Presented are updated results of allogeneic hematopoietic stem cell transplantations (HSCTs) in 25 adult patients with acute lymphoblastic leukemia (ALL) in complete remission (CR) after a reduced intensity conditioning (RIC) combining fludarabine (150 mg/m 2 ) and melphalan (140 mg/m 2 ) with thymoglobulin (4.5 mg/kg or recently 4.0 mg/kg) followed by early initiation of reduction and withdrawal of prophylactic posttransplant immunosuppression.The median post-transplant follow-up was 32 (range, 4-87) months.Stable engraftment of donor's hematopoiesis was achieved in all patients.Acute graft versus host disease (GVHD) as well as the chronic one were equally observed in four cases (16%).Five patients (20%) relapsed with ALL in the median of 9 (range, 3-15) months after HSCT.During the above post-transplant follow-up, 4 recipients (16%) died.Disease progression and posttransplant complications were the cause of death in three (12%) and one (4%) of them, respectively.The probabilities of 2-year event-free (EFS) and overall survival (OS) were 70.3% (95% CI 51.9-88.7%)and 86.1% (95% CI 71.6-100%), respectively.Presented study confirmed our previously reported promising results and this approach may be considered as an alternative to traditional HSCTs performed in high-risk patients with ALL.

Sir, Monitoring BCR-ABL1 kinase activity by assaying CRKL phosphorylation (P-CRKL), a technique first introduced by Gorre et al. 1, has proven to be a reliable method for the assessment of the sensitivity and/or resistance of leukemia cells from CML patients to tyrosine kinase inhibitors (TKIs). In BCR-ABL1-positive cells, P-CRKL is inhibited by TKIs, including imatinib (IM) 1, 2, dasatinib (DAS) 3, and nilotinib (NIL) 4, in a dose-dependent manner. Originally, Western blotting with anti-CRKL antibody was used by several laboratories, including our own, to detect the change in phosphorylation of CRKL 5. These assays usually involve in vitro analyses of mononuclear cells in which the status of BCR-ABL1 kinase inhibition induced by TKI is measured 2, or in vivo analyses using freshly drawn samples 4, 6. A predictive value was established for the extent of inhibition (e.g., for 50% reduction) of P-CRKL from baseline during a defined time interval of treatment 6. Later, a flow cytometry method to measure in vivo P-CRKL was introduced for discriminating between BCR-ABL1 cells that are sensitive or resistant to TKI 7. In addition, flow cytometric determination of SRC family kinase phosphorylation (P-SFK) was used to measure the responses of patients treated with DAS 8. The main purposes of our study were to enable the rapid evaluation of the TKI sensitivity/resistance of cells from CML patients in a routine clinical laboratory, to enable the rapid identification of patients who are likely to benefit from switching to second-line therapy with another TKI, and to justify regular mutation screening. The approach involves assessing the sensitivity of CML patients' leukocytes to TKIs in vitro by monitoring P-CRKL and P-SFK by flow cytometry. Comparative experiments confirmed a close correlation between the results obtained by immunoblotting and by flow cytometry. The BCR-ABL1-positive K562 cell line was used for optimizing the protocols for assaying P–CRKL and P-SFK by flow cytometry. The cells were maintained in RPMI 1640 (with Glutamax; Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 1% penicillin/streptomycin and 10% FBS (Gibco) in all the experiments. The CML patients in this study were treated at Department of Hemato-Oncology, University Hospital Olomouc. The Ethics Committee of the University Hospital approved the collection of samples for the experiments. Informed consent was obtained from all subjects, in accordance with the Declaration of Helsinki. The in vitro test of the sensitivity of K562 cells and/or patients' leukocytes to TKIs was based on the detection of inhibition of phosphorylation of CRKL and SFK after 1-h incubation with or without defined concentrations of IM (Novartis), NIL (Novartis), and DAS (Bristol-Myers Squibb). The preparation of cell lysates and immunoblotting were performed as previously described 5. The staining protocol for K562 cells and the optimized staining protocol for leukocytes are provided in Supplementary Material and Methods; anti-phospho-CRKL (Tyr207) antibody or anti-phospho-SRC family (Tyr416) antibody (both from Cell Signaling Technologies, Beverly, MA; dilution 1:50) were used as primary antibodies; titration for optimal dilution of anti-phospho-CRKL antibody is shown in Figure S1A. Alexa Fluor 488-labeled secondary antibody (goat antirabbit IgG, Invitrogen) was also titrated for optimal amount used for preparations (Figure S1B). The samples were analyzed on a Cytomics FC 500 5-color flow cytometer (with cxp 2.0 software) equipped with 2 lasers (Beckman Coulter, Miami, FL, USA). The cytometer was routinely calibrated with fluorescent beads (Beckman Coulter). K562 cells were gated based on their forward and side scatter characteristics (FSc vs. SSc), and granulocytes from patients were gated into regions as shown in Figure S2A using their forward and side scatter dot plots. A total of 50 000 cells were acquired for each sample. The median fluorescence intensities (MFIs) of P-CRKL- or P-SFK-labeled untreated and TKIs treated samples were compared. To establish the cutoff for sample negativity determination, the MFI of the isotype control antibody was subtracted from the MFI of the treated and untreated samples (K562 cell line). For patient samples, the average MFI of BCR-ABL1-negative healthy subjects (n = 10) was used to set the negative threshold level of P-CRKL and the ‘normal’ median value of P-SFK (red dotted line in the histograms). A comparison of Western blotting and flow cytometry methods for monitoring the phosphorylation status of CRKL and SFK in K562 cells treated with different concentrations of IM, NIL, or DAS is depicted in Figure 1. The results demonstrated the utility of detecting changes in phosphorylation by flow cytometry, as the data obtained from Western blotting and flow cytometry were comparable. A complete inhibition of P-SFK could only be obtained with 250 nm DAS, and not with IM and NIL in these assay conditions, reflecting a partially BCR-ABL1-independent P-SFK in K562 (Figure 1) 5. When we adapted the K562 protocol for staining leukocytes from patients, very low signals were observed, especially in the case of P-CRKL (Figure S2B). A modified protocol with an increased p-formaldehyde concentration above 2% led to increased fragmentation of cells during fixation (data not shown). The addition of blocking reagent (BSA) in combination with a permeabilizing agent (Tween 20) in the washing buffer only slightly improved the signal intensity. The greatest signals were achieved when 90%, instead of 50%, methanol was used, as shown in Figure S2B. This was the case even when compared with ethanol (data not shown). Therefore, the final optimized protocol for immunostaining leukocytes from patients involved p-formaldehyde fixation, followed by 90% methanol permeabilization. This combination, together with 1% BSA and 0.5% Tween 20 in the washing buffer, provided the best staining that was consistent between experiments. Optimization of staining protocol also included measurements of autofluorescence and negative controls (secondary antibody only; Figure S1C). Impact of different p-formaldehyde concentrations on gate settings for leukocyte subpopulations and on immunoreactivity of both measured phospho-epitopes is shown in Figure S3. The optimized p-formaldehyde/methanol method was applied for in vitro assessment of sensitivity/resistance of patients' leukocytes to TKIs. Specifically, 10 μm IM and NIL and 250 nm DAS, concentrations which exceed the drug IC50 and their therapeutic doses, were used. The in vitro testing of P-CRKL level identified three categories of patients, sensitive (100–70% inhibition), partially resistant (70–30% inhibition), and resistant (inhibition lower than 30%; Figure 2a, our own inhibition scale setting). Alternatively, the patients could be characterized based on the extent of inhibition (i.e., the degree of resistance), without additional subdivision to the groups mentioned above. To assess the elevation of SFK activity, we calculated the ratio of the MFI of TKI-untreated CML samples to that of average healthy control samples (Figure 2b). The in vitro inhibition of P-CRKL and P-SFK was assessed by flow cytometry in samples from 27 patients' leukocytes (Table S1), and the P-CRKL and P-SFK statuses in vitro in most cases correlated well with the clinical response of patients to TKI therapy and reflected BCR-ABL1-dependent (such as depicted in Figure 2c) and likely, other cell-intrinsic causes of therapy resistance. Therefore, the assay presented here seems to be a better clinical response predictor to TKI treatment than similar approach investigating the CD34+ CML compartment 9. Monitoring the resistance of CML cells to TKIs in a clinical laboratory should take into account the speed, cost, sensitivity, specificity, and reliability of the test. Most widely used methods are focused on mutation analyses of the BCR-ABL1 kinase domain. Because the resistance of BCR-ABL1-positive cells to TKIs can be caused and modified by various mechanisms intrinsic to the cell, and not only by kinase domain mutations, in vitro tests based on short-term incubation of cells with the drug can be used to complement methods aimed at identifying mutations 1-4. There are different fixation and permeabilization techniques that are used for measuring phosphorylation events in cells by flow cytometry, with specific applications for individual cell populations. Importance of optimized fixative paraformaldehyde concentration and alcohol treatment for flow cytometric detection of phosphorylated epitopes of selected signaling molecules downstream BCR-ABL1 was clearly documented 10. The measurement of CRKL phosphorylation status by flow cytometry was previously reported in imatinib-treated leukocytes or CD34+ cells using formaldehyde/saponin or commercial permeabilization and fixation reagents in combination with phycoerythrin, FITC or Alexa Fluor 647-labeled primary or secondary antibodies 7. In our study, the combination of p-formaldehyde and methanol as fixation and permeabilization reagents, followed by incubation with the specific primary antibody and secondary Alexa Fluor 488-labeled antibody gave optimal results for determining the phospho-status of CRKL and SFK in both K562 cell line and CML patients. However, in contrast to the K562 staining protocol, the greatest immunosignal in patients' leukocytes was achieved with an increase in blocking reagent, with the addition of Tween 20 in the washing buffer and with the usage of 90% methanol for permeabilization. Although lower concentration of paraformaldehyde (0.25%) resulted in increased P-SFK signal in clinical samples 10, it precluded clear separation of leukocyte subpopulations on the FACS dot plot (Figure S2A). Because immunoreactivity of P-CRKL was not sensitive to paraformaldehyde concentration, we chose 2% paraformaldehyde fixation for our assays. In summary, we describe an adaptation of a simple flow cytometry assay to identify the sensitivity or resistance of BCR-ABL1-positive cells to TKI. Compared with Western blot analysis, this technique is quantitative, more efficient, considerably less time-consuming and has the potential to be developed as a routine screening test that can be included in therapeutic decision algorithms. This work was supported by grant No. NT12218-4/2011 (Czech Ministry of Health), IGA_LF_2014_011 and IGA_LF_2014_001 (Palacky University Grant Agency) and by a Bristol-Myers Squibb research grant (to V.D.). Data S1. Material and Methods. Figure S1A. Titration of P-CRKL immunoreactivity for flow cytometric detection of P-CRKL. Figure S1B. Titration of Alexa Fluor 488-labeled secondary antibody using patients' samples. Figure S1C. Determination of background staining levels. Figure S2A. Impact of two different p-formaldehyde (PFA) concentrations on gate settings for leukocyte subpopulations. Figure S2B. The effect of different staining protocols on P-CRKL flow cytometry monitoring of the granulocyte-gated population after their in vitro incubation without (‘0’) or with 10 μM IM and 250 nM DAS. Figure S3. Impact of different PFA concentrations on immunoreactivity of P-CRKL and P-SFK primary antibodies. Table S1. List of CML patients. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Acute promyelocytic leukemia is a unique entity among acute leukemias. Introduction of all-trans retinoic acid and, subsequently, arsenic trioxide in its treatment has markedly improved treatment outcomes for this once frequently fatal disease. Improved outcomes have also been observed in elderly patients, including those in whom standard intensive therapy is contraindicated because of comorbidities.In our center, a total of 60 APL patients were treated in 1993-2013, of whom 9 were aged 60 or more years. Although most of them had significant comorbidities at the time of diagnosis, eight achieved complete remission. At the time of the analysis, six patients were alive and in long-term remission; two patients died of causes other than APL. The median follow-up was 59 months.Included is case report of a patient with a high comorbidity score whose treatment was markedly reduced and individualized.Our experience shows that, in APL patients a curative approach is generally tolerated and should always be attempted regardless of age and comorbidities.

Secondary acute myeloid leukemia (sAML) may arise from the previous clonal disorder of hematopoiesis, usually from myelodysplastic syndrome (MDS) or from chronic myeloproliferative neoplasia (cMPN) or after exposure to a leukemogenic agent (previous chemotherapy or radiotherapy, some immunosuppressive drugs or environmental leukemogenic agents). Secondary origin of AML is associated with unfavorable prognosis and it is not considered to be conventionally curable (with the exception of secondary acute promyelocytic leukemia). The presented study is a retrospective analysis of patients diagnosed and treated at the Department of Hemato-Oncology, University Hospital Olomouc in 1996-2008. Over that period of time, a total 574 patients with AML were diagnosed. Of those, 430 patients were diagnosed as having primary AML; in 86 patients, sAML transformed from myelodysplastic syndrome and 58 patients were followed or treated for various malignancies or were treated with potentially leukemogenic agents because of non-malignant disorders. Patients with secondary AML are older and less commonly treated with curative intention than those with primary AML. According to cytogenetic findings, their prognosis is often worse. Complete hematologic remission is achieved with a low probability, relapse of the disease occurs frequently and overall survival is worse in almost all prognostic subgroups. With the exception of secondary acute promyelocytic leukemia, the prognosis of which does not differ from very good prognosis of the primary forms, secondary AML is not considered a conventionally curable disease.

Low or undetectable numbers of Philadelphia chromosome-positive leukemic stem cells (Ph + CD34 + CD38 neg ) in chronic myeloid leukemia patients in complete cytogenetic remission after tyrosine kinase inhibitor therapy

Abstract Relapse remains the major cause of treatment failure in AML; however, RQ-PCR assays to detect leukemic fusion transcripts have been shown to identify reliably those patients at highest risk of relapse, allowing development of a more individualized treatment approach. In cases lacking a leukemia-specific MRD marker, quantification of genes over-expressed in AML e.g. Wilms’ Tumor gene (WT1) could provide more a precise measurement of disease response and quality of remission, potentially enhancing risk scores such as the one developed by the MRC used to identify those patients most and least likely to benefit from allogeneic transplant in first CR. WT1 is over-expressed in at least 75% of AML cases. Within the European LeukemiaNet we systematically evaluated 9 published and “in house” WT1 assays. Assays were excluded due to demonstrated lack of RNA-specificity or location within the 3′ region of the gene which has been shown to be subject to deletion or mutation in AML. An assay located within the 5′ region associated with superior sensitivity was ultimately selected following parallel testing in 11 labs and evaluated in 238 diagnostic peripheral blood (PB) and 386 bone marrow (BM) samples. WT1 was over-expressed in the majority, with comparable levels in PB and BM (PB - median 4637 WT1 copies/104ABL copies, range 0–1132709; BM - median 7212, 0–750571), as compared to normal BM (median 19.8, 0–213), PB (median 0.01, 0.01–47.6) and PBSCs (median 6.1, 0–39). In cases over-expressing WT1, kinetics of transcript reduction were evaluated following induction. A greater response was associated with a significantly reduced risk of relapse (hazard ratio 0.65 per log reduction (95% CI 0.43–0.96), p=0.03), although this failed to remain significant when adjusted for age, presenting WBC and cytogenetic risk group, which are key variables in the MRC risk index. Indeed, there was a highly significant correlation between larger log reduction in normalized WT1 transcript level and better risk score (p=0.0001). Sequential analysis of PB and BM samples from 15 AML cases with low WT1 expression (<250 copies) showed no significant modulation in transcript level on regeneration after chemotherapy, indicating that in WT1+ AML, transcript levels detected in follow-up samples reliably reflect disease status. This study provides evidence that recognized pre-treatment risk factors for relapse correlate closely with kinetics of response to induction therapy and lend support to the evaluation of early assessment of MRD to develop more robust risk scores, to enhance risk stratification and identify those patients most suited to proceed rapidly to allogeneic transplant.