Regulation of myeloid leukaemia by the cell-fate determinant Musashi.

Chronic myelogenous leukaemia (CML) can progress from a slow growing chronic phase to an aggressive blast crisis phase, but the molecular basis of this transition remains poorly understood. Here we have used mouse models of CML to show that disease progression is regulated by the Musashi-Numb signalling axis. Specifically, we find that the chronic phase is marked by high levels of Numb expression whereas the blast crisis phase has low levels of Numb expression, and that ectopic expression of Numb promotes differentiation and impairs advanced-phase disease in vivo. As a possible explanation for the decreased levels of Numb in the blast crisis phase, we show that NUP98-HOXA9, an oncogene associated with blast crisis CML, can trigger expression of the RNA-binding protein Musashi2 (Msi2), which in turn represses Numb. Notably, loss of Msi2 restores Numb expression and significantly impairs the development and propagation of blast crisis CML in vitro and in vivo. Finally we show that Msi2 expression is not only highly upregulated during human CML progression but is also an early indicator of poorer prognosis. These data show that the Musashi-Numb pathway can control the differentiation of CML cells, and raise the possibility that targeting this pathway may provide a new strategy for the therapy of aggressive leukaemias.

Combining BCR-ABL1 transcript levels at 3 and 6 months in chronic myeloid leukemia: implications for early intervention strategies.

Several groups have shown that that the BCR-ABL1 transcript level measured at 3 or 6 months after starting treatment with tyrosine kinase inhibitors strongly predicts clinical outcomes for patients with chronic myeloid leukemia. In this work, we asked whether the prognostic value of the 3-month transcript level could be improved by combining the 3- and 6-month results. We classified patients treated with imatinib and patients treated with dasatinib according to their transcript levels at 3 months and 6 months. The patients who met the 3-month landmark but failed the 6-month one had outcomes identical to those of patients who met both landmarks, whereas the patients who failed the first landmark but met the second one had prognoses similar to those who failed both landmarks. In summary, early intervention strategies can be based robustly just on the transcript level at 3 months. This trial was registered at www.clinicaltrials.gov as # NCT01460693.

Tyrosine kinase inhibitors impair B-cell immune responses in CML through off-target inhibition of kinases important for cell signaling.

Tyrosine kinase inhibitors (TKIs) have significant off-target multikinase inhibitory effects. We aimed to study the impact of TKIs on the in vivo B-cell response to vaccination. Cellular and humoral responses to influenza and pneumococcal vaccines were evaluated in 51 chronic phase chronic myeloid leukemia (CML) patients on imatinib, or second-line dasatinib and nilotinib, and 24 controls. Following vaccination, CML patients on TKI had significant impairment of IgM humoral response to pneumococcus compared with controls (IgM titer 79.0 vs 200 U/mL, P = .0006), associated with significantly lower frequencies of peripheral blood IgM memory B cells. To elucidate whether CML itself or treatment with TKI was responsible for the impaired humoral response, we assessed memory B-cell subsets in paired samples collected before and after imatinib therapy. Treatment with imatinib was associated with significant reductions in IgM memory B cells. In vitro coincubation of B cells with plasma from CML patients on TKI or with imatinib, dasatinib, or nilotinib induced significant and dose-dependent inhibition of Bruton's tyrosine kinase and indirectly its downstream substrate, phospholipase-C-γ2, both important in B-cell signaling and survival. These data indicate that TKIs, through off-target inhibition of kinases important in B-cell signaling, reduce memory B-cell frequencies and induce significant impairment of B-cell responses in CML.

Antagonistic activities of the immunomodulator and PP2A-activating drug FTY720 (Fingolimod, Gilenya) in Jak2-driven hematologic malignancies.

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.

Preclinical and clinical efficacy of XPO1/CRM1 inhibition by the karyopherin inhibitor KPT-330 in Ph+ leukemias.

As tyrosine kinase inhibitors (TKIs) fail to induce long-term response in blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL), novel therapies targeting leukemia-dysregulated pathways are necessary. Exportin-1 (XPO1), also known as chromosome maintenance protein 1, regulates cell growth and differentiation by controlling the nucleocytoplasmic trafficking of proteins and RNAs, some of which are aberrantly modulated in BCR-ABL1(+) leukemias. Using CD34(+) progenitors from CML, B-ALL, and healthy individuals, we found that XPO1 expression was markedly increased, mostly in a TKI-sensitive manner, in CML-BC and Ph(+) B-ALL. Notably, XPO1 was also elevated in Ph(-) B-ALL. Moreover, the clinically relevant XPO1 inhibitor KPT-330 strongly triggered apoptosis and impaired the clonogenic potential of leukemic, but not normal, CD34(+) progenitors, and increased survival of BCR-ABL1(+) mice, 50% of which remained alive and, mostly, became BCR-ABL1 negative. Moreover, KPT-330 compassionate use in a patient with TKI-resistant CML undergoing disease progression significantly reduced white blood cell count, blast cells, splenomegaly, lactate dehydrogenase levels, and bone pain. Mechanistically, KPT-330 altered the subcellular localization of leukemia-regulated factors including RNA-binding heterogeneous nuclear ribonucleoprotein A1 and the oncogene SET, thereby inducing reactivation of protein phosphatase 2A tumor suppressor and inhibition of BCR-ABL1 in CML-BC cells. Because XPO1 is important for leukemic cell survival, KPT-330 may represent an alternative therapy for TKI-refractory Ph(+) leukemias.

European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013.

Advances in chronic myeloid leukemia treatment, particularly regarding tyrosine kinase inhibitors, mandate regular updating of concepts and management. A European LeukemiaNet expert panel reviewed prior and new studies to update recommendations made in 2009. We recommend as initial treatment imatinib, nilotinib, or dasatinib. Response is assessed with standardized real quantitative polymerase chain reaction and/or cytogenetics at 3, 6, and 12 months. BCR-ABL1 transcript levels ≤10% at 3 months, <1% at 6 months, and ≤0.1% from 12 months onward define optimal response, whereas >10% at 6 months and >1% from 12 months onward define failure, mandating a change in treatment. Similarly, partial cytogenetic response (PCyR) at 3 months and complete cytogenetic response (CCyR) from 6 months onward define optimal response, whereas no CyR (Philadelphia chromosome-positive [Ph+] >95%) at 3 months, less than PCyR at 6 months, and less than CCyR from 12 months onward define failure. Between optimal and failure, there is an intermediate warning zone requiring more frequent monitoring. Similar definitions are provided for response to second-line therapy. Specific recommendations are made for patients in the accelerated and blastic phases, and for allogeneic stem cell transplantation. Optimal responders should continue therapy indefinitely, with careful surveillance, or they can be enrolled in controlled studies of treatment discontinuation once a deeper molecular response is achieved.

Responses to second-line tyrosine kinase inhibitors are durable: an intention-to-treat analysis in chronic myeloid leukemia patients.

Second-generation tyrosine kinase inhibitors (2G-TKIs) are effective at inducing complete cytogenetic responses (CCyRs) in approximately half of chronic myeloid leukemia patients treated while still in the chronic phase and after failing imatinib. It is less clear whether these responses are durable. In the present study, we report the clinical outcome of 119 patients who received a 2G-TKI as second-line treatment while still in the chronic phase. In an intention-to-treat analysis, the 4-year probabilities of overall and event-free survival were 81.9% and 35.3%, respectively. Sixty-two patients discontinued the initial 2G-TKI because of resistance or intolerance. To further explore the durability of cytogenetic responses, irrespective of the need for a third-line TKI, we used the concept of "current CCyR-survival" (c-CCyRS). The c-CCyRS at 4 years was 54.4%. After introduction of a 2G-TKI, 77 patients had a 3-month BCR-ABL1/ABL1 transcript ratio of ≤ 10% and had significantly superior overall survival (91.3% vs 72.1%, P = .02), event-free survival (49.3% vs 13.0%, P < .001), and c-CCyRS (67.2% vs 11.2%, P = .0001) compared with the 33 patients with ratios > 10%. The 3-month molecular response was the only independent predictor for overall survival. Using an intention-to-treat analysis, we have shown that the responses to second-line therapies are durable. Patients destined to fare poorly can be identified early during therapy.

Predictive value of early molecular response in patients with chronic myeloid leukemia treated with first-line dasatinib.

Dasatinib is effective therapy for newly diagnosed patients with chronic myeloid leukemia, but not all patients respond well. We analyzed the outcome of patients treated with dasatinib as first-line therapy to identify patients who are more likely to fare poorly. The 8.6% of patients who at 3 months had a BCR-ABL1/ABL1 ratio > 10% had a significantly worse 2-year cumulative incidence of complete cytogenetic response (58.8% vs 96.6%, P < .001) and molecular responses than the remaining patients with a lower transcript levels. The predictive value of the 3-month transcript level could be improved using the dasatinib-specific transcript level cut-offs, namely, 2.2%, 0.92%, and 0.57% for complete cytogenetic response, 3 log and 4.5 log reductions in the transcript level, respectively. The study was registered at www.clinicaltrials.gov as #NCT01460693.

Graft invariant natural killer T-cell dose predicts risk of acute graft-versus-host disease in allogeneic hematopoietic stem cell transplantation.

Invariant natural killer T (iNKT) cells are powerful immunomodulatory cells that in mice regulate a variety of immune responses, including acute GVHD (aGVHD). However, their clinical relevance and in particular their role in clinical aGVHD are not known. We studied whether peripheral blood stem cell (PBSC) graft iNKT-cell dose affects on the occurrence of clinically significant grade II-IV aGVHD in patients (n = 57) undergoing sibling, HLA-identical allogeneic HSCT. In multivariate analysis, CD4(-) iNKT-cell dose was the only graft parameter to predict clinically significant aGVHD. The cumulative incidence of grade II-IV aGVHD in patients receiving CD4(-) iNKT-cell doses above and below the median were 24.2% and 71.4%, respectively (P = .0008); low CD4(-) iNKT-cell dose was associated with a relative risk of grade II-IV aGVHD of 4.27 (P = .0023; 95% CI, 1.68-10.85). Consistent with a role of iNKT cells in regulating aGVHD, in mixed lymphocyte reaction assays, CD4(-) iNKT cells effectively suppressed T-cell proliferation and IFN-γ secretion in a contact-dependent manner. In conclusion, higher doses of CD4(-) iNKT cells in PBSC grafts are associated with protection from aGVHD. This effect could be harnessed for prevention of aGVHD.

The next questions in chronic myeloid leukaemia and their answers.

PURPOSE OF REVIEW: In this review, we analyze some of the topical issues in the clinical management of chronic myeloid leukaemia (CML). RECENT FINDINGS: In recent years, the management of CML patients has increased in complexity as molecular monitoring has brought to the clinical scene new therapeutic targets and the second-generation tyrosine kinase inhibitors have been licensed for first-line use. SUMMARY: In this article, we will try to answer some of the questions that a practising physician may face in clinical practice, such as: What should be the aim of therapy? What is the best front-line therapy? Which patients should receive an allogeneic stem cell transplant?

A common novel splice variant of SLC22A1 (OCT1) is associated with impaired responses to imatinib in patients with chronic myeloid leukaemia.

Approximately one-third of patients with chronic myeloid leukaemia will fail to achieve or maintain responses to imatinib. Changes in solute carrier family 22 (organic cation transporter), member 1 (SLC22A1, also termed OCT1), the main transporter for imatinib, have been proposed as a possible predictive factor. We analysed SLC22A1 mRNA levels and single nucleotide polymorphisms (SNPs) located in exon 7 in 153 diagnostic whole blood samples from two patient cohorts. The level of SLC22A1 expression did not significantly correlate with imatinib failure or achievement of molecular remission. The SNP 408V>M (g.1222G>A) was present in 65% of patients and was associated in all cases with an eight base-pair insertion (8(+) allele) at the 3' end of exon 7. The latter generates an alternative splice site, leading to a premature stop codon. M420del was found in 33% of patients and never in cis with 8(+) (the 3(-) allele). Significantly longer times to 1% and 0·1% molecular responses (by quantitative reverse transcription polymerase chain reaction) were seen in patients with 8(+) 8(+) or 8(+) N compared to those with the remaining four genotypes (N = no insertion or deletion). Patients lacking 8(+) and 3(-) (NN, 18%) showed the best outcomes overall. Thus, while SLC22A1 expression does not appear to affect response, alterations in its splicing or amino acid sequence may do so.

Three decades of transplantation for chronic myeloid leukemia: what have we learned?

Last year marked 30 years of hematopoietic stem cell transplantation as a curative treatment of chronic myeloid leukemia (CML). Initially studies used stem cells from identical twins but techniques rapidly developed to use cells first from HLA-identical siblings and later unrelated donors. During the 1990s CML became the most frequent indication for allogeneic transplantation worldwide. This, together with the relative biologic homogeneity of CML in chronic phase, its responsiveness to graft-versus-leukemia effect and the ability to monitor low level residual disease placed CML at the forefront of research into different strategies of stem cell transplantation. The introduction of BCR-ABL1 tyrosine kinase inhibitors during the last decade resulted in long-term disease control in the majority of patients with CML. In those who fail to respond and/or develop intolerance to these agents, transplantation remains an effective therapeutic solution. The combination of tyrosine kinase inhibitors with transplantation is an exciting new strategy and it provides inspiration for similar approaches in other malignancies.

Poor adherence is the main reason for loss of CCyR and imatinib failure for chronic myeloid leukemia patients on long-term therapy.

We studied the relation between adherence to imatinib measured with microelectronic monitoring systems and the probabilities of losing a complete cytogenetic response (CCyR) and of imatinib failure in 87 CCyR chronic myeloid leukemia patients receiving long-term therapy. We included in our analysis the most relevant prognostic factors described to date. On multivariate analysis, the adherence rate and having failed to achieve a major molecular response were the only independent predictors for loss of CCyR and discontinuation of imatinib therapy. The 23 patients with an adherence rate less than or equal to 85% had a higher probability of losing their CCyR at 2 years (26.8% vs 1.5%, P = .0002) and a lower probability of remaining on imatinib (64.5% vs 90.6%, P = .006) than the 64 patients with an adherence rate more than 85%. In summary, we have shown that poor adherence is the principal factor contributing to the loss of cytogenetic responses and treatment failure in patients on long-term therapy.

The BCR-ABLT315I mutation compromises survival in chronic phase chronic myelogenous leukemia patients resistant to tyrosine kinase inhibitors, in a matched pair analysis.

The BCR-ABL T315I mutation confers resistance to currently licensed tyrosine kinase inhibitors in chronic myelogenous leukemia. However, the impact of this mutation on survival in early stages of disease, in chronic phase, has never been detailed. Using matched pair analysis, a cohort of 64 patients with chronic phase chronic myelogenous leukemia harboring a T315I mutation and resistant to imatinib mesylate was compared to a similar cohort of 53 chronic phase patients resistant to imatinib, but with no detectable T315I mutation, in the pre-ponatinib era. These patients were matched according to age at diagnosis, interval between disease diagnosis and start of imatinib treatment, and duration of imatinib therapy. Kaplan-Meier survival analyses demonstrated the significant negative impact of the presence of the T315I mutation on overall survival (since imatinib-resistance: 48.4 months for T315I(+) patients versus not reached for T315I(-) ones; P=0.006) and failure-free survival (since imatinib-resistance: 34.7 months for T315I(+) patients versus not reached for T315I(-) patients; P=0.003). In addition, Cox proportional hazard models adjusted on overall survival demonstrated the negative influence of the T315I mutation (P=0.02, HR=2.54). These results confirm early assumptions concerning the poor prognosis of chronic phase chronic myelogenous leukemia patients with the T315I mutation who are not eligible for allogeneic transplantation, and demonstrate the need for more therapeutic options.

Acute myositis secondary to Epstein-Barr virus in the absence of infectious mononucleosis with severe rhabdomyolysis.

SummaryRhabdomyolysis is characterised by muscle breakdown which causes myoglobin light chain release and can result in renal injury. While some of the most common causes of rhabdomyolysis are trauma related, others include toxins, autoimmune processes or viral aetiologies. We present the case of a 20s-year-old man, with no significant medical history, who presented to the emergency department with a 1-week history of weakness, myalgias, nausea, vomiting and subjective fevers. A review of systems and physical exam were otherwise unremarkable, including being negative for sore throat, dysphagia and lymphadenopathy. On presentation, the patient was noted to have dark urine with a creatine kinase value of 452 458 U/L and an elevated creatinine at 7.23 mg/dL. The patient denied any trauma or increased physical activity. His toxin screen and autoimmune workup were negative. The patient's serological workup was significant for acute Epstein-Barr virus (EBV) infection, without additional viral coinfection or mononucleosis. During his hospitalisation course, the patient was managed with supportive care including haemodialysis. The patient made a full renal recovery and was discharged with scheduled outpatient follow-up. This case highlights the recognition of an acute EBV infection causing rhabdomyolysis in the absence of mononucleosis or concomitant infection.

CT-guided microwave ablation of the nonsurgical aspergilloma.

Background: Aspergillosis is a fungal infection that can lead to development of an aspergilloma, especially in patients with a history of cavitary lung disease. It is generally managed with antifungal therapy followed by surgical intervention. There are, however, limited options for the nonsurgical patient. Microwave ablation is already an effective, minimally invasive treatment being used in some lung malignancies and may be an alternative and definitive treatment in the inoperable patient. Methods: Two patients were considered for microwave ablation following their diagnoses of aspergillosis with hemoptysis. We sought to evaluate the efficacy of CT-guided microwave ablation of an aspergilloma in these patients who were not good candidates for surgical intervention. Results: Two male patients presented with hemoptysis and were found to have an aspergilloma. Case 1 was initially treated with antifungals and did not improve. He proceeded with VATS, and the procedure was aborted intraoperatively secondary to a frozen chest cavity. The patient subsequently elected to undergo CT-guided microwave ablation. He did not experience any immediate complications but was hospitalized for hemoptysis several weeks later. He developed alveolar hemorrhage and ultimately succumbed to PEA arrest.Case 2 was without hemoptysis at follow up and chose to pursue microwave ablation for definitive treatment. Case 2 developed post ablation pneumothorax requiring chest tube placement. Follow-up CT chest imaging was consistent with resolution of the aspergilloma. Conclusion: Microwave ablation is a safe and effective therapeutic approach in the treatment of lung malignancy with no severe or death related complications. There are almost no absolute contraindications. Microwave ablation may be utilized as a therapeutic option in the treatment of an aspergilloma in the non-surgical patient. This novel application may challenge the current gold standard of surgical intervention.

EBMT risk score predicts outcome of allogeneic hematopoietic stem cell transplantation in patients who have failed a previous transplantation procedure.

Increasing numbers of allogeneic hematopoietic stem cell transplantation (allo-SCT) are being performed for patients who have failed a previous allogeneic or autologous SCT. We investigated whether the EBMT risk score could predict outcome after a subsequent allo-SCT. We analyzed prognostic factors in 124 consecutive patients who underwent a second transplantation using an allogeneic donor at our institution. Patients with either a first autologous (N = 64) or first allogeneic (N = 60) SCT were included. Age, disease stage, time interval from diagnosis to transplantation, donor type, and donor-recipient sex combination were used to establish a score from 0 to 7 points, from which 3 groups were identified. The 5-year survival probability decreased from 51.7% for risk scores 0-3 (low, n = 25), to 29.3% for risk score 4 (intermediate, n = 42), and only 10.4% for risk scores 5-7 (high, n = 57), P = .001. We propose that the EBMT risk score can identify patients most likely to benefit from a second transplantation.

Efficacy of tyrosine kinase inhibitors (TKIs) as third-line therapy in patients with chronic myeloid leukemia in chronic phase who have failed 2 prior lines of TKI therapy.

We analyzed a cohort of 26 patients with chronic myeloid leukemia who had failed imatinib and a second tyrosine kinase inhibitor but were still in first chronic phase and identified prognostic factors for response and outcomes. The achievement of a prior cytogenetic response on imatinib or on second-line therapy were the only independent predictors for the achievement of complete cytogenetic responses on third-line therapy. Younger age and the achievement of a cytogenetic response on second line were the only independent predictors for overall survival (OS). At 3 months, the 9 patients who had achieved a cytogenetic response had better 30-month probabilities of complete cytogenetic responses and OS than the patients who had failed to do so. Factors measurable before starting treatment with third line therapy and cytogenetic responses at 3 months can accurately predict subsequent outcome and thus guide clinical decisions.

EVI-1 oncogene expression predicts survival in chronic-phase CML patients resistant to imatinib treated with second-generation tyrosine kinase inhibitors.

Activation of the EVI-1 oncogene has been reported in acute myeloid leukemia, chronic myeloid leukemia (CML) in blast crisis, and less commonly, in chronic-phase CML patients. We screened an unselected cohort of 75 chronic-phase CML patients who had failed imatinib for expression of EVI-1 and sought a correlation with subsequent outcome on the second-generation tyrosine kinase inhibitors dasatinib (n = 61) or nilotinib (n = 14). The 8 patients (10.7%) who expressed EVI-1 transcripts detectable by real-time polymerase chain reaction had significantly lower event-free survival, progression-free survival, and overall survival than patients with undetectable transcript. The predictive value of EVI-1 expression was validated in an independent cohort. In a multivariate analysis, EVI-1 expression status and the best cytogenetic response obtained on imatinib were the only independent predictors for overall survival, progression-free survival, and event-free survival. Our data suggest that screening for EVI-1 expression at the time of imatinib failure may predict for response to second-line TKI therapy and consequently aid clinical management.