Daniel Primo

Impact of BCR/ABL gene expression on the proliferative rate of different subpopulations of haematopoietic cells in chronic myeloid leukaemia

Despite the effects of BCR ABL on cell proliferation, no study has compared the proliferative rate of different haematopoietic cell compartments from chronic myeloid leukaemia (CML) with those of normal bone marrow (NBM). We comparatively analysed the cell cycle distribution and BCR/ABL expression in different compartments of BM cells from 15 CML and 11 NBM. Overall, our results showed similar proliferative indices in CML patients and NBM. However, CD34+ myeloid precursors from CML patients displayed an increased proportion of S + G2/M‐phase cells (P = 0·04), while no significant differences were found between CML and NBM for other BM cell subsets analysed. In BM cells separated by fluorescence‐activated cell sorting, decreasing levels of BCR/ABL mRNA were found from CD34+/CD38+ myeloid precursors to myeloblasts; BCR/ABL expression increased afterwards with a peak at the myelocyte/metamyelocyte stage, decreasing in the more mature band/neutrophil compartment. Unexpectedly, BCR/ABL gene expression showed an inverse correlation with the proportion of S + G2/M‐phase cells (R = −0·33; P = 0·04). These results suggest that in CML, BCR/ABL expression is associated with an increased proliferation of CD34+ myeloid haematopoietic progenitor cells but not of other more mature myeloid precursors, as confirmed by the observation of an inverse correlation between the amount of BCR/ABL transcripts and the proportion of S + G2/M‐phase cells.

Lineage involvement in chronic myeloid leukaemia: comparison between MBCR/ABL+ and mBCR/ABL+ cases

The relationship between different Abelson/breakpoint cluster region (BCR/ABL+) gene rearrangements and the involvement of different haematopoietic cell lineages were investigated in 15 chronic myeloid leukaemia patients. Analysis of purified cell populations confirmed the involvement of the neutrophil (89%), monocytic (89%), eosinophil (88%), erythroid (100%), and CD34+ cells (100%) in virtually all patients, without differences between minor BCR/ABL+ and major BCR/ABL+ cases; BCR/ABL+ B‐ and natural killer (NK)‐cells were detected in 43% and 31% of cases, respectively, whereas BCR/ABL+ T‐cells were rare (7%). All three minor BCR/ABL+ patients showed involvement of both B‐ and NK‐cells, which was infrequent (27%, P = 0·06 and 10%, P = 0·01) among major BCR/ABL+ cases.

Bisursodeoxycholate(ethylenediamine)platinum(II): a new autofluorescent compound. Cytotoxic activity and cell cycle analysis in ovarian and hematological cell lines.

The present paper describes for the first time an intrinsic fluorescent square-planar platinum(II) complex carrying two ursodeoxycholate ligands ([Pt(UDC)2(en)], where UDC(-) = ursodeoxycholate), that emits at room temperature once free in solution. Kinetic studies were carried out in aqueous solution and in the presence of different NaCl concentrations: 4 mM (similar to cytoplasmic concentration) and 150 mM (similar to plasmatic concentration). This novel compound was synthesized from a [PtCl2(en)] complex and shows increased cytotoxic activity against both resting and cycling HeLa cells, with no toxicity for cell lines derived from neoplastic haematopoietic cells.

Drug Discovery Testing Compounds in Patient Samples by Automated Flow Cytometry

Functional ex vivo assays that predict a patient’s clinical response to anticancer drugs for guiding cancer treatment have long been a goal, but few have yet proved to be reliable. To address this, we have developed an automated flow cytometry platform for drug screening that evaluates multiple endpoints with a robust data analysis system that can capture the complex mechanisms of action across different compounds. This system, called PharmaFlow, is used to test peripheral blood or bone marrow samples from patients diagnosed with hematological malignancies. Functional assays that use the whole sample, retaining all the microenvironmental components contained in the sample, offer an approach to ex vivo testing that may give results that are clinically relevant. This new approach can help to predict the patients’ response to existing treatments or to drugs under development, for hematological malignancies or other tumors. In addition, relevant biomarkers can be identified that determine the patient’s sensitivity, resistance, or toxicity to a given treatment. We propose that this approach, which better recapitulates the human microenvironment, constitutes a more predictive assay for personalized medicine and preclinical drug discovery.

Reply to Wan et al

tion of BCR/ABL fusion can only be delineated by FISH techniques. There are two points of note in the present case. First, for cryptic insertion of BCR at 9q34, both ours and several previously reported cases showed normal chromosomes 9 and 22 (ie Ph chromosome negative). Therefore, in Ph-negative BCR/ ABL-positive CML, the absence of extra red signal on interphase ES-FISH and 1R2G1F pattern on interphase D-FISH should raise the possibility of cryptic insertion of BCR at 9q34. Second, even despite G-banded metaphase D-FISH, the presence of BCR/ABL fusion could not be definitely ascertained in the present case, as the fusion signal on 9q may be interpreted as reciprocal ABL/ BCR fusion. We propose that, besides molecular detection of BCR/ABL fusion, the simplest way would be to perform ES-FISH or S-FISH to confirm the presence of BCR/ABL gene fusion. TSK Wan SK Ma CK Li LC Chan Department of Pathology, Division of Haematology, The University of Hong Kong, Queen Mary Hospital, Hong Kong; Department of Paediatrics and Adolescent Medicine, Princess Margaret Hospital, Kowloon, Hong Kong

A novel ex vivo high-throughput assay reveals antiproliferative effects of idelalisib and ibrutinib in chronic lymphocytic leukemia

PI3Kδ (idelalisib) and BTK (ibrutinib) inhibitors have demonstrated significant clinical activity in chronic lymphocytic leukemia (CLL) interfering with the cross-talk between CLL cells and the lymph node microenviroment, yet their mechanism of action remains to be fully elucidated. Here, we developed an ex vivo model with the aim of reproducing the effects of the microenvironment that would help shed light on the in vivo mechanism of action of idelalisib and ibrutinib and predict their clinical efficacy in individual patients. First we explored the effects of various cell-extrinsic elements on CLL apoptosis and proliferation and found that the combination of CpG+IL2+HS5 stromal cell line + human serum +CLL plasma and erythrocyte fractions represented the best co-culture conditions to test the effects of the novel inhibitors. Then, using this assay, we investigated the impact of idelalisib and ibrutinib on both survival and proliferation in 30 CLL patients. While both drugs had a limited direct pro-apoptotic activity, a potent inhibition of proliferation was achieved at clinically achievable concentrations. Notably, up to 10% of CLL cells still proliferated even at the highest concentrations, likely mirroring the known difficulty to achieve complete responses in vivo. Altogether, this novel assay represents an appropriate ex vivo drug testing system to potentially predict the clinical response to novel inhibitors in particular by quantifying the antiproliferative effect.