28 melanoma cells). Samples containing a higher number of cells could be quantified by dilution of the sample RNA. The method had an intraassay CV of 15% and an interassay CV of 33%. Most studies have included DNA calibrators for quantification of circulating cancer cells, and to our knowledge, the method presented here is the first that takes advantage of a RNA internal standard for quantification of circulating melanoma cells. A RNA internal standard was also used for detection of circulating breast cancer cells and prostate cancer cells in two recent studies (18)(19). The major advantage of the RNA internal standard is the ability to control both the reverse transcription reaction and the PCR reaction.
Data from the analysis of blood samples from patients with malignant melanoma demonstrated the ability of our method to quantify the MART-1 mRNA found in patient samples. We found that 4 of 12 patients (33%) with malignant melanoma contained MART-1 mRNA in the blood in concentrations of 3–18 x 10-21 mol MART-1 mRNA/mL. In contrast, no MART-1 mRNA was detected in the controls.
Tyrosinase mRNA is the marker used most extensively for the detection of circulating melanoma cells. Other mRNA markers (MART-1, MUC18, and p97) have also been shown to have a potential in detecting these cells (7). Recent studies have demonstrated that inclusion of more than one marker increases the probability of detecting the circulating cancer cells (6). At present, the clinical significance of detecting circulating malignant melanoma cells is under investigation. Several investigators have reported on the prognostic value of the method (5)(7)(8)(20). However, other investigators have questioned the use of RT-PCR detection of circulating melanoma cells using tyrosinase as a marker because large differences in sensitivity have been reported between different laboratories (21)(22). Some of these differences might be explained by differences in the RT-PCR amplification procedures used in the different laboratories. The method presented here might be useful in generating more reliable quantitative data on the presence of cancer cells in the blood of patients.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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fusion mRNA: a study in patients with acute promyelocytic leukemia following peripheral stem cell transplantation. Leukemia 1995;9:889-892.[Web of Science][Medline]
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X174 DNA). The visible bands in the
marker DNA have the following sizes (starting with the top
band), in bp: 1353, 1078, 872, 603, 310, 281 + 271 (running as
one band), 234, and 194. The marker contains two bands, at 118 and 72
bp, that are not visible. (B), RNA isolated from 10
SK-MEL-28 melanoma cells was RT-PCR-amplified with serial dilutions of
the MART-1 RNA internal standard: lane 1, 4.3 x
10-23 mol; lane 2, 2.2 x
10-22 mol; lane 3, 4.3 x
10-22 mol; lane 4, 2.2 x
10-21 mol; lane 5, 4.3 x
10-21 mol; lane 6, 2.2 x
10-20 mol. Lane 7 is marker DNA containing
the DNA fragments described for panel A.
(C), the MART-1 concentration of 10 SK-MEL-28 melanoma
cells is found at the point where the MART-1:internal standard ratio
equals 1 (log ratio equals 0). Bars, SD.