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Biotechnical Methods Section (BTS)

Comparison of competitive-nested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients

Abstract

Real-time RT-PCR has great advantages for estimating transcript levels in a variety of situations. These include relative rapid assay times (hours), reliability and ease of performing replicate analyses. In contrast, competitive PCR is a very labor-intensive procedure requiring a few days to generate useful data. We compared the same samples from CML patients by both methods. Importantly, we used the Bcr-Abl junction plasmid DNA, which is used as a competitor in the manual competitive PCR assay, to generate a standard curve for the real-time assay. This permitted reporting the real-time data as the number of BCR-ABL transcripts per μg of total RNA, which is the same format used for the competitive PCR assay. In this study, a total of 435 peripheral blood and marrow samples from 285 CML patients were analyzed by RT-PCR; these patients were undergoing therapy by STI-571, interferon, and bone marrow transplantation treatment. Most samples also had assay values for the Philadelphia chromosome (Ph), FISH and Western blotting for the Bcr-Abl oncoprotein. Our findings indicated that the real-time assay was less sensitive than the manual competitive RT-PCR assay (t = 5.118; P < 0.001). Of interest, the transcript levels in cell line mixtures with various ratios of K562/KG-1 (BCR-ABL positive/negative) cells were also significantly higher with the competitive RT-PCR assays than real-time RT-PCR, except for levels of BCR-ABL below 200 transcripts per μg of RNA. In both patient and cell line experiments, dividing the BCR-ABL transcripts by the total ABL transcripts virtually eliminated the difference between real-time BCR-ABL transcript values and quantitative competitive BCR-ABL transcript values, indicating that both BCR-ABL and ABL transcripts were underestimated by the real-time assay. In addition, the increased sensitivity of the nested, competitive RT-PCR was readily apparent in patients with minimal residual disease, which by the real-time were negative in the majority of patients but were positive by nested, competitive RT-PCR in 44.6% (n = 29) of samples analyzed (n = 65). These findings indicate that real-time RT-PCR, when normalized for the total ABL transcripts, can be used to monitor CML patients during therapy, but we suggest that nested, competitive RT-PCR be used to determine BCR-ABL/ABL transcript ratios at low transcript values or especially when real-time analyses are negative.

Introduction

Chronic myeloid leukemia (CML) is caused by a Bcr-Abl protein, which results from the fusion of 5′ region of the BCR gene fused to the second exon of the ABL gene. The resulting hybrid protein has an elevated tyrosine kinase activity.1,2 The gold standard for diagnosis of this leukemia is detection of an abnormal chromosome 22, first identified as the Philadelphia chromosome (Ph).3,4 Other assays useful for diagnosis include FISH (fluorescent in situ hybridization),5 Southern blotting for detection of the rearrangement of the BCR gene within the breakpoint cluster region,6 and Western blotting to detect the Bcr-Abl oncoprotein.7,8 These tests are limited in their sensitivity compared to RT-PCR methods and thus become less useful for those patients who have had dramatic responses to therapy and become negative by these tests. Non-quantitative polymerase chain reaction (PCR) methods were developed to monitor patients with minimal residual disease.9,10 Because of the lack of quantitative information, positive detection of BCR-ABL transcripts was at times an uncertain test result, as some patients despite being PCR-positive maintained their minimal disease state and in some cases even progressed to PCR negative.11 Quantititative competitive PCR methods were developed to estimate numbers of transcripts of a particular gene.12 These methods require the use of an internal DNA standard that is an identical sequence to an unknown DNA sample being analyzed except that it contains either a deletion or insertion. Such an internal control has the advantage that the internal standard would use the same primer set as the unknown, but also serve as a competitor in the amplification of the unknown sequence. Fractional log-dilutions of the internal standard yield an equivalence point in the intensity of the resulting two amplimers, separable by agarose gel electrophoresis, which allows an estimation of the amount of transcripts in the unknown sample.

Other quantitative methods have been developed that allow rapid estimation of the number of DNA molecules in a given sample. The method frequently employed is the real-time Taq Man RT-PCR procedure.13,14,15 We compared the sensitivity of this real-time procedure to that of the competitive RT-PCR procedure. Our results indicate that the competitive RT-PCR procedure is uniformly more sensitive than real-time procedure. However, this difference in BCR-ABL transcript levels by the two procedures can be minimized by normalizing to total ABL transcripts in the sample.

Materials and methods

Patients and samples

All peripheral blood or bone marrow samples in this study were derived from CML patients admitted to the MD Anderson Cancer Center from January 1997 to April 2002. Where indicated, only those patient samples that had competitive, nested RT-PCR, real-time RT-PCR, Western blot, cytogenetic analysis and FISH analyses performed on same day were considered in the present study. This allowed a comparison among the results of all of these analyses. All clinical samples were obtained through an approved institutional protocol that obtained informed consent of the patients. CML was diagnosed according to the standard clinical criteria employed within the MD Anderson Cancer Center. Competitive nested RT-PCR, real-time RT-PCR, and Western blot assays for all the patient samples were performed in the Bcr-Abl protein leukemia screening laboratory; cytogenetic analyses and FISH were performed in the cytogenetic laboratory; both are located within the Division of Pathology and Lab Medicine, MD Anderson Cancer Center, Houston, Texas.

Cell lines

K562 is a BCR-ABL b3a2-positive cell line,16 KBM-7 is a BCR-ABL b2a2-positive cell line,17 KG-118 and SMS-SB19 are BCR-ABL-negative hematopoietic cell lines. Extracts of all these cell lines were used in each multiplex RT-PCR and nested RT-PCR assays as positive or negative controls. Cells were grown in RPMI 1640 medium with 10% fetal serum. To compare the cell lines and clinical samples, serial mixtures of K562 and KG-1 cells were prepared in different ratios of cells, as follows: K562: KG-1 in 105/107(1/102); 104/107(1/103); 103/107(1/104); 102/107(1/105); 101/107(1/106); 100/107(1/107) and 0/107 cells for extraction of RNA.

RNA isolation and cDNA synthesis

White blood cells from patients were isolated from 10 to 20 ml peripheral blood or 1–3 ml bone marrow by treatment with two cycles of ammonium chloride buffer. Total RNA was extracted with TriZol (Gibco BRL, Invitrogen, Carlsbad, CA, USA) from about 1 × 106–1 × 107 white blood cells or bone marrow mononuclear cells. This procedure gives less than 0.1% DNA contamination as measured in more than 40 samples in real-time PCR assays performed with an ABL exon 2 primer set on patient samples before and after cDNA synthesis. The difference between ABL transcript levels was not significantly different (P > 0.25). Importantly, the BCR-ABL/ABL ratio values are not significantly different when using the gross ABL transcripts compared to the corrected ABL transcript values. Moreover, real-time PCR performed on cDNA with either ABL 2 or ABL 2–3 primer sets were not significantly different in this same sample set (P > 0.05). Also, patients’ samples having less than 10000 ABL transcripts per μg RNA were not included in this study.

For cDNA synthesis, we first measured the concentration of RNA by a spectrophotometric method (Beckman DU640B, Palo Alto, CA, USA). Between 1 and 5 μg of total RNA in 19 μl H2O was added to 21 μl cDNA mix (95 mM Tris pH 8.3, 142.5 mM KCl, 5.7 mM MgCl2, 19 mM DTT, 1.9 mM each of each dATP, dGTP, dCTP and dTTP at neutral pH, 200 μg/ml pdN6, 14 units/μl MMLV reverse transcriptase and 1.4 units/μl RNase). Synthesis was achieved by incubating at 37ºC for 2 h. Then the reaction was inactivated by heating at 65ºC for 10 min. The cDNA were stored at −20ºC. Competitive nested RT-PCR and real-time RT-PCR were assayed from the same tube of the sample of stored cDNA.

Multiplex RT-PCR

Multiplex RT-PCR was performed to distinguish various BCR-ABL breakpoints as described previously.20 In the PCR protocol, cDNA from K562 cells (b3a2), KBM-7 cells (b2a2); SUP-B15 cells (e1a2) were used as positive controls; KG-1 cells and sterile water (Baxter Co., Deerfield, IL, USA) were used as negative controls. Normal BCR analysis in the multiplex PCR assay was also as an internal control.

Competitive quantitative (nested) RT-PCR

After the BCR-ABL breakpoint was determined, competitive quantitative (and nested if necessary) RT-PCR was performed as described by Cross et al12 for BCR-ABL transcripts (b2a2, b3a2) and total ABL. The positive and negative controls were as mentioned above in multiplex PCR. 2.5 μl of cDNA was used in the PCR protocol.

Real-time quantitative RT-PCR

The real-time PCR primers and TaqMan probes for amplification and detection of BCR-ABL (b3a2, b2a2) and total ABL were designed by the software of ABI PRISM 7700 Sequence detector (Perkin Elmer/Applied Biosystems, Foster City, CA, USA). The primers and the probe are as follows for BCR-ABL junctions: (BCR3)b3 sense: CGT CCA CTC AGC CAC AT; (BCR2)b2 sense: TGC AGA TGC TGA CCA ACT CG; (ABL) a2 antisense: TCC AAC GAG CGG CTT CAC; TaqMan probe for b3a2 and b2a2: CAG TAG CAT CTG ACT TTG AGC CTC AGG GTC T. Primers and probe for ABL exon 2: ABL sense: GTC TGA GTG AAG CCG CTC GT; ABL antisense: GGC CAC AAA ATC ATA CAG TGC A; TaqMan probe for ABL: TGG ACC CAG TGA AAA TGA CCC CAA CC.

Real-time quantitative RT-PCR was performed on an ABI PRISM 7700 Sequence detector (Perkin Elmer/Applied Biosystems). Reaction conditions and primer selection for optimal amplification of both BCR-ABL and ABL were determined as described in the manufacturer's manual; 2.5 μl cDNA templates were added to each tube as with competitive PCR. Real-time PCR standard curves performed with competitive plasmid DNA either in water or in cDNA from non-Bcr-Abl expressing cells (eg KG1 cell line) were not significantly different when performed with three dilutions of plasmid DNA in the same concentration of cDNA from KG1 cells (P > 0.25), indicating that efficiency of PCR from the plasmid was not effected by the presence of excess unrelated cDNA.

Quantitation and normalization in RT-PCR

For competitive BCR-ABL RT-PCR analyses, a BCR-ABL junction plasmid containing a DNA 100 base insert developed by Dr Goldman's group was used as a competitor in nested RT-PCR.12 In comparing the competitive quantitative RT-PCR to the real-time RT-PCR, we chose the same plasmid as used in competitive, quantitative PCR to prepare a standard curve for real-time PCR. The competitor plasmids are 5.25 kb in size and therefore 1 ng equals 1.8 × 108 molecules. After linearization of this plasmid, serial dilutions were prepared, ie 1 × 106, 1 × 105, 1 × 104, 1 × 103, 1 × 102, 1 × 101 and 1 × 100 molecules each in 2.5 μl, respectively. The stock plasmid (1 × 106) was stored at −80ºC; the serial diluted competitor preparations were stored at 4ºC. The diluted plasmids were used for only 1 month. To achieve continuity of the assay results, a cDNA sample with a ratio of in 1/100000 K562/ KG-1 cells and a CML patient sample cDNA previously analyzed 1-month earlier were assayed. In addition to determining BCR-ABL transcript levels, each sample was also assayed for total ABL transcripts, either by competitive or real-time RT-PCR, and the results expressed as a percentage of the ratio of BCR-ABL/ABL, as others have reported.21

Cytogenetic and FISH analyses

Cytogenetic analysis and FISH assays were performed according to established procedures on BM specimens.22

Statistical analysis

Student's t-test was used for comparing BCR-ABL transcript levels and BCR-ABL/ABL ratio percentages between competitive quantitative RT-PCR and real-time RT-PCR. Pearson's correlation coefficient was analyzed to determine possible correlations among competitive quantitative RT-PCR and real-time PCR.

Results

Competitive quantitative RT-PCR had increased BCR-ABL transcript values compared to real-time RT-PCR

We compared the real-time assay results to those obtained with the competitive RT-PCR values, which were performed on the same CML patient samples over a broad range of transcript levels and Ph levels (Table 1). The results showed a significant increase in BCR-ABL transcripts in assays performed by the competitive method compared to the real-time method over the entire range (Table 1, Figure 1a). Of interest, this difference between the two assay methods largely disappeared when the values were divided by the number of total ABL transcripts per μg of total RNA, suggesting that each assay was internally consistent as far as detecting BCR-ABL transcripts and total ABL transcripts (Table 1, Figure 1b). That is, the assay values for total ABL transcripts were also lower by the real-time method compared to the competitive method. Statistical analyses revealed that the transcript levels measured by the two procedures were significantly different (Figure 1a) (t = 5.12, P < 0.001; n = 435) whereas the differences in BCR-ABL transcripts/total ABL transcript were not significantly different (Figure 1b) (t = 0.339, P = 0.367; n = 435). In addition, the linear correlation between the two procedures was much better when the BCR-ABL/ABL transcript ratios (r = 0.85; P < 0.001) were compared (Figure 1d) than when the total transcripts were compared (r = 0.58; P < 0.001) (Figure 1c). The results indicate the real-time method underestimates the level of transcripts in patient samples compared to the competitive method. We compared transcript levels obtained by competitive and real-time RT-PCR from patients having either b2a2 or b3a2 junctions. Again, the data showed that the real-time method underestimated BCR-ABL transcripts having either b2a2 or b3a2 junctions. The differences were significant, as the t- test had P value of less than 0.01 in each sample set.

Table 1 Comparison of competitive RT-PCR, real-time RT-PCR and cytogenetic analyses in CML patients
Figure 1
figure1

Comparison between competitive RT-PCR and real-time RT-PCR for BCR-ABL transcripts (a) or BCR-ABL/ABL ratio as a percentage (b); correlation between the competitive RT-PCR and real-time RT-PCR for BCR-ABL transcripts (c) or BCR-ABL/ABL transcript ratio as a percentage (d).

To determine whether the real-time primer set was responsible for the observed differences between real-time and competitive PCR BCR-ABL transcript values, we performed competitive PCR assays with the real-time primer set on 25 patient samples previously assayed with the competitive primer set. The results revealed no significant differences in the BCR-ABL transcript values when we compared these values to those obtained with the primer set used with our standard competitive PCR (P > 0.05), even though the down-stream 18 base ABL 2 real-time PCR primer at 5′ end has a 2 base mismatch with the insert sequence within the competitor plasmid DNA. Thus, the difference in primers used in the real-time and competitive methods is not responsible for the reduced BCR-ABL transcript values that we have observed with the real-time method (Results not shown).

Comparisons of BCR-ABL transcript levels in mixtures of BCR-ABL-positive and -negative cell lines as assayed by competitive and real-time RT-PCR assays

To assess the differences between the two assay methods in a defined system, we prepared mixtures of BCR-ABL-positive- and -negative cell lines in ratios ranging from one K562 cell per 100 KG-1 cells (105/107) to one K562 cell/1 million KG-1 cells (101/107) (Table 2). As with CML patient samples, large differences in transcript levels were observed in assays performed with competitive and real-time methods, but only at the higher BCR-ABL transcript levels (1/100 to 1/10000). The differences were not observed at low BCR-ABL transcript levels (less than 200 transcripts per μg of total RNA), which coincided with 102/107, 101/107, and 100/107 cell ratios. These data indicate that the real-time RT-PCR assay method underestimates the BCR-ABL transcript levels at relatively high and even modestly high transcript levels but not when the transcript concentration reached the detection limit, suggesting an inherent deficiency in the real-time method in samples with higher transcript content.

Table 2 Comparison of competitive RT-PCR and real-time RT-PCR in mixtures of K562/KG-1 cells for BCR-ABL detection

Nested competitive RT-PCR is required for monitoring minimal residual disease patients

We have found that a significant number of minimal disease patients lack detectable transcripts or have very low transcript levels. Since the real-time assay underestimates the level of BCR-ABL transcripts in all but the lowest level of BCR-ABL transcripts (see Table 2), it is critical to perform the more sensitive competitive RT-PCR assay when the patient reaches a lower transcript level (Table 3). For example, patient 6 had useful values by competitive RT-PCR of 43, 0, 40, 127, 136, 334, 10 and 5 transcripts per μg of total RNA but the real-time assay had values of 160, 0, 0, −, 0, 6, 0, and 0 transcripts (with a gap at assay 4) per μg of total RNA.

Table 3 Comparison of competitive and real-time quantitative RT-PCR results in CML patients

To estimate the frequency of false-negative results (zero values) obtained with real-time PCR, we compared 65 samples that were negative for BCR-ABL transcripts by the real-time method and determined transcript values obtained by the competitive method (Table 4). BCR-ABL transcripts were detected in 29 of 65 samples with the competitive method which were scored as zero with the real-time assay. Transcript levels had average values of 392 transcripts per μg of RNA with a variance of plus or minus 154 as measured by the competitive method. These results indicate that when negative values are obtained by the real-time method, the competitive assay should also be performed. This result is consistent with what is known about PCR methods, since a nested PCR method (35cycles plus 30 cycles) is expected to be more sensitive than the non-nested real-time procedure.

Table 4 Sensitivity of both nested competitive RT-PCR and real-time RT-PCR in CML minimal residual disease patients

Discussion

In this report, we have compared the levels of BCR-ABL transcripts by two quantitative RT-PCR methods. These studies were made possible because each method was designed to measure the number of BCR-ABL transcripts. Thus, we generated standard curves for the real-time assay method using the competitor BCR-ABL junction DNA. This permitted calculations for both the real-time and the competitive methods to be expressed as number of BCR-ABL transcripts per μg of total RNA. We found that the two methods exhibited significant differences in transcript levels for BCR-ABL RNA (Figure 1a). Because differences were noted in both ABL and BCR-ABL transcripts, we observed that the differences between the two assays could be largely eliminated by dividing the BCR-ABL transcript values by the total ABL values (Figure 1b). This normalization effect indicates that the real-time method underestimated transcript levels compared to the competitive method. Of interest, this underestimation effect of the real-time method was also observed in cell line comparisons (Table 2) except at very low BCR-ABL transcript levels (less than 200 transcripts). Because the transcript levels were very similar at lower levels, it appears that the underestimation effect of the real-time method is not due to trivial factors (differences in primer sets between the two assay protocols) but appears to be due to some discrepancy between the real-time fluorescent methodology and the competitive method. This conclusion was verified by comparing competitive RT-PCR transcript values using either real-time or competitive primer sets.

The use of these quantitiative RT-PCR methods to monitor CML patients is controversial.23 It is not at all clear what level of BCR-ABL transcripts can be used as a guidepost to signal relapse in a minimal residual disease patient, who is scoring negative for other assays such as either cytogenetics for the abnormal chromosome or Bcr-Abl protein levels as measured by Western blotting. Others have made estimates of what could be defined as long-term remission using quantitative RT-PCR.24 These values are presented as the percentage of the ratio of BCR-ABL transcripts/total ABL transcripts. It is also clear that because of the imprecision of such a logarithmic assay method, rising or falling trends in transcript levels would provide a more prudent estimation of the therapeutic response that the patient is undergoing. The studies presented here indicate that the two most popular PCR methods being used to assess BCR-ABL levels yield different values and that these differences are likely due to the underestimation of BCR-ABL transcripts by the real-time method compared to the competitive method.

In about 8% of more than 400 patient samples analyzed by real-time RT-PCR, we observed very large reductions in the levels of both BCR-ABL and ABL transcripts, compared to analyses performed by the competitive method (not shown). In these cases, the reductions observed in the real-time assays were much larger (10- to 100-fold) than the reductions observed in the majority of samples analyzed. The reason for these large differences is not known, but since the ABL transcripts in these patient samples were near normal in the competitive assays, it cannot be due to the poor quality of the RNA. Moreover, the pattern of ribosomal RNAs in these samples did not reflect any significant RNA degradation. Of interest, all of these samples had very high BCR-ABL transcript levels, as judged by the competitive method. We suspect that some fluorescent-quenching material was present in these samples, thereby reducing the signal to the real-time fluorescent detection system.

In conclusion, our studies indicate that the competitive quantitative RT-PCR method is more sensitive than the real-time assay method. BCR-ABL transcript values were typically higher at all levels of Ph chromosome-positive cells. Moreover, these differences were also observed throughout a range of mixtures of Bcr-Abl-positive and -negative cell lines.

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Acknowledgements

We would like to thank Dr Goldman for the competitor plasmids, and Dr Feng Lin for advice and suggestions. In addition, we would like to thank Dr Ke Si for help in statistical analysis and we acknowledge expert technical assistance of Qing Wang and Jialing Xu. This research was supported by grants from NIH (CA49639) and the Hendricks Foundation.

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Correspondence to RB Arlinghaus.

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Guo, J., Lin, H., Kantarjian, H. et al. Comparison of competitive-nested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients. Leukemia 16, 2447–2453 (2002). https://doi.org/10.1038/sj.leu.2402730

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Keywords

  • real-time RT-PCR
  • quantitative competitive RT-PCR
  • BCR-ABL transcripts
  • chronic myeloid leukemia

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