Comparison of methods for assessment of minimal residual disease in childhood B-lineage acute lymphoblastic leukemia.

The level of minimal residual disease (MRD) early in treatment of acute lymphoblastic leukemia (ALL) strongly predicts the risk of marrow relapse. As a variety of methods of varying complexity have been separately used for detecting and quantifying MRD, we compared the prognostic utility of three methods measurement of blast percentage on day 14 of treatment, detection of monoclonality on day 14 or day 35, and measurement of MRD by PCR-based limiting dilution analysis on day 14 or day 35. The study group comprised 38 children aged 1-15 with Philadelphia-negative B-lineage ALL who were uniformly treated and followed until relapse or for a minimum of 5 years. We also studied some of the technical factors which influence the ability to detect MRD. Measurement of blast percentage on day 14 by an expert morphologist, detection of monoclonality on day 35, and PCR-based measurement of MRD levels on days 14 and 35 all showed significant ability to divide patients into prognostic groups. Measurement of blast percentage on day 14 by routine morphology or detection of monoclonality on day 14 were not useful. The quality of DNA samples varied greatly, as determined by amplifiability in the PCR. However, virtually all amplifiable leukemic targets in a sample were detectable which suggests that the level of detection achieved by limiting dilution analysis is essentially determined by the amount of DNA which it is practicable to study. We conclude that quantification of MRD at the end of induction provides the full range of prognostic information for marrow relapse but is complex; detection of monoclonality on day 35 is simple and has good positive predictive value; and quantification of MRD on day 14 merits further study. PCR-based methods for measurement of MRD levels should incorporate a correction for variation in DNA amplifiability.

Cardiovascular risk factors in young adult survivors of childhood acute lymphoblastic leukemia.

PURPOSE: To assess cardiovascular risk factors (CVRF) in young adult survivors of childhood acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Twenty-six subjects (median age, 20.9 years; median interval since completion of therapy, 13.3 years) were evaluated. Ten participants had received cranial irradiation (CRT), whereas 16 had received only chemotherapy. Primary outcome measures included body mass index (BMI), blood pressure, fasting lipoprotein, glucose, and insulin levels. Secondary measures included insulin-like growth factor-1 (IGF-1) and IGF binding protein-3 levels, physical activity index, a 7-day dietary recall, tobacco product use, and measurement of the intima-media thickness (IMT) of the common carotid artery. RESULTS: Sixty-two percent (16/26) of participants had at least one CVRF potentially related to their cancer treatment (obesity, dyslipidemia, increased blood pressure, or insulin resistance), with 30% (7/26) having more than two CVRF. Thirty-one percent (8/26) of subjects were obese (BMI > or = 30). Subjects who were treated with CRT (BMI, 30.4 +/- 6.7) had an increased BMI (P = 0.039) in comparison with those who received only chemotherapy (BMI, 25.4 +/- 5.1). Triglyceride and very low-density lipoprotein C levels were significantly higher in those treated with CRT (P = 0.027 and 0.022, respectively). The IGF-1 was inversely correlated with IMT (total group, -0.514, P = 0.009; females only, -0.729, P = 0.003). CONCLUSIONS: Young adult survivors of childhood ALL, especially those treated with CRT, are at risk for obesity and dyslipidemia, insulin resistance, hypertension, and cardiovascular disease. Further investigation of these risks is warranted.

Early resistance to therapy during induction in childhood acute lymphoblastic leukemia.

Many patients with acute lymphoblastic leukemia (ALL) are not cured by current therapy because of the development of drug resistance. It is not clear when resistance develops during the growth of the leukemic clone and whether resistant cells are already present at diagnosis or develop later during treatment. Twenty-two uniformly treated children with ALL were studied throughout induction treatment. The size of the leukemic clone in blood and marrow was estimated by limiting dilution PCR analysis, using the rearranged immunoglobulin heavy chain gene as a molecular marker. The decline in the number of leukemic cells was biphasic in virtually all patients. For both marrow and blood, the logarithmic mean of the number of leukemic cells fell by approximately four orders of magnitude during the first 2 weeks, one order of magnitude during the third week, and not at all during the last two weeks of induction treatment. For marrow, the median of the fraction of leukemic cells in each patient that survived per week of treatment was 0.008 for the first 2 weeks, 0.12 for the third week, and 1.4 for the last 2 weeks; for blood, the corresponding figures were 0.003, 0.14, and 0.69, respectively. In individual patients, the results for marrow and blood showed good correlation. The biphasic decline of leukemic cell number suggests that most leukemic cells were sensitive to treatment and were rapidly killed, leaving behind a minor but substantial population of drug-resistant cells. The most likely explanation for this phenomenon is that these resistant cells were already present at diagnosis, their resistance having originated from genetic or epigenetic mutations during prior growth of the leukemic clone.

Minimal residual disease in childhood acute lymphoblastic leukaemia quantified by aspirate and trephine: is the disease multifocal?

The level of minimal residual disease (MRD) in marrow early in treatment strongly predicts outcome in childhood acute lymphoblastic leukaemia (ALL). Using PCR we studied 30 pairs of aspirates and trephines taken during induction treatment. Consensus PCR primers showed a monoclonal gene rearrangement in eight pairs, polyclonal rearrangement in 18 pairs and a monoclonal rearrangement only in the trephine in four pairs. MRD was quantified by leukaemia-specific primers in 22 pairs. There was a linear relationship between the logarithms of MRD levels of aspirate and trephine, with a residual variance which increased as the level of MRD fell. The mean level of MRD in the trephines was 4.1-fold greater than that in the aspirates, probably due to greater dilution of the aspirates with peripheral blood. The high variance at low levels of MRD could not be explained by measurement variation, which had an MRD-independent value of 0.42 log10 units, and was attributed to sampling variation due to patchiness of disease at low MRD levels. The magnitude of the variation was such that predictions of outcome could well be confounded for many patients. We suggest that MRD sampling variability could be minimized either by taking multiple marrow samples or by measuring MRD in peripheral blood.

The use of monoclonal gene rearrangement for detection of minimal residual disease in acute lymphoblastic leukemia of childhood.

Sensitive quantification of minimal residual disease (MRD) using the polymerase chain reaction (PCR) is strongly predictive of outcome in childhood acute lymphoblastic leukemia (ALL), with MRD levels at the end of induction therapy of >10(-3) predicting a poor outcome. Methods for sensitive quantification are, however, complicated and time-consuming. Detection by PCR of monoclonal immunoglobulin heavy chain (IgH) and T cell receptor (TCR) gene rearrangements is simple and can be used in routine laboratories but is non-quantitative and of lower but uncertain sensitivity. The aim of this study was to determine the value of detection of monoclonality in identification of different levels of MRD. We looked for monoclonality in 64 bone marrow aspirates which had been obtained from 31 patients with B lineage ALL at various times during induction therapy and for which levels of MRD had been determined by limiting dilution analysis using patient-specific PCR primers. Detection of monoclonality identified levels of MRD of > or =10(-3) during induction with a sensitivity of 78% and a specificity of 93%. The positive and negative predictive values were 0.86 and 0.88, respectively. The sensitivity of detection of a monoclonal IgH rearrangement was greater than that for the TCRgamma locus during induction as an IgH rearrangement was detected more often than a TCRgamma rearrangement in patients who had both IgH and TCRgamma rearrangement at diagnosis. Detection of monoclonality is therefore a simple and quick test applicable to the majority of patients with ALL and it may be useful in identifying high-risk patients at the end of induction and in identifying relapsing patients later during therapy.

Estimation of plasma and urinary hemoglobin by a rate spectrophotometric method.

A method is described for estimating plasma and urinary hemoglobin concentrations as low as 3 mg/L. The assay measures at 528 nm the rate of peroxidation of chlorpromazine by hemoglobin and is not affected by ascorbate and bilirubin concentrations up to 500 mumol/L. Results by this method (mean +/- SD: 54.4 +/- 41.6 mg/L; n = 19) correlated well with those by a scanning spectrophotometric method (52.5 +/- 41.6 mg/L; r = 0.96) and a Coulter Instrument method (r = 0.99; Coulter method: 125 +/- 15 g/L; rate method: 122 +/- 15 g/L; n = 10, r = 0.99). The correlation for assays of 20 plasma samples by our method and a tetramethylbenzidine method was also good (r = 0.95) though the latter gave lower results (31.1 +/- 31.6 mg/L) than the chlorpromazine method (50.9 +/- 41.1 mg/L). The chlorpromazine rate method gave an intra- and interday CV of 7.9% and 9.7%, respectively, at a hemoglobin concentration of 31 mg/L.