IMMUNOPHENOTYPE AND PROLIFERATION PROFILE OF NORMAL B CELL PRECURSORS IN REGENERATING BONE MARROW FROM PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS

Background:

During follow up of B cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients, the detection of minimal residual disease is the most important prognostic factor for relapse. The regeneration pattern of normal BCPs in bone marrow (BM) forms the background to which these remaining malignant blasts should be detected.  During the treatment course of BCP-ALL, BCP numbers decrease dramatically. However, in between treatment blocks and after stop of treatment, massive regeneration of BCPs occurs in the BM.

Aims:

BCP regeneration may hamper sensitive detection of MRD. In order to get more insight in the characteristics of regenerating BCPs in the BM of BCP-ALL patients, we compared the immunophenotype and proliferation profile of BCPs in regenerating BM and BM from healthy controls.

Methods:

BM samples of healthy individuals (normal BM), patients with BCP-ALL after induction therapy (day 79) and patients with BCP-ALL one year after stop of therapy (month 36) were analyzed.For immunophenotypic analysis, 8 color flow cytometry was performed. Newly designed Infinicyt Software was used to define multiple maturation stages in the BCP population. For each individual marker, the expression pattern during these maturation stages was compared between regenerating BM and normal BM. To assess proliferation, four BCP subsets were sorted from each BM sample: pre-B-I, pre-B-II-large, pre-B-II-small and immature B cells. To determine the percentage of proliferating cells, propidium iodide (PI) was added and PI expression was measured using flow cytometry. The proliferation history of BCPs in the pre-B-II-small en immature subsets was assessed by performing a K-rearrangement excision circle (KREC)-assay. The ratio between the KREC and the rearranged IGK-gene represented the average amount of replication cycles the BCPs had undergone since IGK-rearrangement.

Results:

The BCP expression patterns of CD19, CD10, CD34, CD58, CD66c, CD38, CD123, CD9, CD81, CD24, TdT, Igκ and Igλ of regenerating BM at day 79 and month 36 were similar to the expression patterns of BM from healthy individuals. As expected, most proliferation in normal and regenerating BM occurred in the pre-B-II-large subset. The average percentages of proliferation in respectively the pre-B-I and pre-B-II-large subsets were 13%±2% and 29%±3% at day 79 and 9%±2% and 30%±3% at month 36. This was comparable to the percentages of proliferating cells in the pre-B-I and pre-B-II-large subsets of normal BM. Regenerating BM at month 36 and normal BM showed no significant proliferation in the preB-II-small and immature BCP subsets. In regenerating BM at day 79, these two subsets were hardly present. KREC-analysis of the pre-B-II-small and immature subsets confirmed that no cell divisions had occurred after IGK-rearrangement in both normal BM and regenerating BM at month 36.

Summary / Conclusion:

Regenerating BM in BCP-ALL patients seems immunophenotypically similar to normal BM. Proliferation in regenerating BM takes mainly place in the pre-B-II-large subset, like in normal BM. The percentage of proliferating cells and the number of cell divisions after IGK-rearrangement in the BCP subsets of regenerating BM are comparable to normal BM. Therefore, the regeneration of BCPs during and after therapy is unlikely the result of enhanced proliferation in the pre-B-I, pre-B-II-large, preB-II-small or immature subset, but might be due to a larger influx of non-committed stem cells into the B cell lineage. Knowledge on regenerating BCPs may support the design of sensitive flow cytometric MRD assays.

Email address: p.theunissen@erasmusmc.nl

Abstrakta v časopise Haematologica 2013, Suppl1

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