Sensitive Measurement of Minimal Residual Disease in Blood by HAT-PCR.

PCR is widely used to measure minimal residual disease (MRD) in lymphoid neoplasms, but its sensitivity is limited. High Adenine/Thymine PCR and High Annealing Temperature PCR (HAT-PCR) is a modified PCR designed to minimize nonspecificity and hence increase sensitivity. It was evaluated in the laboratory and the clinic, using samples from 58 patients. Of these patients, 57 were adolescents or young adults who were participating in the Australasian Leukemia and Lymphoma Group ALL06 trial in which MRD was measured in blood principally by HAT-PCR and in marrow by conventional PCR. HAT-PCR produced significantly less nonspecificity than conventional PCR, and its limit of detection was <10-6 in 90% of patients. In 196 samples, an excellent correlation was found between blood and marrow MRD. Variable partitioning of leukemic cells between blood and marrow was observed. Measurement of MRD in blood by HAT-PCR was noninferior to measurement of MRD in marrow by conventional PCR, in terms of both detecting disease and predicting clinical outcome. At a median follow-up of 3 years and for MRD levels in blood at the end of consolidation treatment, an MRD level of >10-4 cells/L significantly predicted relapse and mortality, whereas undetectable MRD significantly predicted relapse-free survival and overall survival. HAT-PCR is a simple, quick, cheap and sensitive method for measurement of MRD, and its adoption for MRD in blood may be clinically useful.

Patient-Specific Minimal Residual Disease Primers Amplify with Uniformly High Efficiency.

The widespread use of PCR to quantify minimal residual disease has been hampered by the apparently wide variation in amplification efficiency (AE) of PCR primers. A new method to measure AE was developed on the basis of the Ct results of PCR amplification of single copies of a target molecule placed by limiting dilution into wells of a microplate. The mean one copy Ct of a population of primers or of a reference primer was calibrated against the AE determined by the standard method of regression analysis. The AE of a test primer could then be determined by relating its one copy Ct value to the calibrated mean one copy Ct value. This new method was much more precise than direct determination of AE by regression analysis. The AE of minimal residual disease primers, and of primers for eight other genes, was determined to be >95% and often close to 100%. A primer/plasmid standard was produced to enable transfer of the method to other laboratories. The one copy Ct method thus enables AE of a patient-specific primer to be simply and accurately determined.

BCR-ABL1 expression, RT-qPCR and treatment decisions in chronic myeloid leukaemia.

AIMS: RT-qPCR is used to quantify minimal residual disease (MRD) in chronic myeloid leukaemia (CML) in order to make decisions on treatment, but its results depend on the level of BCR-ABL1 expression as well as leukaemic cell number. The aims of the study were to quantify inter-individual differences in expression level, to determine the relationship between expression level and response to treatment, and to investigate the effect of expression level on interpretation of the RT-qPCR result. METHODS: BCR-ABL1 expression was studied in 248 samples from 65 patients with CML by determining the difference between MRD quantified by RT-qPCR and DNA-qPCR. The results were analysed statistically and by simple indicative modelling. RESULTS: Inter-individual levels of expression approximated a normal distribution with an SD of 0.36 log. Expression at diagnosis correlated with expression during treatment. Response to treatment, as measured by the number of leukaemic cells after 3, 6 or 12 months of treatment, was not related to the level of expression. Indicative modelling suggested that interpretation of RT-qPCR results in relation to treatment guidelines could be affected by variation in expression when MRD was around 10% at 3 months and by both expression variation and Poisson variation when MRD was around or below the limit of detection of RT-qPCR. CONCLUSIONS: Variation between individuals in expression of BCR-ABL1 can materially affect interpretation of the RT-qPCR when this test is used to make decisions on treatment.

A DNA real-time quantitative PCR method suitable for routine monitoring of low levels of minimal residual disease in chronic myeloid leukemia.

The BCR-ABL1 sequence has advantages over the BCR-ABL1 transcript as a molecular marker in chronic myeloid leukemia and has been used in research studies. We developed a DNA real-time quantitative PCR (qPCR) method for quantification of BCR-ABL1 sequences, which is also potentially suitable for routine use. The BCR-ABL1 breakpoint was sequenced after isolation by nested short-range PCR of DNA from blood, marrow, and cells on slides, obtained either at diagnosis or during treatment, or from artificial mixtures. PCR primers were chosen from a library of presynthesized and pretested BCR (n = 19) and ABL1 (n = 568) primers. BCR-ABL1 sequences were quantified relative to BCR sequences in 521 assays on 266 samples from 92 patients. For minimal residual disease detectable by DNA qPCR and RT-qPCR, DNA qPCR gave similar minimal residual disease results as RT-qPCR but had better precision at low minimal residual disease levels. The limit of detection of DNA qPCR depended on the amount of DNA assayed, being 10(-5.8) when 5 µg was assayed and 10(-7.0) when 80 µg was assayed. DNA qPCR may be useful and practical for monitoring the increasing number of patients with minimal residual disease around or below the limit of detection of RT-qPCR as the assay itself is simple and the up-front costs will be amortized if sequential assays are performed.

Digital PCR: A brief history.

Digital PCR for quantification of a target of interest has been independently developed several times, being described in 1990 and 1991 using the term "limiting dilution PCR" and in 1999 using the term "digital PCR". It came into use in the decade following its first development but its use was cut short by the description of real-time PCR in 1996. However digital PCR has now had a renaissance due to the recent development of new instruments and chemistry which have made it a much simpler and more practical technique.

Quantification of RNA integrity and its use for measurement of transcript number.

RNA degradation can distort or prevent measurement of RNA transcripts. A mathematical model for degradation was constructed, based on random RNA damage and exponential polymerase chain reaction (PCR) amplification. Degradation, measured as the number of lesions/base, can be quantified by amplifying several sequences of a reference gene, calculating the regression of C(t) on amplicon length and determining the slope. Reverse transcriptase-quantitative PCR (RT-qPCR) data can then be corrected for degradation using lesions/base, amplicon length(s) and the relevant equation obtained from the model. Several predictions of the model were confirmed experimentally; degradation in a sample quantified using the model correlated with degradation quantified using an additional control sample and the ΔΔCt method and application of the model corrected erroneous results for relative quantification resulting from degradation and differences in amplicon length. Compared with RIN, the method was quantitative, simpler, more sensitive and spanned a wider range of RNA damage. The method can use either random or specifically primed complementary DNA and it enables relative and absolute quantification of RNA to be corrected for degradation. The model and method should be applicable to many situations in which RNA is quantified, including quantification of RNA by methods other than nucleic acid amplification.

Durable complete molecular remission of chronic myeloid leukemia following dasatinib cessation, despite adverse disease features.

Patients with chronic myeloid leukemia, treated with imatinib, who have a durable complete molecular response, might remain in complete molecular response after stopping treatment. Previous reports of patients stopping treatment in complete molecular response have included only patients with a good response to imatinib. We describe 3 patients with stable complete molecular response on dasatinib treatment following imatinib failure. Two of the 3 patients remain in complete molecular response more than 12 months after stopping dasatinib. In these 2 patients we used highly sensitive patient-specific BCR-ABL1 DNA PCR to show that the leukemic clone remains detectable, as we have previously shown in imatinib-treated patients. Dasatinib-associated immunological phenomena, such as the emergence of clonal T-cell populations, were observed both in one patient who relapsed and in one patient in remission. Our results suggest that the characteristics of complete molecular response on dasatinib treatment may be similar to that achieved with imatinib, at least in patients with adverse disease features.

Antisense PCR: A simple and robust method for performing nested single-tube PCR.

To overcome the disadvantages of two-round nested PCR, we developed a simple and robust closed single-tube nested PCR method (antisense PCR). The method uses antisense oligonucleotides that carry a 5' tag and that can potentially hybridize to the 3' ends of the outer primers, depending on the annealing temperature. During initial cycles, which are performed at a high annealing temperature, the antisense oligonucleotides do not hybridize and amplification is directed by the outer primers. During later cycles, for which the annealing temperature is decreased, the outer primers hybridize to the antisense oligonucleotides, extend to produce sequences that are mismatched to the amplicon templates, and consequently become inactivated, whereas the inner primers hybridize to the amplicon templates and continue amplification. Antisense quantitative PCR (qPCR) was compared with one-round qPCR for real-time amplification of four PCR targets (BCR, APC, N-RAS, and a rearranged IGH gene). It had equal amplification efficiency but produced much less nonspecific amplification. Antisense PCR enables both endpoint detection and real-time quantification. It can substitute for two-round nested PCRs but may also be applicable to instances of one-round PCR in which nonspecificity is a problem.

Incorporation of measurement of DNA integrity into qPCR assays.

Optimal accuracy of quantitative PCR (qPCR) requires correction for integrity of the target sequence. Here we combine the mathematics of the Poisson distribution and exponential amplification to show that the frequency of lesions per base (which prevent PCR amplification) can be derived from the slope of the regression line between cycle threshold (Ct) and amplicon length. We found that the amplifiable fraction (AF) of a target can be determined from this frequency and the target length. Experimental results from this method correlated with both the magnitude of a damaging agent and with other measures of DNA damage. Applying the method to a reference sequence, we determined the values for lesions/base in control samples, as well as in the AFs of the target sequence in qPCR samples collected from leukemic patients. The AFs used to calculate the final qPCR result were generally >0.5, but were <0.2 in a few samples, indicating significant degradation. We conclude that DNA damage is not always predictable; quantifying the DNA integrity of a sample and determining the AF of a specific qPCR target will improve the accuracy of qPCR and aid in the interpretation of negative results.

Sensitive detection and quantification of minimal residual disease in chronic myeloid leukaemia using nested quantitative PCR for BCR-ABL DNA.

Increasing numbers of patients with chronic myeloid leukaemia (CML) treated with tyrosine kinase inhibitors achieve undetectable levels of BCR-ABL mRNA using sensitive quantitative real-time reverse transcriptase PCR (RT-qPCR) methods and a method to measure minimal residual disease (MRD) in patients with low levels could be of value. Following isolation and sequencing of the patient-specific BCR-ABL breakpoint, a DNA-based nested qPCR assay was established, and MRD was measured by this method and one-round RT-qPCR in 38 samples from 24 patients with CML. Mixing experiments using patient DNA in normal DNA indicated that DNA qPCR could detect BCR-ABL sequences at a limit of approximately 10⁻6. In 22 samples in which MRD was detectable by both methods, comparison of the results of DNA qPCR with the results obtained on the same sample by RT-qPCR showed good correlation. In another 16 samples, BCR-ABL mRNA was not detectable by RT-qPCR. In 8 of the 16 samples, BCR-ABL DNA was detected at levels ranging from 1.1 × 10⁻5 up to 2.8 × 10⁻4 and in the remaining eight samples BCR-ABL was not detected by either method. In one patient, who had stopped imatinib, an almost 1000-fold rise in MRD, to 5.2 × 10⁻4 was observed in sequential samples. Nested DNA qPCR was more sensitive than one-round RT-qPCR and could be used for the monitoring of patients with CML with very low levels of MRD.

Rapid isolation of translocation breakpoints in chronic myeloid and acute promyelocytic leukaemia.

Isolation and sequencing of the translocation breakpoint in chronic myeloid leukaemia-(CML) and acute promyelocytic leukaemia (APML) may help to elucidate the mechanism of translocation and provide a molecular marker for monitoring of minimal residual disease. Amplification across the translocation breakpoint was performed in samples from 91 patients with CML and 15 patients with APML using single-tube multiplex polymerase chain reaction (PCR) involving 308 primers for CML and 40 primers for APML. Nonspecific amplification was removed by a modification of PCR, termed sequential hybrid primer PCR (SHP-PCR), which involved two sequential rounds of PCR, each of which used a low concentration of a specially designed hybrid primer. The resultant amplified material was sequenced. The method as finally developed was simple quick and robust. The translocation breakpoint was successfully isolated and sequenced in all 106 samples. The strategy of highly multiplexed PCR followed by SHP-PCR is thus an effective method for isolating the translocation breakpoint in CML and APML. It may also be applicable to other haematological disorders characterised by translocation, deletion or inversion.

Gene walking using sequential hybrid primer polymerase chain reaction.

We developed a simple and robust method for removing nonspecific amplification produced during gene walking with a gene-specific primer and a degenerate primer. The primary walking polymerase chain reaction (PCR) was followed by two or three PCR rounds, each incorporating a low concentration of a reverse hybrid primer, where the 3' end was bound to a target sequence generated in the preceding PCR round and the 5' end was a new sequence that generated a target sequence for the next PCR round. The low concentration of the hybrid primer and the extent of amplicon stem-loop formation inhibited nonspecific amplification and enabled successful walking along three genes.

Sensitive and specific measurement of minimal residual disease in acute lymphoblastic leukemia.

A sensitive and specific quantitative real-time polymerase chain reaction method, involving three rounds of amplification with two allele-specific oligonucleotide primers directed against an rearrangement, was developed to quantify minimal residual disease (MRD) in B-lineage acute lymphoblastic leukemia (ALL). For a single sample containing 10 microg of good quality DNA, MRD was quantifiable down to approximately 10(-6), which is at least 1 log more sensitive than current methods. Nonspecific amplification was rarely observed. The standard deviation of laboratory estimations was 0.32 log units at moderate or high levels of MRD, but increased markedly as the level of MRD and the number of intact marker gene rearrangements in the sample fell. In 23 children with ALL studied after induction therapy, the mean MRD level was 1.6 x 10(-5) and levels ranged from 1.5 x 10(-2) to less than 10(-7). Comparisons with the conventional one-round quantitative polymerase chain reaction method on 29 samples from another 24 children who received treatment resulted in concordant results for 22 samples and discordant results for seven samples. The sensitivity and specificity of the method are due to the use of nested polymerase chain reaction, one segment-specific and two allele-specific oligonucleotide primers, and the use of a large amount of good quality DNA. This method may improve MRD-based decisions on treatment for ALL patients, and the principles should be applicable to DNA-based MRD measurements in other disorders.

Determining the repertoire of IGH gene rearrangements to develop molecular markers for minimal residual disease in B-lineage acute lymphoblastic leukemia.

Molecular markers for minimal residual disease in B-lineage acute lymphoblastic leukemia were identified by determining, at the time of diagnosis, the repertoire of rearrangements of the immunoglobulin heavy chain (IGH) gene using segment-specific variable (V), diversity (D), and junctional (J) primers in two different studies that involved a total study population of 75 children and 18 adults. This strategy, termed repertoire analysis, was compared with the conventional strategy of identifying markers using family-specific V, D, and J primers for a variety of antigen receptor genes. Repertoire analysis detected significantly more markers for the major leukemic clone than did the conventional strategy, and one or more IgH rearrangements that were suitable for monitoring the major clone were detected in 96% of children and 94% of adults. Repertoire analysis also detected significantly more IGH markers for minor clones. Some minor clones were quite large and a proportion of them would not be able to be detected by a minimal residual disease test directed to the marker for the major clone. IGH repertoire analysis at diagnosis has potential advantages for the identification of molecular markers for the quantification of minimal residual disease in acute lymphoblastic leukemia cases. An IGH marker enables very sensitive quantification of the major leukemic clone, and the detection of minor clones may enable early identification of additional patients who are prone to relapse.

Effect of age on the repertoire of cytotoxic memory (CD8+CD45RO+) T cells in peripheral blood: the use of rearranged T cell receptor gamma genes as clonal markers.

We have established a method to estimate the number of clones in peripheral blood, using rearranged T cell receptor gamma genes as clonal markers, selecting cells at random, and establishing the sizes of the clones to which they belong. Clone sizes were quantified by a clone-specific PCR test based on the VNJ junctional sequence, which typically detects 1-2 copies of its target gene. All clones chosen for study were subsequently quantified in blood, and sizes ranged from 3 x 10(-6) (1 cell in 330,000 CD8+CD45RO+ cells) to 3.5 x 10(-2) permitting numbers of clones to be estimated from the harmonic mean of clone size. Two independent estimates from a healthy young adult (20-30 years old) gave repertoires of 94,000 and 110,000 clones. Two other healthy young adults gave repertoires of 40,000 and 55,000 clones. Repertoires in four healthy active older (>75 years old) adults were more variable but generally lower, being 3600, 5500, 14,000 and 97,000 clones, despite enlarged clones making up >1% of the compartment in the last individual. Overall, young adults had smaller clones (p=0.026, non-directional Mann-Whitney U-test). If the human body contains 5 l of blood, clones have 2 x 10(3)-1.0 x 10(7) cells in blood. These results confirm a diverse repertoire of rearranged T cell receptor gamma genes. The number of clones thus defined are broadly consistent with other estimates of repertoire, despite differences in marker genes used and subsets of cells studied.

In vivo mutagenic effect of very low dose radiation.

Almost all of our knowledge about the mutational effect of radiation has come from high dose studies which are generally not relevant to public exposure. The pKZ1 mouse recombination mutagenesis assay enables study of the mutational effect of very low doses of low LET radiation (microGy to cGy range) in a whole animal model. The mutational end-point studied is chromosomal inversion which is a common mutation in cancer. We have observed 1) a non-linear dose response of induced inversions in pKZ1 mice exposed to a wide dose range of low LET radiation, 2) the ability of low priming doses to cause an adaptive response to subsequent higher test doses and 3) the effect of genetic susceptibility where animals that are heterozygous for the Ataxia Telangiectasia gene (Atm) exhibit different responses to low dose radiation compared to their normal litter-mates.

The linear no-threshold model does not hold for low-dose ionizing radiation.

Almost all of the data on the biological effects of ionizing radiation come from studies of high doses. However, the human population is unlikely to be exposed to such doses. Regulatory limits for radiation exposure are based on the linear no-threshold model, which predicts that the relationship between biological effects and radiation dose is linear, and that any dose has some effect. Chromosomal changes are an important effect of ionizing radiation because of their role in carcinogenesis. Here we exposed pKZ1 mice to single whole-body X-radiation doses as low as 1 microGy. We observed three different phases of response: (1) an induction of inversions at ultra-low doses, (2) a reduction below endogenous inversion frequency at low doses, and (3) an induction of inversions again at higher doses. These results do not fit a linear no-threshold model, and they may have implications for the way in which regulatory standards are presently set and for understanding radiation effects.

Cancer-associated genes can affect somatic intrachromosomal recombination early in carcinogenesis.

The pKZ1 recombination mutagenesis model has provided a sensitive assay where we study somatic intrachromosomal recombination (SICR) as a mutation end-point. SICR is associated with non-homologous end-joining repair of double-strand breaks and can result in chromosomal inversions and deletions, both of which are common chromosomal aberrations identified in cancers. It has been difficult to study the effect of cancer-associated genes on chromosomal changes prior to tumour formation in vivo because of a lack of appropriate test systems. We hypothesised that cancer-associated genes play a role in formation of chromosomal aberrations and that the pKZ1 model would provide a system in which such a role could be studied in the initial steps of carcinogenesis. Transgenic tumour model mice were bred to pKZ1 mice to produce double transgenic animals. SICR inversion events were scored in mouse tissues at an early time, prior to evident tumour formation, and compared with endogenous pKZ1 SICR levels. Over-expression of the c-myc proto-oncogene resulted in a significant 2.1-fold increase in SICR in spleen. Loss of Msh2 and expression of the SV40 T antigen resulted in a significantly reduced SICR frequency (0.3 of the endogenous frequency in pKZ1 mice) in spleen and prostate respectively. Therefore SICR was affected in the case of all three cancer-associated genes studied. We hypothesise that the increase and decrease in SICR in the presence of cancer-associated genes results from incorrect repairing of double-strand breaks. The data presented here suggest that the pKZ1 model may provide a powerful tool for studying the effect of cancer-associated genes on chromosomal changes in the early stages of carcinogenesis.