Summary. We prospectively investigated minimal residual disease (MRD) in 51 children with B-lineage acute lymphoblastic leukaemia (ALL) treated according to the Fralle 93 protocol. PCR follow-up was performed in children in morphological and cytogenetic complete remission, provided an immunoglobulin (IgH) gene rearrangement could be detected using FR3/J H amplimers. MRD was studied according to our previously described methodology, with a few modifications including the use of a consensus J H probe to control for PCR efficiency variations.Out of the initial 51 patients, 34 were assessable for MRD at the end of induction at the time of analysis. MRD levels were as follows: >1/10 3 in 26%, 1/10 3 to 1/10 4 in 50% and <1/10 4 or not detectable in 24%. With a median follow-up of 20 months there were five relapses, all of which occurred in the group of patients with MRD >1/10 3 . To date, none of the patients with MRD р1/10 3 (good molecular responder) has relapsed. Classification according to molecular response at the end of induction did not correlate with the conventional risks groups: there were no statistically significant differences between good and bad molecular responders. Of particular interest is the absence of correlation between WBC at diagnosis and MRD level at the end of induction. We conclude that classification of patients into good and bad molecular responders using PCR seems to be a better prognostic indicator than conventional risk factors in childhood B-lineage ALL. Patients with MRD level >1/10 3 have a particularly poor outcome and should always be considered for alternative therapeutic strategies in the future, whereas in good molecular responders belonging to poor or intermediate risk categories, treatment de-escalation might be contemplated.

Bone marrow samples of 16 patients (two adults and 14 children) with a B lineage acute lymphoblastic leukaemia (ALL), and in whom Ig heavy chain gene rearrangements were detectable at diagnosis using polymerase chain reaction (PCR), were studied during evolution using PCR. The VDJ junctional fragment of the Ig heavy chain rearranged gene was amplified at diagnosis. After length reduction by restriction digestion, the amplified fragment was recovered by chromatography, labelled using a specific hexamer as a primer and directly used as a clonospecific probe. The sensitivity of the PCR ranged from 1:10(4) to 1:10(5) cells, depending on the patient's rearrangement. Residual disease (MRD) was detected in most of the patients achieving a complete remission after induction therapy, regardless of the long-term outcome of treatment. However, in patients remaining in complete remission, the level of MRD showed a tendency to decrease and ultimately become undetectable for variable periods of time, while in patients eventually relapsing there was a trend for MRD to persist at stable levels and even to increase before relapse was clinically evident. We conclude that the use of a simplified methodology for obtaining a clonospecific probe from the Ig heavy chain gene, though less sensitive than the sequencing methodology, is a valuable and readily available tool to monitor MRD in a high proportion of B lineage ALL.

Antisense oligodeoxyribonucleotides (ODNs) are now being extensively investigated in an attempt to achieve cell growth suppression through specific targeting of genes related to cell proliferation, despite increasing evidence of non-antisense cytotoxic effects. In the context of anti-BCR/ABL antisense strategies in chronic myeloid leukemia, we have re-examined the antiproliferative effect of phosphodiester and phosphorothioate ODNs on the leukemic cell line BV173 and on CD34+ bone marrow cells in liquid culture. The 3′ sequences of the ODNs determine their effect. At concentrations of 10 μmol/L (for phosphorothioate ODNs) or 25 μmol/L (for phosphodiester ODNs), all the tested ODNs exert an antiproliferative activity, except those that contain a cytosine residue at either their two most terminal 3′ positions. We show that this antiproliferative effect is due to the toxicity of the d-NMPs (5′ monophosphate deoxyribonucleosides), the enzymatic hydrolysis products of the ODNs in culture medium. The toxicity of the d-NMPs on hematologic cells depends on their nature (d-CMP [2′deoxycytidine 5′-monophosphate] is not cytotoxic), on their concentration (d-GMP [2′-deoxyguanosine 5′-monophosphate], TMP [thymidine 5′-monophosphate], and d-AMP [2′-deoxyadenosine 5′-monophosphate] are cytotoxic at concentrations between 5 and 10 μmol/L), and on the coincident presence of other d-NMPs in the culture medium (d-CMP neutralizes the toxicity of d-AMP, d-GMP, or TMP). The antiproliferative activity of ODNs is thus restricted to conditions where the 3′ hydrolysis process by exonucleases generates significant amounts of d-NMPs with a low proportion of d-CMP. Our results reveal a novel example of a nonantisense effect of ODNs, which should be taken into account when performing any experiment using assumed antisense ODNs.

We established a transient human joining (J)-chain gene expression system in the baby hamster kidney (BHK) cell. The J-chain was detected as a 29-kDa single band on Western blotting. Immunofluorescent staining of the transfectant revealed an exclusive localization of the J-chain in the endoplasmic reticulum (ER). Intracellular transport experiment revealed that incubating conditions favorable for vesicular stomatitis virus glycoprotein (VSV-G) transport did not allow the J-chain to exit from the ER. Analysis of glycosylation status of the J-chain in the transfectant was examined by tunicamycin treatment, endoglycosidase H digestion, and also by treatment with brefeldin A. It was found that an N-glycosylation consensus site of the J-chain was functional, and intracellular J-chain was endoglycosidase H sensitive. These results indicate that, in the absence of any immunoglobulin molecules, J-chain localizes exclusively in the ER. We also tested whether the J-chain could be exported from the ER by perturbing the Ca2+ concentration in the ER. Cultivation of the J-chain transfectant in the presence of ionomycin resulted in the time-dependent secretion of the J-chain. The secreted J-chain was modified by the Golgi resident glycosylation enzymes, indicating that the secreted J-chain passed through the normal exocytic pathway.