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We have developed a simplified fluorescent run-off (FluRO) based IgH PCR strategy in order to facilitate follow-up of large numbers of B-cell precursor (BCP) acute lymphoblastic leukaemias (ALL) in a routine molecular diagnostic laboratory. DNA samples from 26 BCP-ALL and one B-cell line were amplified using IgH FR1 and FR2 consensus primers and analysed in parallel either by ethidium bromide non-denaturing PAGE or, after rendering the PCR products fluorescent with an internal JH consensus primer, by high-resolution analysis on an automated fragment analyser. The latter led to a minimum of one log increase in sensitivity of detection in 62% of alleles from 19 samples (16/28 in FR1; 11/15 in FR2) tested in parallel on log DNA dilutions, and to at least a 10(-2) level of sensitivity of detection in 15/19. The improved resolution allowed an approximate 20% increase in the number of clonal alleles detected, and consequently doubled the incidence of oligoclonality (6/26; 23%). Using these strategies, 6/17 (35%) of children analysed prospectively showed residual IgH positivity in the post induction complete remission bone marrow sample. Both early deaths occurred within this subgroup of patients and of the three of four surviving patients tested, two remained positive 2-3 months later. Although this simplified strategy is, as expected, less sensitive than anti-V-D-J junction specific strategies, it enables detection of a category of 'slow-remitters' which may have prognostic significance at a stage where therapeutic decisions are taken.

Clonal analysis of FACS-purified primitive hematopoietic stem cells and of their progeny as assessed by the progenitors obtained from long-term cultures requires PCR-based approaches, mainly because of the low number of cells available. We have developed a non-radioactive androgen receptor (AR) assay which allows a simple and quantitative evaluation of the clonality of hematopoietic cells and progenitors. In this approach 5' AR primer is labelled by fluorescein and the amplified product is run on a sequencing gel which allows evaluation of the intensity of the fluorescent peaks generated. A computer software then analyzes the reduction of the intensity of the peaks on HpaII-digested samples. In order to determine the feasibility of the technique, we analyzed the clonality of leukemic cells from a patient with an acute-phase CMML which showed a typical clonal pattern of her leukemic DNA sample (WBC = 300 x 10(9)/I) using phosphoglycerate kinase (PGK) analysis. The same sample was then analyzed with either radioactive- or fluorescein-labelled AR primers, showing a typical clonal pattern (complete disappearance of one allele after HpaII digestion). A short-term clonogenic assay was then set up on methylcellulose and clonogenic progenitors were individually analyzed. All 24 colonies tested showed a typical clonal pattern with the disappearance of the same allele on each sample after HpaII digestion, indicating that they all derived from the same leukemic stem cell. Using this approach we then analyzed 94 patients with several hematologic malignancies and quantification of their fluorescent peaks. Fifty-four percent of the patients were clearly heterozygous (ie, a difference of > or = 2 CAG repeats was present between the two copies of the gene) and could be analyzed in an automatic sequencer using the fluorescent primers. Bone marrow mononuclear cells from all patients with acute myeloid leukemia (AML) showed a clonal or oligoclonal pattern at diagnosis whereas a polyclonal pattern was seen when remission was obtained. Similarly, out of 21 patients with a diagnosis of myelodysplastic syndrome (MDS), a clonal pattern was demonstrated in 10 whereas an oligoclonal or non-clonal pattern was shown in 11. These results show that this non-radioactive and safe technology can now be used on a large scale to evaluate the clonality of highly purified hematopoietic stem cells and their progenitors in hematopoietic malignancies and this might allow new insights into the targets of clonal amplification.