Guus Westra

High percentage of CD34-positive cells in autologous AML peripheral blood stem cell products reflects inadequate in vivo purging and low chemotherapeutic toxicity in a subgroup of patients with poor clinical outcome.

In this study, a high CD34% in autologous peripheral blood stem cell (PBSC) products from 71 AML patients was associated directly with a high relapse rate (Pu2009=u20090.006) and inversely with disease-free survival (Pu2009=u20090.003), irrespective whether patients were transplanted or not. The relapse rate at 12 months was 67% in a group with >0.8% CD34+ cells and 34% in a group with ⩽0.8% CD34+ cells. Although the percentage of malignant CD34+ cells in the CD34+ compartment in the relapses of the first group was not high (median 8%), the total number of malignant cells as a percentage of WBC was about 13 times higher than for the patients remaining >12 months in remission. When all patients evaluable were taken together, this frequency of malignant cells correlated strongly with disease-free survival (Pu2009<u20090.001). Both this massive mobilization of normal CD34+ cells and high frequency of malignant cells in the subgroup of patients with CD34 >0.8% and relapse within 12 months indicate an insufficient in vivo purging, as well as low chemotherapeutic bone marrow toxicity. This was confirmed by an inverse correlation between hypoplasia period after the induction therapy and CD34% in PBSC products (Pu2009<u20090.002). It is concluded that a subgroup of patients has been identified that might benefit from a more intensive chemotherapeutic treatment.

In vitro resistance to cytosine arabinoside, not to daunorubicin, is associated with the risk of relapse in de novo acute myeloid leukaemia.

The efficacy of chemotherapy in acute myeloid leukaemia (AML) is limited by clinical drug resistance. We determined in vitro resistance to cytosine arabinoside (ARA‐C), daunorubicin (DNR), mitoxantrone (MITOX), m‐amsacrine (AMSA) and etoposide (VP16) in 49 adults with de novo AML using the MTT assay. Results showed that non‐ responders to chemotherapy were, in vitro, 2.9‐fold more resistant to DNR, but not more resistant to ARA‐C, compared to complete responders. However, complete responders who were in vitro resistant to ARA‐C had a 4‐fold higher risk of relapse (95% CI 1.3–12.5‐fold) compared to complete responders in vitro sensitive to ARA‐C. With a mean follow‐up of 12 months the probability of continuous complete remission (CCR) for patients in vitro sensitive to ARA‐C was 61% at 34 months (95% CI 28–82%), whereas all patients in vitro resistant to ARA‐C relapsed within 18 months from diagnosis. This difference appeared to be independent of other clinical features such as sex, age, white blood cell count, FAB classification, and CD34 expression. In vitro resistance to DNR was not related to the probability of CCR. We conclude that in vitro drug resistance assessed with the MTT assay appears to be associated with short‐ and long‐term clinical outcome in AML. Confirmatory studies comprising a sufficient number of patients for multivariate analyses should prove whether in vitro resistance to ARA‐C will appear to be an independent risk factor.

Interleukin-2 receptor alpha-chain (CD25) expression on leukaemic blasts is predictive for outcome and level of residual disease in AML

We investigated the role of CD25 as a prognostic marker in acute myeloid leukaemia (AML). Seventy-two newly diagnosed patients < or =60 years were retrospectively analysed by flow cytometry for CD25 positivity of AML blasts. Patients with CD25 expression of >10%, when compared to < or =10%, had a significantly shorter overall survival (OS, p=0.0005) and relapse-free survival (RFS, p=0.005). In multivariate analysis CD25 expression is an independent adverse factor for OS and RFS. High CD25 combined with FLT3-ITD positivity resulted in the poorest OS and RFS (p=0.001 and p=0.003, respectively). CD25 expression remained prognostic within the intermediate cytogenetic risk group. In addition, after the first cycle of chemotherapy, a significantly higher MRD frequency was found in patients expressing CD25 above cut-off (p=0.003). Our results show that CD25 expression is an independent adverse prognostic marker in AML patients < or =60 and correlates with MRD.

Novel multiparameter flow cytometry assay using Syto16 for the simultaneous detection of early apoptosis and apoptosis-corrected P-glycoprotein function in clinical samples.

The fluorescent probe Syto16 has been used successfully to measure P‐glycoprotein (Pgp) function and, separately, early apoptosis and cell death. The present study was designed to evaluate whether the combined use of Syto16, the Pgp blocker PSC833, and 7‐AAD allows simultaneous detection of all parameters, with emphasis on applications in acute myeloid leukemia (AML).

ZAP70 in B-CLL cells related to the expression in NK cells is a surrogate marker for mutational status.

The strongest prognostic factor in chronic B‐cell lymphocytic leukemia (CLL) is the mutational status of the immunoglobulin heavy chain variable region (IGHV) genes. Determination of this mutational status is laborious and therefore not applied in routine diagnostics. A search for “surrogate markers” has been conducted over the past few years. One of the most promising surrogate markers is ZAP70, but standardization of the measurement of ZAP70 has proven to be difficult. Conventionally, ZAP70 expression in CLL cells is related to ZAP70 expression in T cells.

Positive selection for CD90 as a purging option in acute myeloid leukemia stem cell transplants

Several studies showed the benefit of purging of acute myeloid leukemia (AML) stem cell transplants. We reported previously that purging by positive selection of CD34+ and CD133+ cells resulted in a 3–4 log tumor cell reduction (TCR) in CD34− and/or CD133− AML, but has been shown to be potentially applicable in only about 50% of cases. Similar to CD34 and CD133, CD90 marks the hematopoietic CD34 positive stem cells capable of full hematopoietic recovery after myeloablative chemotherapy, and therefore, in the present study, we explored whether a similar purging approach is possible using CD90.