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.

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 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.

Importance of minimal residual disease testing during the second year of therapy for children with acute lymphoblastic leukemia.

PURPOSE: A high level of minimal residual disease (MRD) after induction chemotherapy in children with acute lymphoblastic leukemia (ALL) is an indicator of relative chemotherapy resistance and a risk factor for relapse. However, the significance of MRD in the second year of therapy is unclear. Moreover, it is unknown whether treatment intervention can alter outcome in patients with detectable MRD. PATIENTS AND METHODS: We assessed the prognostic value of MRD testing in bone marrow samples from 85 children at 1, 12, and 24 months from diagnosis using clone-specific polymerase chain reaction primers designed to detect clonal antigen receptor gene rearrangements. These children were part of a multicenter, randomized clinical trial, which, in the second year of treatment, compared a 2-month reinduction-reintensification followed by maintenance chemotherapy with standard maintenance chemotherapy alone. RESULTS: MRD was detected in 69% of patients at 1 month, 25% at 12 months, and 28% at 24 months from diagnosis. By univariate analysis, high levels of MRD at 1 month, or the presence of any detectable MRD at 12 or 24 months from diagnosis, were highly predictive of relapse. Multivariate analysis showed that MRD testing at 1 and 24 months each had independent prognostic significance. Intensified therapy at 12 months from diagnosis did not improve prognosis in those patients who were MRD positive at 12 months from diagnosis. CONCLUSION: Clinical outcome in childhood ALL can be predicted with high accuracy by combining the results of MRD testing at 1 and 24 months from diagnosis.