The Importance of New EBMT Criteria on the Diagnosis of Veno-Occlusive Liver Disease in Children.

BACKGROUND: Early recognition and specific therapy facilitate a favorable disease course in hepatic venous-occlusive disease (HVOD) following hematopoietic stem cell transplantation (HCT). Diagnostic and classification criteria, published by the European Society for Blood and Marrow Transplantation (EBMT), better account for clinical differences in disease presentation in pediatric populations. OBJECTIVES: To compare the course of HVOD in children before and after the implementation of new EBMT criteria. MATERIAL AND METHODS: The study retrospectively evaluates 26 HVODs in 179 children treated in a single HCT unit (Slovakia) comparing the period of 2014-2017 using the Baltimore and modified Seattle criteria with the period of 2018-2021, when new EBMT criteria were adopted. RESULTS: No difference in HVOD incidence (11.2% vs. 14.8%, p = 0.46) and in time of diagnosis post-HCT (15.6 days vs. 15.7 days, p = 0.75) was found. With EBMT criteria we observed more frequent anicteric disease at diagnosis (50% vs. 87.5%, p = 0.04), lower serum bilirubin at diagnosis (3.4 mg/dL vs. 1.23 mg/dL, p = 0.045), and non-significant trends of shorter defibrotide treatment (21.7 days vs. 15.6 days, p = 0.73), decreased mortality (30% vs. 6.2%, p = 0.10) and shorter hospitalization (73.1 days vs. 59.6 days, p = 0.54). CONCLUSIONS: Different time periods around the implementation of new criteria are evaluated, underling that pediatric EBMT criteria for post-transplant HVOD diagnosis appear more sensitive.

Detection of Unknown and Rare Pathogenic Variants in Antithrombin, Protein C and Protein S Deficiency Using High-Throughput Targeted Sequencing.

The deficiency of natural anticoagulants­antithrombin (AT), protein C (PC), and protein S (PS)­is a highly predisposing factor for thrombosis, which is still underdiagnosed at the genetic level. We aimed to establish and evaluate an optimal diagnostic approach based on a high-throughput sequencing platform suitable for testing a small number of genes. A fast, flexible, and efficient method involving automated amplicon library preparation and target sequencing on the Ion Torrent platform was optimized. The cohort consisted of a group of 31 unrelated patients selected for sequencing due to repeatedly low levels of one of the anticoagulant proteins (11 AT-deficient, 13 PC-deficient, and 7 PS-deficient patients). The overall mutation detection rate was 67.7%, highest in PC deficiency (76.9%), and six variants were newly detected­SERPINC1 c.398A > T (p.Gln133Leu), PROC c.450C > A (p.Tyr150Ter), c.715G > C (p.Gly239Arg) and c.866C > G (p.Pro289Arg), and PROS1 c.1468delA (p.Ile490fs) and c.1931T > A (p.Ile644Asn). Our data are consistent with those of previous studies, which mostly used time-consuming Sanger sequencing for genotyping, and the indication criteria for molecular genetic testing were adapted to this process in the past. Our promising results allow for a wider application of the described methodology in clinical practice, which will enable a suitable expansion of the group of indicated patients to include individuals with severe clinical findings of thrombosis at a young age. Moreover, this approach is flexible and applicable to other oligogenic panels.

Allogeneic Hematopoietic Stem Cell Transplantation for Chronic Myelomonocytic Leukemia: Clinical and Molecular Genetic Prognostic Factors in a Nordic Population.

Chronic myelomonocytic leukemia (CMML) is an aggressive disease in which survival after allogeneic hematopoietic stem cell transplantation (HCT) remains relatively poor. An assessment of prognostic factors is an important part of treatment decision making and has the potential to be greatly improved by the inclusion of molecular genetics. However, there is a significant knowledge gap in the interpretation of mutational patterns. This study aimed to describe outcomes of allogeneic HCT in patients with CMML in relation to clinical and molecular genetic risk factors. This retrospective study included 64 patients with CMML who underwent allogeneic HCT between 2008 and 2018, with a median follow-up of 5.4 years. Next-generation sequencing using targeted myeloid panels was carried out on saved material from 51 patients from the time of transplantation. Kaplan-Meier and Cox regression were used for analysis of overall survival (OS), and cumulative incidence with competing risks and Fine and Gray models were used for analysis of relapse and nonrelapse mortality (NRM). Mutations were detected in 48 patients (94%), indicating high levels of minimal residual disease (MRD) positivity at transplantation, even among those in complete remission (CR) (n = 14), 86% of whom had detectable mutations. The most frequently mutated genes were ASXL1 (37%), TET2 (37%), RUNX1 (33%), SRSF2 (26%), and NRAS (20%). Risk stratification using the CMML-specific Prognostic Scoring System molecular score (CPSS-Mol) resulted in 45% of patients moving to a higher risk-group compared with risk stratification using the CPSS. High leucocyte count (≥13 × 109/L), transfusion requirement, and previous intensive chemotherapy were associated with higher incidence of relapse. Being in CR was not linked to better outcomes. Neither ASXL1 nor RUNX1 mutation was associated with a difference in OS, relapse, or NRM, despite being high risk in the nontransplantation setting. TET2 mutations were associated with a significantly higher 3-year OS (73% versus 40%; P = .039). Achieving MRD-negative CR was rare in this CMML cohort, which may explain why we did not observe better outcomes for those in CR. This merits further investigation. Our analyses suggest that the negative impact of ASXL1 and RUNX1 mutations can be overcome by allogeneic HCT; however, risk stratification is complex in CMML and requires larger cohorts and multivariate models, presenting an ongoing challenge in this rare disease.

Short stature and SHOX (Short stature homeobox) variants-efficacy of screening using various strategies.

BACKGROUND: SHOX mutations have previously been described as causes of Léri-Weill dyschondrosteosis (LWD), Langer mesomelic dysplasia (LMD), and idiopathic short stature. The loss of X chromosome-Turner syndrome or mosaic 45,X/46,XX or 46,XY-also leads to the heterozygous loss of SHOX in patients with short stature only or with features similar to LWD. The aim of this study was to assess the efficacy of the targeted screening for SHOX variants, which involved different methods in the laboratory analysis of short stature. We determined the significance and positive predictive value of short stature for the detection of SHOX variants. METHODS: Targeted screening for variants in SHOX involving MLPA, sequencing, karyotyping and FISH was performed in the short stature cohort (N = 174) and control cohort (N = 91). The significance of short stature and particular characteristics for the detection of SHOX variants was determined by Fisher's exact test, and the probability of SHOX mutation occurrence was calculated using a forward/stepwise logistic regression model. RESULTS: In total, 27 and 15 variants influencing SHOX were detected in the short stature and control cohorts, respectively (p > 0.01). Sex chromosome aberrations and pathogenic CNV resulting in diagnosis were detected in eight (4.6%) and five (2.9%) patients of the short stature group and three (3.3%) and one (1.1%) individuals of the control group. VUS variants were discovered in 14 (8.0%) and 11 (12.1%) individuals of the short stature and control groups, respectively. MLPA demonstrated the detection rate of 13.22%, and it can be used as a frontline method for detection of aberrations involving SHOX. However, only mosaicism of monosomy X with a higher frequency of monosomic cells could be reliably discovered by this method. Karyotyping and FISH can compensate for this limitation; their detection rates in short stature group were 3.55% and 13.46% (N = 52), respectively. FISH proved to be more effective than karyotyping in the study as it could reveal cryptic mosaics in some cases where karyotyping initially failed to detect such a clone. We suggest adding FISH on different tissue than peripheral blood to verify sex-chromosome constitution, especially in cases with karyotypes: 45,X; mosaic 45,X/46,XX or 46,XY; 46,Xidic(Y) detected from blood; in children, where mosaic 45,X was detected prenatally but was not confirmed from peripheral blood. The correlation of short stature with the occurrence of SHOX mutations was insignificant and short stature demonstrates a low positive predictive value-15.5% as unique indicator for SHOX mutations. The typical skeletal signs of LWD, including Madelung deformity and disproportionate growth, positively correlate with the findings of pathogenic SHOX variants (p < 0.01) by Fisher's exact test but not with the findings of VUS variants in SHOX which are more prevalent in the individuals with idiopathic short stature or in the individuals with normal height.

Searching for genetic variants associated with thrombophilia.

Thrombotic states are inherited or acquired predisposition for thrombosis in the human vascular system. Nowadays Leiden mutation and mutation in prothrombin G20210A contributing to congenital thrombophilia are routinely tested. These mutations have a high prevalence in the population. Congenital deficiencies of protein S, protein C and antithrombin III are rare thrombophilia with lower population frequency, but higher risk of thromboembolic event. The genetic causes are mutations in the genes, which encode these proteins. The choice of proper molecular genetic testing depends on the difference in the detection of well-known single nucleotide polymorphism or unknown/rare variant. For the detection of causative variant FV Leiden and prothrombin G20210A are mostly used PCR-RFLP, reverse Strip Assay®, allele-specific PCR, TaqMan real-time PCR and SNaPshot®. Precise patient selection should precede the genetic testing of rare variants in anticoagulant proteins. It is appropriate to use methodology of massive parallel sequencing supplemented by a methodology for the detection of larger gene rearrangements - MLPA. We are successfully employing this approach in our institute. This methodology is faster with larger analytic capacity compared to commonly used direct sequencing by Sanger method.

First-line imatinib in elderly patients with chronic myeloid leukaemia from the CAMELIA registry: Age and dose still matter.

We retrospectively evaluated the role of age and dosage in 372 CML patients (170 women, 202 men) treated with first-line imatinib (IMA) from the records of the CAMELIA registry. The median follow-up of the patients was 82.3 (18.0-177.3) months. The treatment results of 80 elderly patients aged over 65 years at diagnosis were compared in analysis "A" with those of 292 younger patients and in analysis "B" with those of 90 patients younger than 40 and 202 patients aged 40-64. The elderly patients had statistically adverse values of the Sokal, ELTS, and ECOG scores and Charlson comorbidity index in both analyses (p from = 0.012 to ≤ 0.001). Despite a more frequent use of a daily dose lower than 400 mg - in 31 elderly patients (38.8%) than in 45 younger ones (15.4%) (p < 0.001), there were no statistically significant differences in the achievement of optimal haematological, cytogenetic, and molecular responses according to the ELN criteria in both the analyses, A and B. The comparisons of overall survival with CML-related death (OSCML) and event-free survival (EFS) were insignificant inanalysis A (p = 0.07 and 0.396, respectively) but progression-free survival (PFS) differed significantly (p = 0.007). In analysis B OSCML and PFS differed significantly (p = 0.027 and 0.003) but EFS was similar (p = 0.351). Elderly patients with a sustained dose of IMA of 400 mg/day have insignificantly better OS, PFS, and EFS compared to patients treated with a lower dosage of IMA. The results in the treatment of the elderly CML patients were comparable with those of the younger ones in terms of the probabilities of the achievement of optimal ELN responses. However, the results for the survival probabilities were influenced by age and the IMA dosage.

Granulocyte transfusions collected after steroid priming for severe infections during neutropenia: A single center experience.

BACKGROUND: There is no universally accepted opinion on the use of granulocyte transfusions collected using apheresis (GTA) in neutropenic patients and severe infection. PATIENTS AND METHODS: The efficacy and safety of GTAs transfused at a single center over 10 years were analyzed retrospectively. GTAs were harvested from voluntary unrelated donors after priming with methylprednisolone using continuous apheresis and hydroxyethylstarch as sedimentation agent. RESULTS: 41 patients with neutropenia and hematologic malignancy (15 females and 26 males aged 22-69 (median 45.5)) were given a median 3.5 GTAs per patient (range: 1-17) containing a median 1.39×1010 granulocyte/GTA (range: 0.65-2.81). The indications for GTA use were soft tissue inflammation, sepsis, and pneumonia in 30, 22, and 14 cases, respectively. After GTA complete (30 patients: 73.2%) or partial (6 patients: 14.6%) healing of the infection was achieved. The success rate was 91.7% in soft tissue infections, 66.7% in invasive fungal infections, and 68% in sepsis. Septic shock (documented in 12 cases) was associated with a poor response (P<0.03; Chi-square test). Clinical worsening was observed in six cases (14.6%); four patients died. No significant short-term side effects of GTA treatment were recorded. CONCLUSIONS: In our study GTAs collected after steroid priming and used for the treatment of infection during severe neutropenia have shown comparable efficacy with several previously reported trials. However retrospective fashion of our study and inhomogeneous group of patients do not allow any firm conclusions. Prospective studies (including patients' registries) are needed for the better clarification of the role and the dose of GTAs necessary for the successful infection management during neutropenia.

Interferon-α Revisited: Individualized Treatment Management Eased the Selective Pressure of Tyrosine Kinase Inhibitors on BCR-ABL1 Mutations Resulting in a Molecular Response in High-Risk CML Patients.

Bone marrow transplantation or ponatinib treatment are currently recommended strategies for management of patients with chronic myeloid leukemia (CML) harboring the T315I mutation and compound or polyclonal mutations. However, in some individual cases, these treatment scenarios cannot be applied. We used an alternative treatment strategy with interferon-α (IFN-α) given solo, sequentially or together with TKI in a group of 6 cases of high risk CML patients, assuming that the TKI-independent mechanism of action may lead to mutant clone repression. IFN-α based individualized therapy decreases of T315I or compound mutations to undetectable levels as assessed by next-generation deep sequencing, which was associated with a molecular response in 4/6 patients. Based on the observed results from immune profiling, we assumed that the principal mechanism leading to the success of the treatment was the immune activation induced with dasatinib pre-treatment followed by restoration of immunological surveillance after application of IFN-α therapy. Moreover, we showed that sensitive measurement of mutated BCR-ABL1 transcript levels augments the safety of this individualized treatment strategy.

Spontaneous splenic rupture in a patient with acute promyelocytic leukaemia during induction chemotherapy.

BACKGROUND: Acute promyelocytic leukaemia (APL) is a subtype of acute myeloid leukaemia with high curability rates. However, it is often accompanied by severe coagulopathy and bleeding risk and thus represents a potentially fatal haematological emergency requiring immediate treatment. Spontaneous splenic rupture is a rare event in all haematological malignancies. Only two clinical cases have been described so far in a setting of APL. CASE REPORT: We report a patient with APL without preceding splenomegaly who underwent urgent splenectomy for spontaneously occurring splenic rupture during induction chemotherapy. After surgery the patient completed induction chemotherapy and achieved complete remission. CONCLUSION: This is the second case of spontaneous splenic rupture without preceding splenomegaly in a patient with APL during induction chemotherapy described so far. Our case demonstrates that emergent splenectomy can be lifesaving even in the unfavourable condition of patient with severe immune deficiency.

Comparison of reduced conditionings combining fludarabine with melphalan or 3-day busulfan in patients allografted for myeloid neoplasms.

In the present study we compared outcomes of patients with myeloid neoplasms undergoing allogeneic hematopoietic stem cell transplantation after fludarabine-based regimens with melphalan (FM140) or 3-day busulfan (FB3). The FM140 and FB3 combinations were administered to 21 and 27 patients, respectively. Efforts for early reduction (from day +30 to 60) and discontinuation (until day +100 to 130) of prophylactic immunosuppression were a component of the post-transplant approach. Following FB3 patients suffered from more severe stomatitis (P = 0.013). In contrast, other manifestations of regimen-related toxicity were more frequent in the FM140 group (P = 0.048). There were no statistically significant differences in the development of graft-versus-host disease, non-relapse mortality, post-transplant remission rate, or relapse incidence. Two-year disease-free survival rates were comparable in the two cohorts (66 vs. 55 %; P = 0.751), and so were the overall survival rates (64 vs. 62 %; P = 0.715). The outcomes of allografted patients with myeloid neoplasms were comparable after the FM140 and FB3 regimens. Relatively high therapeutic response in both groups may have been influenced by early reduction and discontinuation of prophylactic immunosuppression followed by effective immunological control of the malignant clone.

Enlarged memory T-cell pool and enhanced Th1-type responses in chronic myeloid leukemia patients who have successfully discontinued IFN-α monotherapy.

A small proportion of chronic myeloid leukemia patients treated with interferon-α (IFN-α) monotherapy are able to discontinue the treatment without disease relapse although residual leukemia cells are present. Recently, we showed that these patients have increased amount of NK-cells and a distinct blood cytokine profile. We now aimed to study the function of NK- and T-cells in order to understand the role of the immune system in maintaining the treatment response after IFN-α discontinuation. The study included 13 patients: 5 patients were still treated with IFN-α monotherapy (IFN-ON, median treatment time 163 months) and 8 had stopped the treatment successfully (IFN-OFF, median time without therapy 42 months). Detailed immunophenotype and cytokine production of NK- and T-cells was analyzed with flow cytometry. In addition, the cytotoxicity of NK-cells was studied using K562 as target cells and both the degranulation and direct killing was measured. Compared to healthy controls, IFN-OFF patients had increased proportion of CD4(+) effector memory (CCR7(-)CD45RA(-); median 23% vs. healthy 16%, p = 0.009) and CD8(+) central memory T-cells (CCR7(+)CD45RA(-); median 26% vs. healthy 14%, p = 0.004). Further, upon stimulation the IFN-γ/TNF-α cytokine secretion by CD4(+) T-cells was significantly enhanced in IFN-OFF patients (median 13.7% vs. healthy 7.8%, p = 0.01), and CD4+ effector and central memory cells were the main cytokine producers. No similar increase was observed in IFN-ON group (6.5%). In addition, the proportion of NK-cells was significantly increased in IFN-OFF patients (median IFN-OFF 24%, healthy 13%, p = 0.04), but their direct killing of K562 cells was impaired. The cytotoxicity of NK-cells was also diminished in IFN-ON patients. To conclude, in addition to elevated NK-cell count, IFN-OFF patients have increased amount of memory T-cells, which are able to induce strong cytokine response upon stimulation. This activity may contribute to the maintenance of prolonged remission after successful IFN-α discontinuation.

Imatinib and pegylated IFN-α2b discontinuation in first-line chronic myeloid leukemia patients following a major molecular response.

OBJECTIVES: Previous studies indicate that 40-50% of patients with chronic myeloid leukemia in prolonged complete molecular remission may discontinue imatinib therapy without imminent relapse. The combination of pegylated interferon-alpha (Peg-IFN-α2b) and imatinib may increase the rate of successful discontinuation. METHODS: In this pilot study, we prospectively stopped imatinib from patients (n = 12) who had achieved major molecular response (MMR) after ≥12 months of treatment with either imatinib or imatinib+Peg-IFN-α2b. Molecular monitoring was carried out monthly for BCR-ABL1. In addition, analyses of lymphocyte immunophenotype, function, and plasma cytokines were performed. RESULTS: In the monotherapy group, 5/6 patients lost MMR within 4 months. One patient remains to date in MR(4.0) 61 months after discontinuation. In the combination therapy group, 2/6 patients relapsed within 4 months while still receiving Peg-IFN-α2b. Four of six patients were able to discontinue both treatments, but three of these patients relapsed after 3 months. One patient is still in sustained MR(4.0) at 58 months off all treatment. All relapsed patients re-responded to imatinib. The two successfully discontinued patients had either an increased number of NK-cells or functionally active T-cells. CONCLUSIONS: A higher frequency of relapsed patients in our study in comparison with other studies may be due to the shorter duration of imatinib treatment prior to discontinuation. However, in selected patients with an active immune system, even a short duration of TKI therapy (<2 yr) may allow for therapy discontinuation but this needs to be confirmed in larger prospective studies.

A significant proportion of patients with chronic myeloid leukemia and suboptimal response according to European Leukemia Net criteria have excellent prognosis without treatment change.

BACKGROUND: The Recommendations of the European Leukemia Net (ELN) have become an essential tool in the management and prognosis of patients with chronic myeloid leukemia (CML) treated with tyrosine kinase inhibitors (TKIs). However, the definition of suboptimal response remains under discussion. METHODS: We used conventional cytogenetics for the detection of clonal changes in Ph-positive and negative clones. RT-PCR and sequencing were carried out on peripheral blood leukocytes to detect the type of BCR-ABL1 transcript. The BCR-ABL1 mutational status was assessed using sequencing of RT-PCR products. High performance capillary electrophoresis for determination of imatinib (IMA) plasma concentration was used. RESULTS: A retrospective study of 110 patients diagnosed with chronic-phase (CP) CML treated with IMA or 2(nd) generation TKIs in the years 2000-2009 focused on analysis of patients with suboptimal response according to ELN criteria. 40 patients were administered IMA as first-line therapy and 70 had been pretreated with interferon-alpha (IFN-α) with or without Ara-C and/or hydroxyurea (HU) for a median 12 months (range, 1-92 months). After adjusting for the ELN criteria, major molecular response (MMR) was achieved after median 34 and 39 months in 66.7% and 41.7% of patients after the first and second-line IMA therapy with suboptimal response defined as lack of achievement of MMR at the 18(th) month of treatment, respectively. In comparison to patients with optimal response, patients with suboptimal response did not show significant differences in overall survival (OS) or progression-free survival (PFS). Cytogenetic assays demonstrated additional chromosome abnormalities (ACAs): chromosome 8 trisomy in a Ph-negative clone during the IMA treatment (in 1 case) and der(9q) in Ph-positive clone (in 2 cases); in patients receiving first-line IMA only chromosome 8 trisomy was observed which was associated with myelodysplastic syndrome - this was the only case where hematopoietic stem cell transplantation (HSCT) was performed. During the treatment with IMA in both subgroups no regulatory mutations in the ABL kinase domain were confirmed. CONCLUSION: We believe that the category of suboptimal response should be redefined or withdrawn from the ELN 2009 recommendations for management of CML patients treated with TKIs. Patients with suboptimal response who have no additional risks (additional cytogenetic abnormalities or BCR-ABL1 regulatory mutations) may remain on IMA treatment while patients with these risks should be switched to the 2(nd) generation TKIs.

Interferon-alpha in chronic myeloid leukemia revisited: a long-term retrospective study in Central and Northern Moravia.

AIMS: We assessed the long-term outcome of consecutive patients in the chronic phase of chronic myeloid leukemia (CML) treated with interferon-alpha (INF-α) in Central and Northern Moravia between 1989 and 2006. METHODS: A retrospective study focused on the response, prognostic factors and side-effects of INF-α. RESULTS: 118 patients (67 males and 51 females, median age 50 years; range 18-71) were analyzed. The median follow-up was 82.6 months (12.4-212.6). Thirty-six patients (30.5%) achieved major cytogenetic response (CyR) in median of 18.3 months (3.7-47.3) and maintained it for a median of 64.0 months (7.0-176.0). Sixty-one patients treated with INF-α for more than 12 months had an overall survival (OS) of 137.0 months (95% CI 117.6-156.4). Eighteen (29.5%) achieved complete CyR (CCyR). 109 patients discontinued the treatment with INF-α because of hematologic or cytogenetic resistance in 53 (48.7%), progression of CML in 31 (28.4%) and intolerance to INF-α in 17 (15.6%) patients. The percentage of peripheral blasts, leukocyte count (>50x10(9)/L), splenomegaly, anemia (Hgb≤110 g/L) and Sokal score had statistical impact on the OS in univariate assessment but only the Sokal score remained significant in multivariate analysis. Additional cytogenetic abnormalities at diagnosis were associated with poor prognosis. CONCLUSIONS: In most patients, treatment with INF-α had to be stopped because of a failure to induce response, progression of CML or side-effects but nearly one third of patients treated at least for one year had a long-term benefit from INF-α. The best prognosis was associated with achievement of CCyR and negativity of BCR-ABL in nested RT-PCR.

Treatment of chronic myelomonocytic leukemia with 5-azacytidine: case reports.

Epigenetic therapy with hypomethylating agent (5-azacytidine; AZA) is common in the management of specific subtypes of myelodysplastic syndrome (MDS), but there are only few studies in chronic myelomonocytic leukemia (CMML) patients. In this paper our experience with 3 CMML patients treated with AZA is described. In one patient transfusion independency was observed after 4 treatment cycles; in one case a partial response was recorded, but a progression to acute myeloid leukemia (AML) after 13 AZA cycles has appeared. In one patient, AZA in reduced dosage was administered as a bridging treatment before allogeneic stem cell transplantation (ASCT), but in the control bone marrow aspirate (before ASCT) a progression to AML was recorded. Future studies are mandatory for evaluation of new molecular and clinical features which could predict the efficiency of hypomethylating agents in CMML therapy with respect to overall survival, event-free survival, quality-adjusted life year, and pharmacoeconomy.

Biological therapy and the immune system in patients with chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells that has been recognized as a disease responsive to immunotherapy. Despite the huge success of the tyrosine kinase inhibitors (TKIs), CML remains for the most part incurable, probably due to treatment resistance of leukemic stem cells, which are responsible for rapid disease relapse after discontinuation of therapy. Only allogeneic stem cell transplantation enables disease eradication. In addition to the Bcr-Abl1 oncoprotein, TKIs also inhibit off-target kinases (e.g. c-kit, Src, Tec), some of them having physiological functions in immune responses. In vitro studies have implied immunomodulatory effects of TKIs and interferon-alpha (IFN-α), but comprehensive information from in vivo analyses is missing. This review summarizes the recent advances in the field of immunology of CML, including basic information about leukemia-associated antigens and peptide vaccines, that could lead to the incorporation of TKIs and IFN-α in future therapeutic, potentially curative, interventions for CML.

Operational cures after interferon-alpha in patients with chronic myeloid leukemia in Central and Northern Moravia.

We assessed long-term outcome of 118 consecutive patients in chronic phase of chronic myeloid leukemia (CML) treated with interferon-alpha (IFN-α) in the Central and Northern Moravia region between 1989 and 2006 with focus on operational cure. The median follow-up was 82.6 months (range 12.4-212.6). Eighteen (15.3%) patients achieved complete cytogenetic response (CCyR) after median 16.7 (3.7-40.8) months. Nine of these patients (7.6%) achieved BCR-ABL negativity in nested reverse transcriptase-polymerase chain reaction ["complete" molecular response (CMR)] and 6 of them have been operationally cured without any treatment for median 6 (4-10) years, while 2 continue with IFN-α and 1 died from CML-unrelated cause. Operationally cured patients had a significantly lower percentage of initial peripheral promyelocytes, blasts, and erythroblasts than the rest of patients treated for more than 12 months (P=0.01-0.03). Unlike patients with sole CCyR, the majority of whom lost CCyR despite continuing IFN-α therapy and required imatinib, patients who achieved CMR had excellent long-term outcome.

Identification of e6a2 BCR-ABL fusion in a Philadelphia-positive CML with marked basophilia: implications for treatment strategy.

AIM: This is a case report of a 51 year old male with marked splenomegaly, basophilia, severe thrombocytopenia, anemia and high SFKL phosphorylation downstream of Bcr-Abl, investigated for association of the e6a2 BCR-ABL fusion gene and marked basophilia. The treatment strategy implications in patients with Philadelphia positive CML are described. METHODS: RT-PCR and sequencing were carried out on the peripheral blood leukocytes to detect the type of BCR-ABL transcript. The BCR-ABL mutational status was assessed using sequencing of the RT-PCR products. The in vitro test of sensitivity to TKIs was based on detecting inhibited phosphorylation of the Crkl and Phospho-Src family kinases (SFK, Tyr416) using immunodetection. RESULTS: The cytogenetics revealed 90% of Ph+ (Philadelphia) cells in the bone marrow aspirate with no additional clonal chromosomal abnormalities at diagnosis. This correlated with an accelerated phase of the CML. Sequencing analysis of reverse transcribed and PCR amplified BCR-ABL transcript revealed a rare e6a2 fusion, with no evidence for Bcr-Abl kinase domain mutation. Western blot analysis showed high phosphorylation (activation) of Crkl and the Src family of kinases (P-SFK). In vitro test of sensitivity of the patients' leukemic cells to imatinib demonstrated sensitivity of Bcr-Abl tyrosine kinase to imatinib, as assessed by a decrease in phosphorylated Crkl and the disappearance of P-SFK, suggesting that P-Src reflects only the Bcr-Abl-dependent Src activity. The initial treatment strategy was reduced imatinib and search for an unrelated hematopoietic stem cell donor (according to the ELN recommendations). The patient was allografted with peripheral stem cells from an HLA- identical male donor but on day +70 graft failure occurred. He was allografted again with the peripheral stem cells from an HLA-identical female donor, engrafted on day +15 and showed 100% donor chimerism with no evidence of the e6a2 BCR-ABL fusion transcript on day +30. CONCLUSION: The clinical disease course in patients with the rare e6a2 BCR-ABL transcript variant is aggressive. This may be the result of increased kinase activity due to partial loss of the guanine exchange factor/dbl-like domain which mediates the interaction with several Ras-like G-proteins involved in cell proliferation, signal transduction, and cytoskeletal organization. For the above reasons, these patients should receive stem cell transplant immediately after a short course of treatment with imatinib/ dual Src/Abl kinase inhibitor or they should be registered in clinical trials with experimental agents.