ACCURATE QUANTITATION OF RESIDUAL B-PRECURSOR ACUTE LYMPHOBLASTIC-LEUKEMIA BY LIMITING DILUTION AND A PCR-BASED DETECTION SYSTEM - A DESCRIPTION OF THE METHOD AND THE PRINCIPLES INVOLVED

The detection of residual leukemia cells in the bone marrow of patient s during morphologic remission has been greatly facilitated by use of the polymerase chain reaction (PCR) to amplify leukemia-specific seque nces. While the current PCR strategies for estimating the amount of re sidual leukemia claim a detection sensitivity of one leukemia cell amo ngst 10(5) or 10(6) normal cells, a rigorous assessment of the relativ e error associated with these techniques has not been presented. We ha ve developed a method of estimating the amount of residual leukemia in remission marrows that is analogous to the limiting dilution assays u sed to determine the frequency of immunocompetent cells in a responder cell population. Using this method we measured the fraction of all-or -none (i.e. positive or negative) reactions of the PCR amplification o f the leukemia-specific IgH gene rearrangement in replicate samples of serial dilutions of DNA obtained from diagnostic bone marrow specimen s from 15 children with B-precursor acute lymphoblastic leukemia (ALL) . A sigmoid curve representing the fraction of positive PCR reactions at a given dilution of leukemia DNA was found to be the best fit to th e data. The narrowness of the log-linear region of this curve prevents the direct application of the analysis methodology that has previousl y been described for limiting dilution assays. However, the residual l eukemia burden during morphological remission in these 15 patients and in two additional patients who experienced relapse could be estimated by the described dilution analysis method using the best-fit equation . Furthermore, the data generated for diagnostic, remission and relaps e marrow samples exhibited a small interspecimen variation. The result s suggest that this method can reliably estimate residual leukemia ove r a range of five orders of magnitude. Although the PCR reaction appea rs to be one of the most sensitive methods for detecting residual leuk emia, all techniques based on this procedure, including our own, must exhibit limitations inherent to the amplification process. Our estimat es of relative error suggest that a realistic limit for the PCR estima tion of residual leukemia lies in the range of one leukemia cell per 1 0(5) normal cells. The suggested method is rapid, technically simple a nd relatively inexpensive. Furthermore, the principles that it is base d upon can be applied to any PCR-based strategy.