Improvement of clonality detection rate in multiple myeloma using fluorescent IgH PCR with different sets of primers.

The IgH rearrangement provides a useful marker of clonality in B-cell malignancies and amplification of this rearrangement is the method of choice to monitor the residual tumor cells in multiple myeloma (MM). The critical point of this analysis was the false-negative rate observed at diagnosis in patients presenting tumor cells well above the limit of detection. The aim of this study was therefore to increase the clonality detection rate by IgH polymerase chain reaction (PCR). Bone marrow DNA from 37 MM patients were analyzed at diagnosis. IgH PCR with agarose gel detection was performed between framework regions FR3 and FR1, both in combination with 5 different primers in FR4. Fluorescent IgH PCR with highly resolutive capillary electrophoresis was used to improve the detection and to size clonal PCR products. Sixty-two percent of the clonal rearrangements were initially detected with JHD primer specific to the JH segments 1,2,4,5. The use of JH3 and JH6 homologous primers increased the detection rate to 78%, whereas a consensus JH primer only reached 67% of positivity. The lowest detection rates were obtained with JHExt and JH3 with a detection of respectively 43 and 14%. However, three rearrangements were exclusively amplified by JHExt and two additional cases were detected by JH3. The combined use of primers yielded the best score with 89% of positivity. With Genescan analysis, two additional cases showed a monoclonal rearrangement improving the detection rate to 95%. The use of multiple sets of primers along with a highly sensitive genescan analysis makes possible the follow-up of minimal residual disease for most MM patients.

Collection of circulating stem cells and their use for autograft in adults with acute lymphoblastic leukaemia.

The level of peripheral blood granulocyte-monocytic precursors (PB CFU-GM) was studied serially in 10 adult patients with acute lymphoblastic leukaemia (ALL) in early complete remission after induction chemotherapy. The patients were distributed into 2 main groups according to the morphological French-American-British classification: ALL2 and ALL3. Collection of circulating stem cells (CSC) by cytapheresis was performed in 7 of these 10 patients with satisfactory results, except in 2 ALL2 patients, both of whom had chromosome translocation, which could have been a contributing factor. It appears, moreover, that even with 2 or 3 inductions courses, the more intensive the chemotherapy regimen in previously non-treated patients, the higher the peak of PB CFU-GM and the better the collection of CSC. The measurement of CFU-GM is certainly a less effective indicator of pluripotent stem cells in blood than in bone marrow, and it is probably necessary, but sufficient, to inject 5 times more CFU-GM than normally injected in marrow transplants to allow prompt and stable engraftment. The feasibility of this new graft technique seems for the moment undeniable, the main problem in ALL is the risk of relapse.

[Collection of circulating stem cells during remission after chemotherapy in acute leukemia]. / Collection de cellules souches circulantes en sortie d'aplasie post-chimiothérapique dans les leucémies aiguës.

The level of circulating myeloid progenitor cells (CFU-G), considered to be a good index of the quantity of circulating hemopoietic stem cells, was measured in the peripheral blood of 5 patients with acute leukemia as they entered first remission. High levels of circulating CFU-G were found in 4 of these 5 patients, depending on the intensity and the number of courses of induction chemotherapy. Repeated cytaphereses were done on 3 of these patients in order to collect and to cryopreserve circulating stem cells, to be used later for autologous transplantation. We propose a model which calculates the number of cytaphereses sufficient to obtain a level of 10(5) CFU-G/kg of weight, considered necessary to achieve a good hemopoietic reconstitution after transplantation.