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.

The identification of a novel HLA-B*35:279 allele in a Czech patient is described. This allele is identical to the B*35:03:01 variant except the G/A nucleotide exchange at position 652 of the HLA-B gene that corresponds to the amino acid substitution from valine to isoleucine in alpha 3 domain of the HLA-B antigen.

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.

Myelodysplastic syndromes (MDS) are the heterogenous group of diseases with a different risk of development to acute leukemia (AL). The allogeneic stem cell transplantation (alloSCT) is one of the treatment possibilities that is more accessible thanks to availability of recent conditioning regimes. Many questions are still open. The goal of the retrospective analysis was the evaluation of alloSCTs performed in two transplantation centres to estimate the importance of alloSCT for MDS patients (pts), to evaluate prognostic parametres and the course of alloSCTs and overall survival (OS). Patients characteristics: The cohort consisted of 30 pts, male:female ratio was 1:1, transplanted in 1997 – 2010, the age were 23 – 63 year (median 56). According the WHO 2001 classification the pts were initially classified as: 2 (7%) 5q- sy, 2 (7%) RARS, 9 (30%) RCMD, 6 (20%) RAEB I, 11 (36%) RAEB II, according to IPSS as: 2 (6%) low risk, 11 (37%) intermediate-1, 11 (37%) intermediate-2, 6 (20%) high risk. In 13 cases (43%) alloSCT was performed in leukemic phase, in majority of cases after induction/consolidation chemotherapy. 6 (20%) pts underwent alloSCT during the progression into more advanced MDS and 11 (37%) in phase of stabile disease. 11 pts (37%) underwent myeloablative conditioning regime and 19 (63%) pts reduced intensity regime. 14 transplantations were from sibling donors, 16x from unrelated donors, only in 1 case was used bone marrow. Median of follow-up from MDS diagnosis was 33 months, from alloSCT 15 months. Transplantation was performed in median of 11 months from diagnosis of MDS. 11 pts died in the whole cohort – the reason was the progression of disease (4x), infection complications (4x), GVHD and current infection (3x). It dealt initially about the most risky MDS, 8 (27%) transformed into AL, according IPSS 4 (13%) were initially high risk, 7 (23%) intermediate (int-1 and int-2). Median from diagnosis was 12 and from alloSCT 3 months. Other pts have received long time remission and in the low risk MDS transfusion dependence was removed and the quality of life was outstandingly improved. At the moment 19 (63%) pts live with median of follow-up 49 month from MDS diagnosis and 27 months from alloSCT. It is possible curatively influence MDS or secondary AL in favour of patient exploiting aloSCT. Consideration of this curative possibility should have become routine part of diagnostic-prognostic algorithm in MDS patients.

Improved survival has been observed in poor-risk diffuse large B-cell lymphoma (DLBCL) patients treated with high-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) in first complete remission. Retrospective studies have suggested that HDT with ASCT can improve survival also in partial responders but some doubts about the advantage of intensive therapy in such patients still remain. We evaluated retrospectively the results of HDT and ASCT in 55 patients with confirmed DLBCL treated between May 1999 and July 2006. Thirty-six patients (65%) showed partial remission (PR) and 19 patients (35%) reached complete remission (CR) after induction treatment with (44%) or without (56%) concomitant rituximab (R) immunotherapy. After HDT and ASCT, 69% of patients fulfilled the criteria of CR, 22% had unconfirmed CR (CRu), 7% remained in PR and 1 patient (2%) relapsed. Twenty patients in PR after the induction treatment reached CR after ASCT, 12 other PR patients achieved CRu. The 5-year event-free survival (EFS) of the 55 transplanted patients was 76% (95% confidence interval /CI/, 63% to 89%) and the 5-year overall survival (OS) was 85% (95% CI, 73% to 97%). The EFS and OS rates differed significantly only between patients younger than 40 years and older groups (p=0.022 and p=0.046, respectively). On univariate analysis of prognostic factors, EFS and OS were not affected by any of the following: age, sex, stage, subtype of DLBCL, initial lactate dehydrogenase, beta-2-microglobulin and serum thymidine kinase levels, International Prognostic Index (IPI) and age-adjusted IPI scores, induction treatment with or without rituximab and type of primary therapeutic response (CR vs PR). These results show that first-line HDT and ASCT for adults up to the age of 65 years with poor-risk DLBCL is a feasible and effective treatment option even in the era of R-chemotherapy in CR as well as for patients in PR.

The group of 20 patients treated for non-Hodgkin lymphomas and multiple myeloma was examined by stomatologists before planned autologous transplantation of stem cells. After tooth sanation and removal of the sources of local irritation and potential foci of odontogenic focal infection the patients were observed in the course of 4 to 5 weeks after transplantation for the occurrence of oral mucositis. Oral mucositis of 1st to 2nd degree (according to NCI-CTC classification) became manifest in the course of hemato-oncological treatment in all patients in relation to the type of chemotherapy. After successful termination of hemato-oncological treatment a spontaneous disappearance of oral mucositis developed. Low seriousness of oral complications of hemato-oncological therapy confirms the importance of careful stomatological preparation of the patient before planned autologous transplantation.